Methods For Screening Of Novel Functions Of Receptor Like Kinases

  *US20100170006A1*
  US20100170006A1                                 
(19)United States 
(12)Patent Application Publication(10)Pub. No.: US 2010/0170006 A1
 Yang et al.(43)Pub. Date:Jul.  1, 2010

(54)METHODS FOR SCREENING OF NOVEL FUNCTIONS OF RECEPTOR LIKE KINASES 
    
(75)Inventors: Zhenbiao Yang,  Riverside, CA (US); 
  Stephen Karr,  Camarillo, CA (US) 
    
 Correspondence Address: 
 Joseph R. Baker, APC
Gavrilovich, Dodd & Lindsey LLP 
 
 4660 La Jolla Village Drive, Suite 750 
 San Diego, CA 92122  (US) 
    
(73)Assignee:THE REGENTS OF THE UNIVERSITY OF CALIFORNIA,  Oakland, CA (US), Type: US Company 
(21)Appl. No.: 12/641,641 
(22)Filed: Dec.  18, 2009 
 Related U.S. Application Data 
(60)Provisional application No. 61/138,902, filed on Dec.  18, 2008.
 
 Publication Classification 
(51)Int. Cl. A01H 001/00 (20060101); A01H 005/00 (20060101); C12N 015/63 (20060101); C40B 040/06 (20060101); C40B 050/00 (20060101); C12N 005/04 (20060101); C12Q 001/02 (20060101); C40B 040/02 (20060101)
(52)U.S. Cl. 800/279; 800/278; 800/298; 800/301; 435/320.1; 506/16; 506/23; 435/419; 435/29; 506/14

        

(57)

Abstract

The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases.
 Claim(s),  Drawing Sheet(s), and Figure(s)
 
 


CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Ser. No. 61/138,902, filed Dec. 18, 2008, the disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases.

BACKGROUND

[0003] Receptor-like kinases (RLKs) form a large monophyletic gene family of approximately 600 members in plants (Shiu and Bleecker, Plant receptor-like kinase gene family: diversity, function and signaling. Science STKE, re22, 2001; and Shiu and Bleecker, Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proceeding of the National Academy of Science U.S.A. 98:10763-10768, 2001). They consist of proteins that contain a single extracellular domain that is thought to be the site of ligand binding, connected to a single kinase domain, via a single transmembrane domain. Upon ligand binding the kinase domain is capable of generating a phosphorylation signaling cascade. Because of the sheer size of this gene family and of the potential functional redundancy among closely related gene family members, not much is known about the function of many of these important signaling genes. What little that was known shows that RLKs have many diverse roles in plants such as, hormone perception, plant defense, plant development and cell growth.

SUMMARY

[0004] The disclosure provides a method of identifying the function of receptor-like kinases (RLKs) that modulate plant function and morphology comprising: identifying a family of RLKs that comprise at least 50% sequence identity in the extracellular and transmembrane domains; using a set of PCR primer pair, generating from a cDNA library of RLKs a plurality of RLKs lacking a functional kinase domain (DN-RLKs); cloning the DN-RLKs into a plant species to obtain recombinant plants comprising at least one DN-RLK from the plurality of DN-RLKs; expressing the DN-RLKs; and identifying recombinant plants having morphological or functional traits different than a wild-type plant species. In one embodiment, the family of RLKs has at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity between members of the family. In another embodiment, the PCR primer pair comprise a first primer comprises a sequence corresponding to the extracellular domain end of the coding sequence and the second primer comprises a sequence that truncates the kinase domain or induces a mutation in the kinase domain that results in a domain lacks kinase activity. The plant species can be any plant species including crop plants. In one embodiment the plant species is Arabidopsis sp.
[0005] The disclosure also provides transgenic plants generated by the methods of the disclosure. In one embodiment, the transgenic plant comprises improved growth characteristics, pathogen resistance, plant height or metabolic activity compared to a wild-type plant.
[0006] The disclosure also provides a method of generating a transgene comprising a dominant-negative receptor-like kinases (RLKs) that modulate plant function and morphology comprising: identifying a family of RLKs that comprise at least 50% sequence identity in the extracellular and transmembrane domains; using a set of PCR primer pair, generating from a cDNA library of RLKs a plurality of RLKs lacking a functional kinase domain (DN-RLKs); cloning at least one DN-RLK from the plurality of DN-RLKs into a vector.
[0007] The disclosure also provides a method for modulating plant height, organ shape, metabolism, growth characteristics or pathogen resistance comprising the step of expressing a transgene of the disclosure in a plant, wherein the transgene encodes a receptor-like kinase (RLK) protein lacking an active receptor domain or kinase domain and wherein expression of the transgene modulates plant height, organ shape, metabolism, growth characteristics or pathogen resistance.
[0008] The disclosure also provides a method for enhancing the plant height, organ shape, metabolism, growth characteristics or pathogen resistance of a plant, comprising the steps of: (a) introducing a transgene of the disclosure into a plant, wherein the transgene encodes a receptor-like kinase protein lacking an active receptor domain or kinase domain and wherein expression of the transgene enhances the plant height, organ shape, metabolism, growth characteristics or pathogen resistance of the crop plant; and (b) growing the transgenic plant under conditions in which the transgene is expressed to enhance the plant height, organ shape, metabolism, growth characteristics or pathogen resistance of the plant.
[0009] The disclosure also provides a library of dominant-negative RLK-encoding polynucleotides wherein the polynucleotide encodes a dominant-negative RLK lacking a receptor domain or kinase domain, the library obtained by the method of the disclosure. In one embodiment the library comprise an RLK having at least 90%, 95%, 98%, 99% or 100% identity to a sequence found in the AGI accession number of Table 1.
[0010] The disclosure also provides a method of making a library of dominant-negative RLK encoding polynucleotides comprising: (a) identifying a family of RLKs having at least 50% identity to one another; (b) mutating the RLKs having identity to disrupt function ligand binding function or kinase function; and (c) cloning the mutant RLKs. The method can further comprise transforming plant cells with the mutant RLKs, growing the cells and identifying desirable phenotypes.
[0011] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0012] FIGS. 1A-J shows distance mapping tree of the extracellular domains of all receptor-like kinases (RLKs) in Arabidposis thaliana.
[0013] FIG. 2 Examination of partial distance map for the wall-associated kinase family 1.5 showing nearest neighbor protein identities. 50% was used for the cutoff point.
[0014] FIG. 3 Model of dominant negative (DN) receptor-like kinase action in vivo.
[0015] FIGS. 4A-B shows a flow chart and demonstration. A) Flowchart of gene expression database directed experiment design for DNRLKs. B) Actual demonstration of using Genevestigator gene expression data for programmed cell death (PCD) to examine senescence phenotype of DN-1.5-11 (DNWAKL14).
[0016] FIGS. 5A-F shows root and seedling growth. A-C) Examination of root hairs from 7-day old seedlings grown on MS media. A) WT, B) DN-1.12-23 (At5g01890) showing root hair branching, and C) SALK053567C (At3g28040) homozygous line for 1.12-23 subfamily member showing similar branched root hair phenotype. D-F) UV-confocal microscope images of 3-day old dark grown hypocotyls grown on MS media without supplemented sucrose. D) WT, E) DN-1.1-4 (At3g14350) showing block-like epidermal cells, and F) SALK077702 (At1g53730) showing enhanced block-like epidermal cells.

DETAILED DESCRIPTION

[0017] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the gene” includes reference to one or more genes and equivalents thereof, and so forth.
[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described.
[0019] Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.
[0020] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
[0021] All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which are described in the publications, which might be used in connection with the description herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. The disclosures of International Application No. PCT/US09/65766, filed Nov. 24, 2009, and International Application No. PCT/US09/65777, filed Nov. 24, 2009, are incorporated herein by reference in their entirety.
[0022] There are over 400 receptor-like kinases (RLKs) in Arabidposis that have predicted transmembrane domains and extracellular domains larger than 100 amino acids, for many of which the function is unknown or unclear. In order to better understand the functions of these RLKs the disclosure provides an approach whereby kinase-free versions of the RLKs (i.e., the dominant negative: DN) were generated and over-expressed in Arabidposis and subsequent changes in phenotypes were examined (Shpak et al., Dominant-negative receptor uncovers redundancy in the Arabidposis ERECTA leucine-rich repeat receptor-like kinase signaling pathway that regulates organ shape. The Plant Cell, 15:1095-1110, 2003). This approach works in two ways. One, the kinase free RLK may homo- or heterodimerize with the endogenous RLKs and the result would be a termination of the phosphorylation cascade, or secondly it could compete for and bind up ligand(s) that are required for signaling of the endogenous RLKs and again diminish any downstream signaling (see, e.g., FIG. 3). To date, 100 kinase free RLK constructs have been generated and 72 of these stably transformed into Arabidposis as homozygous lines. This covers over 63% of all the RLKs in kinase-free (DN) constructs and over 45% coverage in homozygous lines. These homozygous lines were then investigated for morphological, developmental and stress response phenotypes.
[0023] The dominant negative (DN) approach described herein can be used to study many different classes of receptor-like kinases in Arabidposis. This approach has allowed for the investigation of many important functions of RLKs such as nutrient sensing and response to abiotic stress. The disclosure demonstrates that the dominant negative effect shown in LRR-RLKs was not limited to just this family of RLKs but appears to work in the other classes as well.
[0024] A method of the disclosure provides a method of identifying the function of receptor-like kinases (RLKs) that modulate plant function and morphology comprising identifying a family of RLKs that comprise at least 50% sequence identity in the extracellular and transmembrane domains; using a set of PCR primer pair, generating from a cDNA library of RLKs a plurality of RLKs lacking a functional kinase domain (DN-RLKs); cloning the DN-RLKs into a plant species to obtain recombinant plants comprising at least one DN-RLK from the plurality of DN-RLKs; expressing the DN-RLKs; and identifying recombinant plants having morphological or functional traits different than a wild-type plant species. In one embodiment, the family of RLKs has at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity between members of the family. In another embodiment, the PCR primer pair comprise a first primer comprises a sequence corresponding to the extracellular domain end of the coding sequence and the second primer comprises a sequence that truncates the kinase domain or induces a mutation in the kinase domain that results in a domain lacks kinase activity. The plant species can be any plant species including crop plants. In one embodiment the plant species is Arabidposis sp.
[0025] As described more fully below, percent identity and alignment can be performed using commercially and generally available sequence algorithms. The percent identity can be modified to range from 50% to more than 99% (and any value there between). As set forth in Table 1 a large number of sequences are available in general databases related to RLKs. These sequences can be utilized from such databases, screened and categorized into families using the percent identity. Typically the identity of the extracellular and transmembrane domains are used as a criteria for identifying a family member; however, the criteria can use one or the other or both such domain and may further include the kinase domain.
[0026] Once a family is characterized a set of primers can be designed based upon the sequences having identity across all family member or which utilize a set of degenerate primers having a degree of identity. One primer will have identity to the coding sequences of the extracellular domain (e.g., proximal or equal to the terminal end) and the other primer will have identity to the transmembrane domain or kinase domain, such that amplification of the primer pair by PCR techniques will generate a product having the extracellular and transmembrane domain, but may be lacking a kinase domain or may have induced mutation to generate a non-functional kinase domain such that the amplified product comprises a dominant-negative RLK (DN-RLK) polynucleotide encoding a DN-RLK polypeptide. The DN-RLK polynucleotide can then be cloned into a suitable vector for expression in a desired plant cell or cell type.
[0027] The vector can then be used to transform a plant cell of interest to generate a transgenic plant. Expression of the vector can be measured using various techniques as described more fully below. The function of the expressed DN-RLK can be detected by functional, phenotypical and morphological changes in the transgenic plant compared to a wild-type plant.
[0028] By comparing the DN-RLK and knockout lines confirmed that the DN-RLK was responsible for the observed phenotype, which was stronger than the knockout. This was also the case with the DN-ERECTA mutant in Shpak et al., where they observed a similar phenotype to the ERECTA knockout (Shpak et al., 2003, supra). They also showed that there was functional redundancy of the ERECTA receptor by expressing the DN in an ERECTA knockout, this phenotype was more severe than the single mutant suggesting that the DN was interfering with ERECTA-like receptors and adverting functional redundancy problems. The disclosure further demonstrates that the most common morphological phenotypes when grown on soil affected the leaf size and shape and an increase in the time it took for the plants to flower. It is also important to note that under normal conditions the majority (76.4%) of the DN-RLKs showed no detectable phenotype. This was a logical observation as RLKs may function in many diverse ways: development, pathogen response, light response or nutrient response, to name just a few and under normal conditions these RLKs may not be expressed or necessary until a cue elicits their action. The disclosure provides sensitizing screens and bioinformatics that allowed for the discovery of novel phenotypes. The DN-RLK provided by the disclosure is an excellent resource for future investigations of receptor-like kinase functions in Arabidposis as well as agronomically important species like rice or corn.
[0029] The dominant negative receptor kinases methods and compositions provided by the disclosure allow for the perturbation of the function of many subfamily members at once. The preliminary steps involved compiling all of the known RLKs (˜600) from the publicly available databases (TAIR and PlantsP) and journal articles (Shiu and Bleecker, supra). These were then aligned using the extracellular and transmembrane domains only and a distance map was generated (FIG. 1). This distance map was used to group RLKs into over 250 subfamilies (Table 1). Subfamily categories were determined by a nearest neighbor alignment that looks at the percent shared identity to the adjacent RLKs, all neighbors with over 50% identity were classified as being in the same subfamily (FIG. 2), this alignment is available on the website, (http:˜˜bioinfo.ucr.edu/projects/RLK/Analyses/Final/DecisionTree.html). FIG. 2 is an example of how the nearest neighbor distance map was used to generate the RLK subfamilies. In this example a section of the family was used to demonstrate how the protein similarities in the extracellular domain were used to generate the subfamilies. Subfamily 1.5-7 (Group 1.5-7) contains four genes (At4g31100, At1g19390, At1g17910, At4g31110) that are all greater then 50% identical to each other but less then 50% identical to subfamily 1.5-6 (At1g79680, At1g69730) and 1.5-2 (At1g16260). This method was used on all the RLKs to generate the subfamilies used in this study (FIG. 2).
[0030] Upon further investigation RLKs without predicted transmembrane domains (137 RLKs) or of less than 450 amino acids in length (122 RLKs) or in the class of receptor-like cytoplasmic kinases (113 RLCKs), were removed which left 430 RLKs that constituted 157 RLK subfamilies. It was these 157 subfamilies that were used to generate the 72 dominant negative RLK lines.
[0031] The disclosure is based in part upon the hypothesis that the overexpression of dominant negative would act as either a ligand trap by binding up free ligands to a catalytically inactive RLK and/or form a dimer with the native RLK but be unable to propagate a signal because there was no active kinase domain to transphosphorylate (FIG. 3). In subfamilies with many members the dominant negative can homo/heterodimerize with other subfamily members and attenuate the signal and thereby allow for determination of the function of that RLK subfamily.
[0032] Furthermore, gene expression data (via Genevestigator) was used to better target searches for RLK gene function (FIG. 4). The meta analyzer tool available on the Genevestigator website, https:˜˜www.genevestigator.ethz.ch, to enter in the AGI numbers of all of the RLKs (the maximum allowed at one time is 100) and analyze the expression patterns in each of three categories: developmental stages, tissue regions and biotic and abiotic elicitors (these can be: hormones/chemicals, light, nutrients as well as pathogens). This approach allowed a look at DN-RLK lines that showed no apparent phenotype when grown under normal growth conditions and to use sensitized screening to elucidate phenotypes. This approach also allowed us to look for other RLKs that may have similar functions based on similar expression patterns.
[0033] Seventy-two different DN-RLK constructs, which represents 72 subfamilies of RLKs that effectively encompass 45.9% of the RLKs were generated in Arabidposis that fit the initial cutoff criteria (Table 2). Initially the expression levels of the DN-RLKs were examined to determine if the expression levels of the DN-RLKs were detectible and expressed above wild type levels using semi-quantitative RT-PCR. In all cases the DN-RLK transgenic lines had higher then wild type gene expression. For each experiment the maximum number of independent lines used was five unless there were only fewer than those amounts.
[0034] Of the 72 DN-RLK subfamilies examined on soil only 23.6% (17 out of 72) showed a developmental or morphological phenotype. When using more selective growing conditions (nutrient deprivation, light regimes or detailed root examination) many more phenotypes were found, with about 64% (37 out of 58, 14 were not examined) showing a phenotype (Table 2). Previously, it was shown that the dominant negative approached worked but this was limited to the family of receptor-like kinases called leucine-rich repeat (LRR) RLKs (Steak et al., 2003). Over half of the DN-RLKs examined (39 of 72) were not LRR-RLKs (Table 2). It appears that the DN approach will also work on non-LRR-RLKs, which makes it an excellent tool for examining RLK function.
[0035] FIG. 5 examines two dominant negative constructs that showed morphological phenotypes that were then confirmed using knockout mutants. The first DN-RLK (1.12-23, At5g01890) was from a LRR-RLK subfamily containing 3 members. All independent lines exhibited a root hair phenotype where the root hairs were shorter and thicker then wild type and were branched (FIG. 5B). A homozygous knockout line was obtained from the ARBC (At3g28040, SALK 093189) and this mutant also had this same root hair phenotype, only not a severe as the DN (FIG. 5C). The difference in severity of phenotype is probably due to the DN having a stronger effect than the single knockout. This again illustrates the utility of the DN approach for overcoming functional redundancy. The other DN-RLK construct is a member of the Strubbelig Receptor Family (SRF) and exhibited a change in hypocotyl epidermal cell size and shape. This gene subfamily only contains two members (At3g14350 and At1g53730). In the wild type the epidermal cells are long and rectangular, however in the DN the epidermal cells are smaller and more square-like (FIGS. 5D/E).
[0036] The most common morphological phenotypes observed when grown on soil were changes in leaf shape, size or number as well as a delay in flowering time compared to the wild type. Out of the 72 DN-RLK constructs only two showed a reduction in leaf size (1.3-9, At5g49760; 1.5-5, At1g16110) while fives showed and increase in leaf size (1.1-2, At3g21630; 1.1-4, At3g14350; 1.9-1, At5g38990; 1.9-7, At1g34300; 1.12-30, At5g62710) (Table 2). A delay in flowering time over one week more than the wild type plants was the most common morphological phenotype with 6 different DN-RLK constructs showing a delay in flowering phenotype (1.2-31, At2g28250; 1.7-10, At1g70520; 1.9-1, At5g38990; 1.9-7, At1g34300; 1.9-8, At4g32300; 1.14-5, At1g78940) (Table 2).
[0037] When seedlings were grown under limiting conditions (e.g., nutrient deprivation) on Petri dishes the phenotypes of all of the DN-RLKs was very reproducible from one experiment to the next. The most variability of phenotypes from one growing period to the next was when the DN-RLKs were grown on soil. This may be due to the differences in temperature, light quality and watering frequency from one time to the next. In cases where there are many different independent lines (>10) for a DN-RLK construct a gradation in the severity of the phenotype was observed. This may be due to differences in DN-RLK expression levels based on the region of the transgenes insertion into the genome. Otherwise the phenotypes of the DN-RLK constructs are very reproducible and consistent when growth conditions can be rigorously maintained.
[00001] [TABLE-US-00001]
  TABLE 1
 
Receptor-like kinases from Arabidopsis arranged by PlantsP family
  and subfamily, based on extracellular domain. Names were from TAIR
  website. For those with no name currently: PK = protein kinase; LRR =
  leucine rich repeat receptor-like kinase. Transmembrane domain (TMD)
  prediction was determined using the HMMTOP (http://hmmtop.enzim.hu/) and
  the region in parentheses was the amino acid residues predicted to be in
  the membrane. Size is the predicted amino acid number for the protein.
  AGI# is the TAIR classification. PNAS is the functional classification
  found in Shiu and Bleecker (2001). Tree position is the location of the
  RLK in the distance map (FIG. 2.1)
      TMD Predicted   Size       Tree
  ID #   Name   (HMMTOP)   (aa)   AGI#   PNAS   Position
 
  Family 1.Other
  1.Other-1   PK   N   351   At4g11890   DUF26   489
  1.Other-2   PK   N   377   At5g60080   NF   N.A.
  1.Other-2   PK   N   398   At5g60090   NF   N.A.
  1.Other-3   PK   N   312   At5g11400   RLCK II   593
  1.Other-3   PK   N   336   At5g11410   RLCK II   592
  1.Other-4   PK   Y (9-31; 156-178)   361   At5g61570   LRR III   330
  1.Other-4   PK   Y (4-26)   359   At5g07620   LRR III   331
  1.Other-5   PK   Y (7-30; 85-108)   359   At5g42440   LRR X   396
  1.Other-5   PK   Y (7-29)   332   At5g46080   N.A.   91
  1.Other-6   PK   Y (54-78)   445   At2g30940.1   TAKL   125
  1.Other-6   PK   Y (54-78)   447   At2g30940.2   TAKL   125
  1.Other-7   PK   Y (4-27)   380   At3g26700   RLCK IX   572
  1.Other-8   PK   N   557   At3g08760   N.A.   N/A
  1.Other-9   LRR   Y (6-29; 192-210;   518   At4g20790   LRR VI   587
      217-235)
  1.Other-   LRR   Y (6-23; 173-190;   502   At5g39390   LRR XII   547
  10     203-220)
  1.Other-   LRR   Y (297-320; 370-393)   666   At5g45800   LRR VII   339
  11
  1.Other-   ERL P   Y (602-621)   1048   At5g10020   LRR III   334
  12
  1.Other-   LRR   Y (553-570)   1007   At2g27060   LRR III   335
  12
  1.Other-   InRPK1   Y (614-638)   977   At4g20940   LRR III   333
  12
  1.Other-   EPL P   Y (8-31; 246-263;   633   At2g46850   N.A.   539
  13     284-307)
  1.Other-   Duel PKD   Y (718-737)   851   At2g32800   L-Lectin   535
  14
  1.Other-   PK   N   350   At1g52540   N.A.   540
  15
  Family
  1.1
  1.1-1   SRF8   N   338   At4g22130   LRR V   94
  1.1-2   PK   Y (6-23; 234-252;   617   At3g21630   LysM   285
      372-389)
  1.1-2   RLK   Y (121-145; 237-260)   657   At1g51940   LysM   286
    (LysM)
  1.1-3   PK   Y (243-262; 506-525)   654   At3g01840   N.A.   603
  1.1-3   RLK   N   612   At2g23770   LysM   605
    (LysM)
  1.1-3   RLK   Y (121-145; 237-260)   651   At2g33580   LysM   604
    (LysM)
  1.1-4   SRF7   Y (288-312)   717   At3g14350.1   LRR V   93
  1.1-4   SRF7   Y (251-275)   680   At3g14350.2   LRR V   93
  1.1-4   SRF7   Y (288-312)   689   At3g14350.3   LRR V   93
  1.1-4   SRF6   Y (291-314)   719   At1g53730   LRR V   92
  1.1-5   SRF5   Y (267-291)   693   At1g78980   LRR V   99
  1.1-5   SRF4   Y (233-257)   646   At3g13065   LRR V   98
  1.1-6   SRF2   Y (294-318)   735   At5g06820   LRR V   100
  1.1-7   SRF3   Y (7-29; 36-58;   776   At4g03390   LRR V   95
      317-339)
  1.1-7   SRF9   Y (8-26; 342-360;   768   At1g11130   NF   N.A.
    (SUB)   472-490)
  1.1-7   SRF1   Y (9-28; 312-331)   772   At2g20850   LRR V   96
  Family
  1.2
  1.2-1   Pto KI 1 P   N   406   At2g43230   RLCKVIII   69
  1.2-1   Pto KI 1 P   N   408   At3g59350.1   RLCKVIII   70
  1.2-1   Pto KI 1 P   N   366   At3g59350.2   RLCKVIII   70
  1.2-2   Pto KI 1 P   N   361   At1g06700   RLCKVIII   67
  1.2-2   Pto KI 1 P   N   366   At2g30740   RLCKVIII   66
  1.2-3   Pto KI 1 P   N   338   At2g30730   RLCKVIII   68
  1.2-4   Pto KI 1 P   N   365   At2g41970   RLCKVIII   75
  1.2-5   Pto KI 1 P   N   363   At1g48210   RLCKVIII   73
  1.2-5   Pto KI 1 P   N   388   At1g48220   RLCKVIII   76
  1.2-5   Pto KI 1 P   N   364   At3g17410   RLCKVIII   74
  1.2-6   Pto KI 1 P   N   365   At2g47060.1   RLCKVIII   71
  1.2-6   Pto KI 1 P   N   397   At2g47060.2   RLCKVIII   71
  1.2-6   Pto KI 1 P   N   361   At3g62220   RLCKVIII   72
  1.2-7   APK1A P   N   375   At1g24030   RLCKVII   46
  1.2-8   PK   N   442   At2g07180   RLCKVII   20
  1.2-8   PK   Y (268-287)   450   At1g72540   RLCKVII   24
  1.2-8   PK   Y (175-197)   408   At5g56460   RLCKVII   22
  1.2-9   PK   N   202   At1g61590   RLCKVII   23
  1.2-10   PK   N   462   At2g05940   RLCKVII   18
  1.2-10   PK   N   457   At5g35580   RLCKVII   17
  1.2-10   PK   N   424   At2g26290   RLCKVII   19
  1.2-11   PK   Y (274-291)   410   At5g47070   RLCKVII   30
  1.2-11   PK   N   388   At4g17660   RLCKVII   29
  1.2-12   APK1A P   Y (238-257)   490   At3g01300   RLCKVII   6
  1.2-12   APK1A P   N   493   At5g15080   RLCKVII   7
  1.2-13   APK1A P   Y (81-100)   376   At3g28690   RLCKVII   8
  1.2-14   LMBR1   Y (18-41; 133-156;   310   At3g08930.1   NF   N.A.
      177-201;
      222-245; 276-297)
  1.2-14   LMBR1   Y (6-28; 45-62;   526   At3g08930.2   NF   N.A.
      89-111; 126-150;
      235-257; 349-372;
      399-423;
      438-461; 492-513)
  1.2-14   PK   N   435   At2g39110   RLCKVII   27
  1.2-14   PK   N   420   At5g03320   RLCKVII   26
  1.2-15   PK   N   399   At1g74490   RLCKVII   13
  1.2-16   APK2B   N   426   At2g02800.1   RLCKVII   10
  1.2-16   APK2B   N   426   At2g02800.2   RLCKVII   10
  1.2-16   APK2B   N   426   At1g14370   RLCKVII   9
  1.2-17   PK   N   412   At1g26970   RLCKVII   11
  1.2-17   APK1A P   N   387   At1g69790   RLCKVII   12
  1.2-18   APK1A   N   410   At1g07570.1   RLCKVII   2
  1.2-18   APK1A   N   410   At1g07570.2   RLCKVII   2
  1.2-18   APK1A/B P   Y (11-27)   423   At2g28930   RLCKVII   1
  1.2-19   PK   N   389   At5g02290.1   RLCKVII   3
  1.2-19   PK   N   389   At5g02290.2   RLCKVII   3
  1.2-20   BIK1   N   395   At2g39660   RLCKVII   4
  1.2-20   APK2B P   N   389   At3g55450   RLCKVII   5
  1.2-21   APK1A P   Y (280-297)   414   At2g17220.1   RLCKVII   14
  1.2-21   APK1A P   Y (279-296)   413   At2g17220.2   RLCKVII   14
  1.2-21   PK   Y (278-294)   419   At4g35600   RLCKVII   16
  1.2-22   PK   Y (284-303)   423   At1g07870   RLCKVII   35
  1.2-22   PK   N   424   At2g28590   RLCKVII   34
  1.2-23   PK   N   386   At3g20530   RLCKVII   36
  1.2-23   RLK   N   389   At1g61860   RLCKVII   40
  1.2-24   RLK   N   585   At1g20650   RLCKVII   42
  1.2-24   APK2B P   N   381   At1g76370   RLCKVII   41
  1.2-25   PK   N   379   At3g24790   RLCKVII   39
  1.2-26   PBS1   N   456   At5g13160   RLCKVII   32
  1.2-26   PK   N   378   At5g02800   RLCKVII   33
  1.2-26   PK   N   513   At5g18610   RLCKVII   31
  1.2-27   PK   Y (247-266)   558   At3g02810   RLCKVII   43
  1.2-27   PK   Y (260-279)   414   At3g07070   RLCKVII   37
  1.2-27   PK   Y (258-275)   636   At5g16500   RLCKVII   44
  1.2-28   PK   N   410   At5g01020   RLCKVII   21
  1.2-28   TSL   Y (399-416)   688   At5g20930   N.A.   N.A.
  1.2-29   PK   Y (6-30; 259-281)   744   At2g20300   Extensin   78
  1.2-29   NF   NF   ??   At4g02101   Extensin   79
  1.2-29   PK   Y (568-585; 629-652)   1113   At5g56890   Extensin   77
  1.2-30   PK   N   484   At1g76360   RLCKVII   15
  1.2-31   RERK1 L   Y (71-90; 103-122;   565   At2g28250   N.A.   82
      392-411)
  1.2-32   CDG1   N   432   At3g26940   RLCKVII   45
  1.2-33   PK   N   343   At2g28940   RLCKVII   28
  1.2-34   PBS1 P   N   405   At4g13190   RLCKVII   38
  Family
  1.3
  1.3-1   PK   Y (7-28)   261   At5g54590.1   LRRI   225
  1.3-1   PK   Y (8-30)   440   At5g54590.2   LRRI   225
  1.3-2   AtPK2324L   Y (7-26)   663   At1g49730.1   URK1   275
  1.3-2   AtPK2324L   Y (7-26; 256-275;   450   At1g49730.2   URK1   275
      322-341)
  1.3-2   AtPK2324L   Y (200-219; 266-285)   394   At1g49730.3   URK1   275
  1.3-2   PK   Y (8-25; 258-275)   663   At3g19300   URK1   276
  1.3-3   CRPK1L-1   Y (0-27; 339-356;   824   At5g24010   CrRLK1L-1   198
      408-432)
  1.3-3   PK   Y (407-426; 472-488)   834   At2g23200   CrRLK1L-1   207
  1.3-3   CRPK1L-1   Y (408-432; 463-480)   815   At2g39360   CrRLK1L-1   206
  1.3-3   PK   Y (8-24; 386-402;   849   At1g30570   CrRLK1L-1   202
      431-455)
  1.3-4   CRPK1L-1   Y (6-23)   829   At5g59700   CrRLK1L-1   196
  1.3-4   PK   Y (8-25; 404-428;   830   At3g46290   CrRLK1L-1   195
      441-465)
  1.3-5   PK   Y (21-43; 439-461;   871   At2g21480   CrRLK1L-1   199
      476-493)
  1.3-5   PK   Y (23-45; 440-462;   878   At4g39110   CrRLK1L-1   200
      477-494)
  1.3-6   CRPK1L-1   Y (424-446; 499-516)   842   At5g61350   CrRLK1L-1   201
  1.3-6   PK (THE1)   Y (7-26; 314-338;   855   At5g54380   CrRLK1L-1   197
      418-442)
  1.3-7   FERONIA   Y (11-28; 447-470;   895   At3g51550   CrRLK1L-1   205
      485-502)
  1.3-7   PK   N   850   At3g04690   CrRLK1L-1   203
  1.3-7   PK   Y (7-23)   858   At5g28680   CrRLK1L-1   204
  1.3-8   LRR   Y (55-77; 88-104;   1032   At5g01950   LRR   211
      643-665)       VIII-1
  1.3-8   LRR   Y (546-570)   939   At1g06840   LRR   212
            VIII-1
  1.3-8   LRR   Y (537-561)   935   At5g37450   LRR   213
            VIII-1
  1.3-8   LRR CLV1 P   Y (376-394)   783   At3g53590   LRR   210
            VIII-1
  1.3-9   RLK (LRR-   Y (8-25; 514-537;   953   At5g49760   LRR   214
    VIII-1)   558-582)       VIII-1
  1.3-9   RLK (LRR-   Y (7-26; 562-585;   946   At5g49770   LRR   215
    VIII-1)   616-634)       VIII-1
  1.3-9   LRR   Y (612-633; 683-702)   1006   At5g49780   LRR   216
            VIII-1
  1.3-9   LRR   ND   ND   At1g79620.1   LRR   217
            VIII-1
  Family
  1.4
  1.4-1   PK   Y (7-31 395-411   776   At2g39180   CR4L   86
      432-448)
  1.4-1   PK   Y (24-43 83-100)   775   At3g09780   CR4L   87
  1.4-1   ACR4   Y (17-39 437-455)   895   At3g59420   CR4L   88
  1.4-2   PK   Y (6-28)   751   At5g47850   CR4L   89
  1.4-2   NF   NF   ND   At2g55950   CR4L   90
  Family
  1.5
  1.5-1   CRCK3   N   510   At2g11520   RLCK IV   220
  1.5-2   WAKL8   Y (316-337; 446-463)   720   At1g16260   WAKL   178
  1.5-3   WAKL2   Y (346-363; 472-489)   748   At1g16130   WAKL   169
  1.5-3   WAKL4   Y (368-392; 498-515)   779   At1g16150   WAKL   170
  1.5-4   WAKL22   Y (6-23; 351-368;   751   At1g79670.1   WAKL   171
      476-493)
  1.5-4   WAKL22   Y (6-25; 314-331;   714   At1g79670.2   WAKL   171
      439-456)
  1.5-5   WAKL6   Y (362-379; 488-505;   642   At1g16110   WAKL   168
      536-553;
      584-601)
  1.5-5   WAKL5   Y (340-361; 467-484;   711   At1g16160   WAKL   167
      515-534)
  1.5-5   WAKL1   Y (359-376; 485-502;   730   At1g16120   WAKL   165
      533-552)
  1.5-5   WAKL3   Y (322-338; 444-460;   690   At1g16140   WAKL   166
      491-510)
  1.5-6   WAKL9   Y (373-397; 503-520)   792   At1g69730   WAKL   176
  1.5-6   WAKL10   Y (8-25; 359-383;   769   At1g79680   WAKL   177
      489-506)
  1.5-7   WAKL17   Y (369-390; 500-517)   786   At4g31100   WAKL   172
  1.5-7   WAKL18   Y (9-26; 345-362;   756   At4g31110   WAKL   173
      472-489)
  1.5-7   WAKL13   Y (7-26; 381-400;   764   At1g17910   WAKL   175
      510-527)
  1.5-7   WAKL11   Y (378-397; 507-524)   788   At1g19390   WAKL   174
  1.5-8   WAK1   Y (362-379; 488-505;   642   At1g21250   WAK   184
      536-553;
      584-601)
  1.5-8   WAK4   N   738   At1g21210   WAK   180
  1.5-8   WAK2   Y (332-350; 371-389)   732   At1g21270   WAK   181
  1.5-8   WAK5   N   733   At1g21230   WAK   179
  1.5-8   WAK3   Y (343-361; 382-400)   741   At1g21240   WAK   183
  1.5-9   WAKL16   Y (6-24; 29-47;   433   At3g25490   WAKL   185
      76-93)
  1.5-10   WAKL20   Y (7-24; 293-316;   657   At5g02070   WAKL   186
      418-435)
  1.5-10   WAKL15   N   639   At3g53840   WAKL   187
  1.5-11   WAKL14   Y (24-46; 283-306)   708   At2g23450.1   WAKL   192
  1.5-11   WAKL14   Y (24-46; 283-306)   708   At2g23450.2   WAKL   192
  1.5-11   WAKL21   Y (8-26; 248-272;   622   At5g66790   WAKL   193
      283-299)
  1.5-12   PK   Y (256-275)   636   At1g69910   LRK10L-1   194
  1.5-13   PK   N   605   At1g18390   LRK10L-1   189
  1.5-14   PK   Y (14-31)   686   At5g38210   LRK10L-1   190
  Family
  1.6
  1.6-1   PK   Y (8-26; 35-54)   452   At5g20050   N.A.   148
  1.6-1   PK   Y (268-287)   450   At1g72540   RLCKVII   24
  1.6-2   PK   Y (32-54)   676   At1g55200   PERKL   63
  1.6-2   PK   Y (35-57)   753   At3g13690   PERKL   64
  1.6-2   PK   Y (110-127; 393-410)   669   At5g56790   PERKL   65
  1.6-3   PK   Y (21-45)   437   At4g34500   TAKL   124
  1.6-4   PK   Y (26-50)   512   At3g59110   TAKL   116
  1.6-4   PK   Y (25-48; 345-362)   494   At2g42960   TAKL   115
  1.6-5   GPK1   Y (21-40)   467   At3g17420   TAKL   119
  1.6-5   PK   Y (21-40)   484   At5g18500   TAKL   120
  1.6-6   PK   Y (24-48; 210-227)   386   At1g01540.1   TAKL   122
  1.6-6   PK   Y (24-48)   472   At1g01540.2   TAKL   122
  1.6-6   PK   Y (26-49; 218-235)   329   At4g01330   TAKL   121
  1.6-6   PK   Y (22-46)   492   At4g02630   TAKL   123
  1.6-7   PK   Y (179-196; 227-250)   625   At1g11050   RKF3L   163
  1.6-7   RKF3   Y (7-24; 169-186;   617   At2g48010   RKF3L   164
      213-231)
  1.6-8   PK   Y (58-82; 199-216)   509   At1g52290   PERKL   50
  1.6-9   PERK3   Y (124-144)   509   At3g24540   PERKL   47
  1.6-10   PERK4   Y (151-170)   633   At2g18470   PERKL   54
  1.6-11   PERK5   Y (187-209)   670   At4g34440   PERKL   53
  1.6-11   PERK7   Y (175-198)   699   At1g49270   PERKL   51
  1.6-11   PERK6   Y (186-210)   700   At3g18810   PERKL   52
  1.6-11   PERK1   Y (140-162; 336-353)   652   At3g24550   PERKL   48
  1.6-12   PERK12   Y (247-266)   720   At1g23540   PERKL   58
  1.6-12   PERK11   Y (263-282)   718   At1g10620   PERKL   59
  1.6-12   PERK13   Y (236-255)   710   At1g70460   PERKL   57
  1.6-13   PERK10   Y (329-352)   760   At1g26150   PERKL   60
  1.6-13   PERK8   Y (237-259)   681   At5g38560   PERKL   62
  1.6-14   TMK1   Y (6-23; 481-505;   942   At1g66150   LRR IX   281
      539-556)
  1.6-14   LRR   N   886   At1g24650   LRR IX   283
  1.6-14   TMK1L   Y (483-500; 643-660)   943   At2g01820   LRR IX   282
  1.6-14   LRR   Y (475-494; 517-534)   928   At3g23750   LRR IX   284
  Family
  1.7
  1.7-1   LRR   Y (7-24; 89-106)   112   At3g14840   LRR   470
            VIII-2
  1.7-2   PK   N   372   At4g00960   DUF26   424
  1.7-3   PK   N   390   At1g16670   LRR   476
            VIII-2
  1.7-3   PK   N   393   At3g09010   LRR   475
            VIII-2
  1.7-4   PK   Y (235-254)   425   At1g70740   DUF26   481
  1.7-5   LRR   Y (16-35; 562-581;   1049   At1g29740   LRR   471
      600-619)       VIII-2
  1.7-5   LRR   Y (13-35)   940   At1g29730   LRR   472
    (RKF1)         VIII-2
  1.7-6   LRR   Y (624-643; 723-742)   1014   At1g07650   LRR   468
            VIII-2
  1.7-6   LRR   Y (571-590; 603-622;   1030   At1g53430   LRR   467
      840-859)       VIII-2
  1.7-6   LRR   Y (10-29; 576-595;   1035   At1g53440   LRR   466
      608-627)       VIII-2
  1.7-7   LRR   Y (7-24; 89-106)   112   At3g14840   LRR   470
            VIII-2
  1.7-7   LRR   Y (6-23; 569-586;   953   At1g53420   LRR   469
      607-624)       VIII-2
  1.7-8   LRR   N   1032   At1g56140   LRR   480
            VIII-2
  1.7-8   LRR   Y (7-24; 605-624;   1032   At1g56130   LRR   478
      637-656)       VIII-2
  1.7-8   LRR   Y (618-637; 650-673)   1045   At1g56120   LRR   479
            VIII-2
  1.7-9   CRK16   N   352   At4g23240   DUF26   419
  1.7-10   CRK2   Y (260-284; 327-344)   649   At1g70520   DUF26   485
  1.7-10   CRK1   Y (6-23)   600   At1g19090   DUF26   484
  1.7-10   CRK3   Y (259-283; 296-312)   646   At1g70530   DUF26   483
  1.7-10   CRK42   Y (192-216; 260-282)   591   At5g40380   DUF26   482
  1.7-10   PK   Y (256-273; 387-404)   625   At4g28670   DUF26   486
  1.7-11   CRK24   Y (96-115; 132-149)   416   At4g23320   DUF26   421
  1.7-12   CRK10/RLK4 P   Y (11-28)   669   At4g23180   DUF26   406
  1.7-12   CRK25   Y (8-25; 252-270;   675   At4g05200   DUF26   407
      283-300)
  1.7-12   CRK4   Y (289-306; 361-378)   676   At3g45860   DUF26   411
  1.7-13   CRK6/RLK5   Y (7-24; 211-228;   674   At4g23140.1   DUF26   403
      289-306)
  1.7-13   CRK6/RLK5   Y (7-24; 211-228;   680   At4g23140.2   DUF26   403
      289-306)
  1.7-14   CRK7   Y (248-265; 274-291)   659   At4g23150   DUF26   405
  1.7-14   CRK8   Y (577-593; 600-616;   1262   At4g23160   DUF26   404
      854-870;
      877-894)
  1.7-15   CRK19   Y (7-26; 263-285;   645   At4g23270   DUF26   412
      308-327)
  1.7-15   CRK20   Y (6-23; 254-277;   656   At4g23280   DUF26   408
      324-341)
  1.7-16   RLK4, 5, 6L   Y (6-23; 431-453;   830   At4g23310   DUF26   409
      488-505)
  1.7-17   CRK5/RLK6   Y (252-271; 280-299)   659   At4g23130.1   DUF26   410
  1.7-17   CRK5/RLK6   Y (252-271; 280-299)   663   At4g23130.2   DUF26   410
  1.7-18   CRK29   Y (6-23; 287-309;   679   At4g21410   DUF26   426
      402-419)
  1.7-18   CRK41   Y (13-30)   665   At4g00970   DUF26   423
  1.7-18   CRK28   Y (7-24, 289-311;   711   At4g21400   DUF26   425
      330-347)
  1.7-19   CRK21   Y (192-209; 329-346)   600   At4g23290.1   DUF26   420
  1.7-19   CRK21   Y (12-29; 282-299;   690   At4g23290.2   DUF26   420
      419-436)
  1.7-20   CRK14   Y (131-148; 159-183)   542   At4g23220   DUF26   399
  1.7-21   CRK32   Y (6-23; 262-279;   656   At4g11480   DUF26   415
      366-383)
  1.7-21   CRK31   Y (6-23; 221-238;   666   At4g11470   DUF26   414
      278-301)
  1.7-22   CRK34   Y (6-23; 548-569;   931   At4g11530   DUF26   400
      663-680)
  1.7-23   CRK33   Y (7-24; 242-259;   636   At4g11490   DUF26   413
      266-290)
  1.7-23   CRK22   Y (6-24; 291-315;   660   At4g23300   DUF26   402
      409-426)
  1.7-23   CRK30   Y (6-24; 286-304;   700   At4g11460   DUF26   416
      326-343)
  1.7-24   CRK17   Y (8-26; 289-308;   998   At4g23250   DUF26   418
      385-402;
      941-962)
  1.7-24   CRK18   Y (208-227; 304-323)   579   At4g23260   DUF26   417
  1.7-24   CRK12   Y (6-25)   648   At4g23200   DUF26   398
  1.7-25   CRK40   Y (6-25; 289-308;   654   At4g04570   DUF26   430
      329-345)
  1.7-25   CRK36   Y (6-24; 282-302;   658   At4g04490   DUF26   428
      325-342)
  1.7-25   CRK37   Y (6-24; 288-307;   646   At4g04500   DUF26   429
      338-357)
  1.7-25   CRK38   Y (6-23; 238-255;   648   At4g04510   DUF26   432
      280-299)
  1.7-25   CRK39   Y (6-22; 291-310;   659   At4g04540   DUF26   431
      333-350)
  1.7-26   LPK   Y (424-441; 512-528)   850   At3g16030   SD-1   449
  1.7-26   LPK   N   587   At1g67520   SD-1   448
  1.7-27   S-Locus   Y (447-464; 588-605)   852   At4g03230   SD-1   435
    LPK
  1.7-27   S-Locus   Y (8-25; 468-486;   849   At4g11900   SD-1   464
    LPK   699-716)
  1.7-28   S-Locus   Y (18-42; 395-412;   830   At1g11280.1   SD-1   460
    LPK   445-462)
  1.7-28   S-Locus   Y (8-32; 385-402;   820   At1g11280.2   SD-1   460
    LPK   435-452)
  1.7-28   S-Locus   Y (8-32; 385-402;   808   At1g11280.3   SD-1   460
    LPK   435-452)
  1.7-28   S-Locus   Y (186-205; 241-260)   598   At1g61460   SD-1   463
    LPK
  1.7-28   S-Locus   Y (6-29; 367-386;   802   At1g61550   SD-1   454
    LPK   421-440)
  1.7-28   S-Locus   Y (7-26; 377-394;   804   At1g61500   SD-1   451
    LPK   427-446)
  1.7-28   S-Locus   Y (20-37; 386-403;   821   At1g61400   SD-1   457
    LPK   436-453)
  1.7-28   S-Locus   Y (369-386; 419-436)   792   At1g61440   SD-1   458
    LPK
  1.7-28   S-Locus   Y (7-26; 375-392;   806   At1g61430   SD-1   456
    LPK   425-444)
  1.7-28   S-Locus   Y (7-26; 371-390;   807   At1g61420   SD-1   452
    LPK   426-445)
  1.7-28   S-Locus   Y (7-26; 371-390;   809   At1g61480   SD-1   453
    LPK   426-445)
  1.7-28   S-Locus   Y (7-26; 57-76;   804   At1g61490   SD-1   450
    LPK   83-100; 378-397;
      426-445)
  1.7-29   S-Locus   Y (6-27; 379-400;   805   At1g61380   SD-1   461
    LPK   429-450)
  1.7-29   S-Locus   Y (7-31; 378-395;   821   At1g61360   SD-1   462
    LPK   428-446)
  1.7-29   S-Locus   Y (22-39; 396-415;   831   At1g61390   SD-1   455
    LPK   450-468)
  1.7-29   S-Locus   Y (6-27; 380-399;   814   At1g61370   SD-1   459
    LPK   434-453)
  1.7-30   S-Locus   Y (6-23; 439-461;   815   At4g27300   SD-1   433
    LPK   492-509)
  1.7-31   S-Locus   Y (6-26)   840   At1g11410   SD-1   437
    LPK
  1.7-31   S-Locus   Y (69-86; 99-116)   901   At1g11340   SD-1   436
    LPK
  1.7-32   S-Locus   Y (10-27)   850   At4g21380   SD-1   440
    LPK
    (ARK3)
  1.7-32   S-Locus   Y (11-30; 444-463;   844   At4g21370   SD-1   441
    LPK (SRKaP)   486-502)
  1.7-32   S-Locus   Y (10-26)   843   At1g65790   SD-1   439
    LPK
    (ARK1)
  1.7-32   S-Locus   Y (11-28; 394-411;   847   At1g65800   SD-1   438
    LPK   440-457)
    (ARK2)
  1.7-33   S-Locus   Y (9-29)   772   At4g27290   SD-1   434
    LPK
  1.7-34   S-Locus   Y (446-464; 687-704)   842   At1g61610   SD-1   447
    LPK
  1.7-34   S-Locus   Y (7-26; 393-410;   849   At4g21390   SD-1   446
    LPK   439-458)
  1.7-35   S-Locus   Y (435-457; 497-514)   830   At1g11350   SD-1   445
    LPK
  1.7-35   S-Locus   Y (6-23; 424-441;   1635   At1g11300   SD-1   442
    LPK   479-496;
      1252-1269; 1309-1326)
  1.7-34   S-Locus   Y (445-466; 684-701)   840   At1g11330   SD-1   444
    LPK
  Family
  1.8
  1.8-1   LRR   Y (545-569)   895   At5g48740   LRRI   223
  1.8-2   LRR   Y (531-554)   934   At2g37050   LRRI   221
  1.8-2   LRR   Y (533-557)   929   At1g67720   LRRI   222
  1.8-3   RLK   Y (316-340; 373-390)   675   At1g51830   LRRI   247
  1.8-4   RLK   Y (6-25; 514-532;   843   At1g05700   LRRI   266
      549-567)
  1.8-4   SIRK P   Y (6-22; 519-538;   876   At2g19190   LRRI   265
    (light-   569-585)
    responsive)
  1.8-4   LRR   Y (516-533; 564-581)   876   At4g29990   LRRI   264
    (light
    repressible)
  1.8-4   LRR   Y (6-23)   881   At2g19210   LRRI   262
    (light
    repressible)
  1.8-4   LRR   Y (6-24)   877   At2g19230   LRRI   263
    (light
    repressible)
  1.8-4   LRR   Y (438-455; 516-540)   881   At1g51790   LRRI   270
    (light
    repressible)
  1.8-5   LRR   Y (512-536; 561-585)   863   At4g29450   LRRI   268
    (light
    repressible)
  1.8-5   LRR   Y (6-22; 508-530;   911   At4g29180   LRRI   267
    (light   555-571)
    repressible)
  1.8-6   LRR   Y (6-23; 512-536;   894   At1g51800   LRRI   252
    (light   595-612)
    repressible)
  1.8-6   PK   Y (413-429; 460-483)   837   At1g51870   LRRI   254
  1.8-6   RLK (LRR-   Y (511-528; 589-606)   890   At1g51860   LRRI   253
    I)
  1.8-6   LRR   Y (462-484; 517-541)   880   At1g51880   LRRI   255
    (light
    repressible)
  1.8-6   LRR   Y (490-514; 615-632)   888   At1g51890   LRRI   256
    (light
    repressible)
  1.8-6   PK   Y (6-23; 460-477;   876   At1g51910   LRRI   257
      508-531)
  1.8-7   LRR   Y (447-469; 506-529)   884   At2g28990   LRRI   242
    (light
    repressible)
  1.8-7   LRR   Y (408-432; 477-494)   786   At2g28970   LRRI   241
    (light
    repressible)
  1.8-8   LRR   Y (7-24)   898   At4g20450   LRRI   239
    (light
    repressible)
  1.8-9   LRR   Y (6-22; 510-529;   872   At2g29000   N.A.   N.A.
      562-579)
  1.8-9   LRR   Y (8-24; 509-532;   880   At2g28960   LRRI   237
    (light   578-594)
    repressible)
  1.8-10   LRR   Y (10-27; 464-483;   880   At3g21340   LRRI   250
    (light   519-543)
    repressible)
  1.8-10   LRR   Y (7-24; 518-542;   888   At1g49100   LRRI   251
    (light   579-596)
    repressible)
  1.8-10   LRR   Y (7-24; 479-503;   851   At2g04300   LRRI   249
    (light   539-556)
    repressible)
  1.8-11   LRR   Y (505-529; 572-591)   884   At1g51805   N.A.   N.A.
    (light
    repressible)
  1.8-11   LRR   N   843   At1g51810   LRRI   248
    (light
    repressible)
  1.8-11   LRR   Y (506-530; 573-592)   885   At1g51820   LRRI   244
    (light
    repressible)
  1.8-11   LRR   Y (486-510; 555-572)   865   At1g51850   LRRI   245
    (light
    repressible)
  1.8-12   LRR   Y (459-478; 503-522)   868   At5g59670   LRRI   236
    (light
    repressible)
  1.8-12   LRR   Y (8-27; 515-537;   866   At5g16900   LRRI   240
    (light   568-587)
    repressible)
  1.8-12   LRR   Y (6-23; 463-480;   878   At3g46330   LRRI   232
    (light   517-534)
    repressible)
  1.8-12   LRR   Y (362-378; 517-541)   892   At5g59650   LRRI   233
  1.8-12   LRR   Y (509-531)   882   At5g59680   LRRI   234
    (light
    repressible)
  1.8-12   LRR   Y (6-22; 462-481;   856   At1g07560   LRRI   243
    (light   496-520)
    repressible)
  1.8-13   LRR   Y (508-530; 561-578)   868   At2g14510   LRRI   258
    (light
    repressible)
  1.8-13   LRR   Y (506-527; 558-575)   864   At1g07550   LRRI   260
    (light
    repressible)
  1.8-13   LRR   Y (7-23; 526-548;   886   At2g14440   LRRI   259
    (light   579-595)
    repressible)
  1.8-14   LRR   Y (6-23; 452-469   889   At3g46340   LRRI   226
    (light   511-535)
    repressible)
  1.8-14   LRR   N   871   At3g46350   LRRI   227
  1.8-14   LRR   N   838   At3g46420   LRRI   231
  1.8-15   LRR   Y (7-31; 508-532;   883   At3g46400   LRRI   230
    (light   581-598)
    repressible)
  1.8-15   LRR   Y (427-450; 492-509)   793   At3g46370   LRRI   229
    (light
    repressible)
  Family
  1.9
  1.9-1   PK   Y (441-464; 530-553)   880   At5g38990   CrRLK1L-1   208
  1.9-1   PK   Y (441-465; 522-546)   873   At5g39000   CrRLK1L-1   209
  1.9-2   PK   Y (444-465; 496-513)   806   At5g39030   CrRLK1L-2   129
  1.9-2   PK   Y (6-22; 438-462;   813   At5g39020   CrRLK1L-2   128
      475-491)
  1.9-3   PR55K P   Y (11-28)   579   At5g38250   LRK10L-2   132
  1.9-3   PR55K P   Y (14-30)   588   At5g38240   LRK10L-2   131
  1.9-4   PR5K P   Y (465-484; 565-584)   853   At4g18250   Thaumatin   139
  1.9-4   PK   Y (744-763; 794-811)   1109   At1g66980   LRK10L-2   135
  1.9-4   PR5K P   N   799   At1g70250   Thaumatin   140
  1.9-4   PR5K   Y (6-23; 277-297;   665   At5g38280   Thaumatin   138
      329-346)
  1.9-5   PK   Y (71-93; 133-155)   470   At5g24080   SD-2   144
  1.9-6   RLK4   N   402   At4g00340   SD-2   142
  1.9-7   Lec   Y (11-35; 390-407;   829   At1g34300   SD-2   146
    Binding   422-439)
    PK
  1.9-7   Lec   Y (6-23; 449-466;   764   At2g41890   SD-3   602
    Binding   483-500)
    PK
  1.9-7   Lec   Y (6-23)   748   At5g60900   SD-2   141
    Binding
    PK
  1.9-8   Lec   Y (6-25; 431-450;   821   At4g32300   SD-2   145
    Binding   537-556)
    PK
  1.9-8   Lec   Y (6-25; 442-464;   870   At5g35370   SD-2   147
    Binding   519-540)
    PK
  1.9-9   S-locus   Y (440-463; 494-512)   828   At2g19130   SD-2   143
    LecRK
  Family
  1.10
  1.10-1   RLK   N   756   At1g21590   LRR VI   102
  1.10-1   RLK   N   794   At1g77280   LRR VI   101
  1.10-1   PK   N   705   At5g63940   LRR VI   103
  1.10-2   PK   N   321   At4g35030   LRR VI   104
  1.10-2   PK   N   617   At2g16750   LRR VI   105
  1.10-3   PK   N   467   At5g10520   LRR VI   111
  1.10-3   PK   Y (327-344)   461   At3g05140   LRR VI   110
  1.10-3   PK   N   456   At5g65530   LRR VI   112
  1.10-3   PK   Y (157-173)   511   At5g18910   LRR VI   109
  1.10-4   PK   N   552   At5g37790   LRR VI   106
  1.10-4   PK   N   467   At1g66460   LRR VI   107
  1.10-5   PK   N   416   At5g57670   LRR VI   114
  1.10-6   PK   Y (128-147)   392   At2g18890   LRR VI   113
  1.10-7   PK   N   429   At5g35960   LRR VI   108
  Family
  1.11
  1.11-1   LecRK   Y (19-38; 284-308;   675   At5g65600   L-Lectin   533
      407-424)
  1.11-1   LecRK 3 P   Y (6-24; 269-292;   651   At5g10530   L-Lectin   532
      344-363)
  1.11-2   LecRK   Y (270-289; 326-345)   652   At5g06740   L-Lectin   534
  1.11-3   LecRK   Y (95-119; 314-338;   711   At5g03140   L-Lectin   529
      369-393)
  1.11-3   LecRK   Y (113-135; 307-331)   691   At5g42120   L-Lectin   531
  1.11-3   LecRK   Y (4-21; 82-106;   715   At3g53380   L-Lectin   528
      316-339)
  1.11-3   LecRK 3 L   Y (7-26; 306-325;   681   At5g55830   L-Lectin   530
      374-393)
  1.11-4   LecRK 3 L   Y (18-41; 72-89;   686   At4g04960   L-Lectin   527
      287-310)
  1.11-4   LecRK 3 L   Y (13-30; 302-325)   656   At1g15530   L-Lectin   507
  1.11-4   LecRK   Y (6-24; 37-55;   649   At4g28350   L-Lectin   526
      76-94)
  1.11-5   PK   Y (6-22; 296-313;   675   At2g37710   L-Lectin   502
      351-368)
  1.11-5   LecRK 3 P   Y (7-25; 240-261;   677   At3g53810   L-Lectin   503
      292-313)
  1.11-6   LecRK   Y (6-23; 38-55;   674   At4g02410   L-Lectin   504
      86-103; 248-265;
      298-317)
  1.11-6   LecRK 3 L   Y (6-23; 40-59;   669   At4g02420   L-Lectin   505
      90-109; 245-264;
      295-312)
  1.11-7   LecRK   Y (253-270; 287-311)   669   At4g29050   L-Lectin   500
  1.11-7   LecRK 3 L   Y (7-23; 228-245;   656   At1g70130   L-Lectin   499
      277-301)
  1.11-7   LecRK   Y (233-256; 287-311)   666   At1g70110   L-Lectin   501
  1.11-7   LecRK 3 P   Y (7-31; 75-93;   684   At3g55550   L-Lectin   506
      236-254; 289-313)
  1.11-8   LecRK   Y (102-125; 293-315)   668   At5g59270   L-Lectin   524
  1.11-8   LecRK 3 P   Y (6-23; 305-322;   674   At5g59260   L-Lectin   523
      360-377)
  1.11-9   LecRK 3 L   Y (7-23; 69-93;   623   At2g29250   L-Lectin   536
      241-263; 303-327)
  1.11-9   LecRK 3 L   Y (6-23; 244-261;   627   At2g29220   L-Lectin   537
      304-321)
  1.11-   LecRK   Y (236-258; 289-308)   718   At5g60300.1   L-Lectin   520
  10
  1.11-   LecRK   Y (236-258; 289-308)   718   At5g60300.2   L-Lectin   520
  10
  1.11-   LecRK   Y (233-255; 286-305)   668   At5g60270   L-Lectin   522
  10
  1.11-   LecRK   Y (7-24; 241-262;   682   At3g45330   L-Lectin   512
  10     293-310)
  1.11-   LecRK   Y (6-23; 296-317;   604   At3g45390   L-Lectin   513
  10     425-442)
  1.11-   LecRK   Y (234-256; 287-306)   669   At3g45440   L-Lectin   517
  10
  1.11-   LecRK   Y (240-262; 293-312)   675   At5g60320   L-Lectin   514
  10
  1.11-   LecRK   Y (234-256; 281-303)   657   At5g60280   L-Lectin   518
  10
  1.11-   LecRK   Y (234-256; 287-306)   664   At3g45410   L-Lectin   515
  10
  1.11-   LecRK   Y (296-315; 346-365)   667   At3g45420   L-Lectin   516
  010
  1.11-   LecRK   Y (174-195; 226-247)   613   At3g45430   L-Lectin   519
  010
  1.11-   LecRK 3 P   Y (235-257; 288-307)   616   At5g60310   L-Lectin   521
  010
  1.11-   LecRK 3 P   Y (7-24; 85-102;   682   At5g01540   L-Lectin   510
  011     310-333; 360-377)
  1.11-   LecRK 3 P   Y (311-335; 373-395)   693   At3g08870   L-Lectin   511
  011
  1.11-   LecRK 3 P   Y (306-330)   691   At5g01560   L-Lectin   509
  011
  1.11-   LecRK 3 P   Y (304-328)   688   At5g01550   L-Lectin   508
  011
  1.11-   LecRK 3 P   Y (227-246; 277-300)   523   At3g59730   L-Lectin   496
  012
  1.11-   LecRK1 P   Y (7-25; 234-257;   664   At2g43690   L-Lectin   498
  012     278-301)
  1.11-   LecRK   Y (278-302; 339-356)   658   At2g43700   L-Lectin   497
  012
  1.11-   LecRK   Y (27-44; 65-82;   661   At3g59700   L-Lectin   495
  012     238-255; 280-304)
  1.11-   LecRK 3 P   Y (248-265; 276-300)   659   At3g59740   L-Lectin   493
  012
  1.11-   LecRK 3 P   Y (196-215; 246-268)   626   At3g59750   L-Lectin   494
  012
  1.11-   PK   N   337   At3g46760   L-Lectin   525
  013
  Family
  1.12
  1.12-1   PK   Y (11-29; 131-148)   355   At1g78530   LRR XIII   378
  1.12-2   PK   Y (9-27; 62-84)   376   At5g13290.1   N.A.   336
  1.12-2   PK   Y (9-27; 62-84)   331   At5g13290.2   N.A.   336
  1.12-3   RPK1   Y (199-220; 241-261)   540   At1g69270   N.A.   287
  1.12-4   LRR   Y (15-32; 285-309)   641   At2g31880   LRR XI   374
  1.12-5   LRR   Y (11-29; 230-247;   605   At3g28450   LRR X   386
      297-314)
  1.12-5   CLV1 P   Y (4-21; 221-245)   601   At1g27190   LRR X   385
  1.12-5   LRR   Y (215-239; 351-368)   591   At1g69990   LRR X   384
  1.12-5   LRR   Y (6-25; 227-246;   620   At5g48380   LRR X   387
      358-375)
  1.12-6   IMK2   Y (18-35; 459-483)   836   At3g51740   LRR III   328
  1.12-6   MRLK   Y (373-395; 533-555;   719   At3g56100   LRR III   329
      572-589)
  1.12-7   LRR   Y (646-667; 686-705)   985   At3g02130   N.A.
  1.12-8   LRR   Y (512-536; 557-574)   882   At1g12460   LRR VII   346
  1.12-8   LRR   Y (6-22; 518-540;   890   At1g62950   LRR VII   345
      605-627)
  1.12-9   LRR   N   1036   At5g53890   LRR X   393
  1.12-9   LRR   Y (20-44)   1095   At1g72300   LRR X   395
  1.12-9   LRR   N   1008   At2g02220   LRR X   394
  1.12-   LRR   Y (20-37)   966   At1g34420   LRR X   383
  10
  1.12-   LRR   Y (9-28; 541-560;   872   At5g06940   N.A.   601
  10     645-662)
  1.12-   RLK   Y (535-558)   890   At2g41820   LRR X   382
  10
  1.12-   LRR   Y (6-24)   1133   At1g17230   LRR XI   353
  11
  1.12-   LRR   Y (14-30; 707-725;   1124   At2g33170   LRR XI   351
  11     753-772)
  1.12-   LRR   Y (8-27)   1102   At5g63930   LRR XI   352
  11
  1.12-   LRR   Y (7-26)   953   At5g56040   LRR XI   357
  12
  1.12-   LRR   N   1045   At1g34110   LRR XI   358
  12
  1.12-   CLV1 L   Y (7-28)   1141   At3g24240   LRR XI   355
  12
  1.12-   LRR   Y (12-31)   1135   At5g48940   LRR XI   354
  12
  1.12-   PK   Y (8-25)   1089   At4g26540   LRR XI   356
  12
  1.12-   LRR   Y (6-29; 770-793;   1123   At1g73080   LRR XI   372
  13     831-848)
  1.12-   InRPK P   Y (738-761)   1088   At1g17750   LRR XI   373
  13
  1.12-   LRR   Y (30-48; 709-727)   1045   At4g08850.1   LRR XII   551
  14
  1.12-   LRR   Y (30-47; 709-726;   1009   At4g08850.2   LRR XII   551
  14     991-1008)
  1.12-   LRR   Y (13-32)   1120   At1g35710   LRR XII   552
  14
  1.12-   LRR   Y (875-894;   1249   At4g20140   LRR XI   367
  15     1008-1026)
  1.12-   LRR   Y (875-898;   1252   At5g44700   LRR XI   368
  15     1005-1023)
  1.12-   FLS2   Y (7-23; 807-823;   1173   At5g46330   LRR XII   550
  15     869-885)
  1.12-   EMS1   Y (827-846)   1192   At5g07280   LRR X   392
  15
  1.12-   LRR   Y (753-772)   1136   At4g36180   LRR VII   340
  16
  1.12-   LRR   Y (484-503; 754-772;   1140   At1g75640   LRR VII   341
  16     943-961)
  1.12-   LRR   Y (609-629)   976   At1g09970.1   LRR XI   369
  17
  1.12-   LRR   Y (609-629)   977   At1g09970.2   LRR XI   369
  17
  1.12-   IKU2   Y (448-465; 616-635)   991   At3g19700   LRR XI   370
  17
  1.12-   LRR   Y (7-24; 624-641;   977   At1g72180   LRR XI   365
  17     743-760)
  1.12-   LRR   Y (624-641)   996   At1g28440   LRR XI   363
  18
  1.12-   HAESA/RLK5   Y (625-648)   999   At4g28490   LRR XI   362
  18
  1.12-   LRR   Y (633-653)   993   At5g65710   LRR XI   364
  18
  1.12-   Pre RLK5   Y (628-650; 681-704)   1005   At5g25930   LRR XI   371
  18
  1.12-   LRR   Y (6-23; 594-611;   966   At5g49660   LRR XI   366
  18     721-738)
  1.12-   BAM1   Y (642-661)   1003   At5g65700   LRR XI   347
  19
  1.12-   LRR   Y (589-613; 678-701)   895   At5g51350   LRR IV   608
  19
  1.12-   LRR   Y (585-604; 635-658)   960   At2g25790   N.A.   600
  19
  1.12-   CLV1   Y (641-659; 749-766)   980   At1g75820   LRR XI   349
  19
  1.12-   BAM3   Y (6-24; 659-678;   992   At4g20270   LRR XI   350
  19     767-784)
  1.12-   BAM2   Y (638-657; 748-765)   1002   At3g49670   LRR XI   348
  19
  1.12-   CLV1 L   Y (6-23; 649-666;   1029   At1g08590   LRR XI   360
  20     697-714)
  1.12-   RPK5   Y (6-25; 634-653;   1013   At4g28650   LRR XI   359
  20     682-699)
  1.12-   PXY RLK   Y (6-25)   1041   At5g61480   LRR XI   361
  20
  1.12-   LRR Xa21   Y (601-619; 642-666)   1010   At3g47570   LRR XII   545
  21
  1.12-   LRR Xa21   Y (71-93; 643-665;   1009   At3g47090   LRR XII   544
  21     696-715)
  1.12-   LRR Xa21   Y (643-667; 698-722)   1011   At3g47580   LRR XII   543
  21
  1.12-   LRR Xa21   Y (654-678; 715-734)   1025   At3g47110   LRR XII   548
  21
  1.12-   LRR Xa21   Y (605-624; 651-670)   1031   At5g20480   LRR XII   546
  21
  1.12-   ERL1   Y (8-26; 556-574;   966   At5g62230   LRR XIII   380
  22     585-603)
  1.12-   LRR   N   980   At2g24130   LRR XII   549
  22
  1.12-   ER   Y (7-23; 551-567;   976   At2g26330   LRR XIII   381
  22     582-599)
  1.12-   ERL2   Y (523-542; 551-570)   932   At5g07180   LRR XIII   379
  22
  1.12-   LRPKm1   Y (606-630; 749-766;   967   At5g01890   LRR VII   342
  23     787-805)
  1.12-   LRPKm   Y (598-622; 740-757)   964   At3g56370   LRR VII   343
  23
  1.12-   LRR   Y (7-24; 643-667;   1016   At3g28040   LRR VII   344
  23     784-801)
  1.12-   BRL3   Y (773-796)   1164   At3g13380   LRR X   389
  24
  1.12-   BRL P   Y (17-34)   1106   At1g74360   LRR X   397
  24
  1.12-   BRI1   Y (6-24; 792-811)   1196   At4g39400   LRR X   390
  24
  1.12-   BRL1   Y (6-22; 774-797)   1166   At1g55610   LRR X   388
  24
  1.12-   BRL P   Y (757-776)   1143   At2g01950   LRR X   391
  24
  1.12-   LRR   Y (13-30)   648   At4g30520   LRR II   158
  25
  1.12-   LRR   Y (6-23; 234-258;   634   At2g23950   LRR II   157
  25     292-309)
  1.12-   LRR   Y (11-28; 247-270)   635   At3g25560.1   LRR II   159
  26
  1.12-   LRR   Y (11-28; 248-271)   636   At3g25560.2   LRR II   159
  26
  1.12-   LRR   Y (11-28; 237-259)   632   At1g60800   LRR II   161
  26
  1.12-   LRR   Y (14-33; 247-269)   638   At5g16000   LRR II   160
  26
  1.12-   LRR   Y (8-27; 240-264;   614   At5g45780   LRR II   162
  26     295-314)
  1.12-   SERK1   Y (235-259)   625   At1g71830   LRR II   150
  27
  1.12-   SERK2   Y (8-27; 239-262)   628   At1g34210   LRR II   149
  27
  1.12-   SERKL4   Y (10-29)   620   At2g13790   LRR II   152
  27
  1.12-   SERK3   Y (223-246)   615   At4g33430   LRR II   151
  27   (BAK1)
  1.12-   SERKL5   Y (120-139; 217-236)   601   At2g13800   LRR II   153
  27
  1.12-   LRR   Y (9-26; 226-247)   613   At5g10290   LRR II   155
  28
  1.12-   RLK   Y (220-241)   617   At5g65240   LRR II   154
  28
  1.12-   LRR   Y (30-47)   614   At5g63710   LRR II   156
  29
  1.12-   LRR   Y (7-31; 241-265)   604   At5g62710   LRR XIII   377
  30
  1.12-   LRR   Y (239-263)   592   At1g31420   LRR XIII   375
  30
  1.12-   SERK1 P   Y (237-261; 272-288)   589   At2g35620   LRR XIII   376
  30
  Family
  1.13
  1.13-1   LRR   Y (270-293)   672   At2g36570   LRR III   322
  1.13-2   LRR   N   669   At5g67200   LRR III   297
  1.13-2   LRR   Y (275-299; 433-450)   670   At1g68400   LRR III   323
  1.13-2   LRR   Y (251-275)   652   At1g60630   LRR III   301
  1.13-2   LRR   Y (7-24; 280-302)   669   At5g43020   LRR III   299
  1.13-2   RKL1 P   Y (5-29; 293-317)   660   At3g50230   LRR III   298
  1.13-3   LRR   Y (261-285)   640   At3g08680.1   LRR III   313
  1.13-3   LRR   Y (261-285)   640   At3g08680.2   LRR III   313
  1.13-3   LRR   Y (257-281)   658   At2g26730   LRR III   315
  1.13-3   RLK   Y (21-45; 76-100;   654   At5g58300   LRR III   314
      281-305)
  1.13-3   LRR   Y (6-25; 222-241;   640   At5g05160   LRR III   321
      266-290)
  1.13-4   LRR   Y (7-24; 251-275;   627   At3g02880   LRR III   325
      391-408)
  1.13-4   LRR   Y (244-268)   625   At5g16590   LRR III   324
  1.13-4   RKL1   Y (268-291)   655   At1g48480   LRR III   326
  1.13-4   RLK902   Y (265-288)   647   At3g17840   LRR III   327
  1.13-5   RKL1 P   Y (258-282)   638   At4g23740   LRR III   316
  1.13-5   LRR   N   587   At1g64210   LRR III   318
  1.13-5   LRR   Y (7-24; 252-276;   614   At5g24100   LRR III   320
      325-344)
  1.13-5   LRR   Y (235-259)   601   At5g53320   LRR III   319
  1.13-6   PRK1 P   Y (8-25; 269-287;   659   At5g20690   LRR III   295
      351-370)
  1.13-6   PRK1 P   Y (251-270; 367-385)   633   At3g42880   LRR III   294
  1.13-7   LRR   Y (23-43; 166-188;   686   At1g50610   LRR III   292
      280-304)
  1.13-7   LRR   Y (9-26; 210-229;   676   At4g31250   LRR III   293
      244-268)
  1.13-7   LRR   Y (253-272)   644   At1g72460   LRR III   296
  1.13-7   LRR   Y (20-38; 172-196;   679   At3g20190   LRR III   291
      278-302)
  1.13-7   LRR   Y (245-267)   647   At2g07040   LRR III   290
  1.13-7   PRK1   Y (9-26; 257-276;   657   At5g35390   LRR III   289
      362-379)
  1.13-8   LRR   N   680   At5g51560   LRR IV   491
  1.13-8   LRR   Y (5-22; 77-94;   691   At2g45340   LRR IV   490
      311-328; 489-505)
  1.13-8   LRR   Y (12-331; 607-626)   688   At4g22730   LRR IV   492
  1.13-9   LRR   Y (6-25; 280-299;   662   At3g57830   LRR III   306
      365-386)
  1.13-9   LRR   Y (276-298)   646   At2g42290   LRR III   305
  1.13-   LRR   Y (14-38; 336-360)   751   At5g67280   LRR III   310
  10
  1.13-   LRR   Y (336-358)   744   At2g15300   LRR III   311
  10
  1.13-   LRR   Y (339-363)   757   At4g34220   LRR III   312
  10
  1.13-   LRR   Y (9-31; 333-355)   773   At2g23300   LRR III   308
  10
  1.13-   LRR   Y (329-352)   768   At4g37250   LRR III   309
  10
  1.13-   LRR   Y (317-336; 609-628)   702   At1g25320   LRR III   302
  11
  1.13-   LRR   Y (315-339)   719   At1g67510   LRR III   307
  11
  1.13-   LRR   N   716   At2g01210   LRR III   303
  11
  1.13-   LRR   Y (305-329)   685   At1g66830   LRR III   304
  11
  1.13-   RHG1 P   N   359   At5g41680.1   LRR III   317
  12
  1.13-   RHG1 P   N   333   At5g41680.2   LRR III   317
  12
  Family
  1.14
  1.14-1   PK   N   351   At4g11890.1   DUF26   489
  1.14-1   PK   N   352   At4g11890.2   DUF26   489
  1.14-1   PK   Y (21-38)   354   At4g11890.3   DUF26   489
  1.14-2   PK   N   341   At5g23170   CR4L   85
  1.14-3   PK   Y (262-280)   470   At1g28390   CR4L   83
  1.14-3   PK   N   362   At3g51990   CR4L   84
  1.14-4   PK   Y (571-588)   697   At1g72760   RLCK IX   562
  1.14-4   PK   Y (97-114; 474-491;   733   At1g17540   RLCK IX   563
      610-627)
  1.14-5   PK   Y (432-451; 555-574)   680   At1g78940   RLCK IX   559
  1.14-5   PK   N   758   At1g16760   RLCK IX   560
  1.14-5   PK   N   780   At3g20200   RLCK IX   561
  1.14-6   PK   N   731   At5g35380   RLCK IX   557
  1.14-6   PK   N   700   At2g07020   RLCK IX   558
  1.14-6   PK   N   816   At2g24370   RLCK IX   553
  1.14-7   PK   N   703   At5g26150   RLCK IX   555
  1.14-7   PK   Y (99-116; 560-577;   703   At5g12000   RLCK IX   556
      608-627)
  1.14-8   PnPK1 L   N   845   At5g61550   RLCK IX   566
  1.14-8   PK   Y (533-550)   835   At4g25160   RLCK IX   564
  1.14-8   PK   Y (513-530)   819   At5g51270   RLCK IX   565
  1.14-8   PK   N   796   At5g61560   RLCK IX   567
  1.14-9   U-Box PK   Y (59-81)   801   At2g19410   RLCK IX   568
  1.14-   U-Box PK   N   834   At2g45910   RLCK IX   569
  10
  1.14-   U-Box PK   N   805   At3g49060   RLCK IX   570
  10
  1.14-   U-Box PK   N   765   At5g65500   RLCK IX   571
  10
  Family
  1.15
  1.15-1   PK   Y (145-168)   499   At3g56050   RLCK I   579
  1.15-1   PK   Y (7-24; 143-160)   489   At2g40270.1   RLCK I   578
  1.15-1   PK   Y (7-24; 136-153)   482   At2g40270.2   RLCK I   578
  1.15-2   LRR   Y (13-32; 230-253)   553   At5g07150   LRR VI   575
  1.15-2   RLK   Y (8-26; 320-343)   686   At4g18640   LRR VI   576
  1.15-2   LRR   Y (151-167; 312-334)   668   At5g45840   LRR VI   577
  1.15-3   PK   Y (142-166)   484   At5g58540.1   RLCK I   574
  1.15-3   PK   N   242   At5g58540.2   RLCK I   574
  1.15-3   PK   Y (6-23)   341   At5g58540.3   RLCK I   574
  1.15-4   LRR   Y (388-412; 533-557;   802   At3g03770   LRR VI   584
      584-606)
  1.15-4   LRR   Y (103-127; 396-420)   812   At5g14210   LRR VI   585
  1.15-4   LRR   Y (9-25; 300-316;   747   At1g14390   LRR VI   582
      354-377)
  1.15-4   LRR   Y (301-317; 354-377)   753   At2g02780   LRR VI   583
  1.15-4   LRR-VI   N   680   At5g63410   LRR VI   586
  1.15-5   ER P   Y (417-440)   864   At4g39270.1   LRR IV   606
  1.15-5   ER P   Y (417-440)   694   At4g39270.2   LRR IV   606
  1.15-5   LRR   Y (8-27; 447-471)   915   At2g16250   N.A.   607
  1.15-6   LRR   Y (13-30; 282-299)   664   At5g41180   LRR VI   581
  1.15-6   LRR   Y (6-24)   664   At1g63430   LRR VI   580
  Family
  1.16
  1.16-1   PK   Y (19-36)   422   At1g63500   N.A.   N.A.
  1.16-2   PK   N   489   At4g00710   N.A.   N.A.
  1.16-2   PK   N   483   At1g01740   N.A.   N.A.
  1.16-2   PK   N   487   At5g41260   N.A.   N.A.
  1.16-2   PK   N   489   At5g46570   N.A.   N.A.
  1.16-3   PK   N   490   At3g54030   N.A.   N.A.
  1.16-3   PK   N   507   At1g50990   N.A.   N.A.
  1.16-3   PK   N   477   At3g09240   N.A.   N.A.
  1.16-3   PK   N   499   At5g01060   RLCK II   590
  1.16-3   PK   N   489   At5g59010   RLCK II   589
  1.16-3   PK   N   512   At4g35230   RLCK II   588
  1.16-4   PK   N   465   At2g17090   RLCK II   591
  1.16-4   PK   N   328   At2g17170   N.A.   N.A.
  Family
  1.17
  1.17-1   PK   N   269   At3g57770   RLCK III   597
  1.17-2   PK   Y (177-194; 258-275)   355   At3g57730   N.A.   N.A.
  1.17-2   PK   Y (253-272)   351   At3g57710   RLCK III   596
  1.17-2   PK   Y (70-89)   359   At3g57720   RLCK III   595
  1.17-2   PK   N   334   At3g57750.1   N.A.   N.A.
  1.17-2   PK   N   334   At3g57750.2   N.A.   N.A.
  No
  Family
  No   PK   N   342   At4g10390   N.A.   610
  Fam-1
  No   PK RLK   Y (48-70)   349   At1g33260.1   N.A.   609
  Fam-1
  No   PK RLK   Y (48-70)   348   At1g33260.2   N.A.   609
  Fam-1
  No   PK   N   389   At1g67470   RLCK III   598
  Fam-2
  No   PK   Y (225-241)   372   At1g65250   RLCK III   599
  Fam-2
  No   PK   N   356   At3g57640   N.A.
  Fam-2
  No   PK   Y (7-26; 35-52;   418   At4g32000   RLCK X   274
  Fam-3     65-84)
  No   PK   Y (7-23; 71-94)   427   At1g80640   RLCK X   271
  Fam-3
  No   PK   Y (15-34)   383   At2g25220   RLCK X   273
  Fam-3
  No   PK   Y (6-25)   372   At5g11020   RLCK X   272
  Fam-3
  No   PK   Y (23-44)   492   At1g56720.1   TAKL   118
  Fam-4
  No   PK   Y (23-44)   492   At1g56720.2   TAKL   118
  Fam-4
  No   PK   Y (9-31)   466   At1g09440   TAKL   117
  Fam-4
  No   PK   Y (31-51)   683   At2g45590   RLCK XI   279
  Fam-5
  No   PK   Y (41-60)   651   At4g25390.1   RLCK XI   278
  Fam-5
  No   PK   Y (41-60)   497   At4g25390.2   RLCK XI   278
  Fam-5
  No   PK   Y (31-50)   654   At5g51770   RLCK XI   277
  Fam-5
  No   RKF1 P   Y (8-32; 576-593;   1006   At1g29750.1   LRR   474
  Fam-6     606-630;       VIII-2
      856-880)
  No   RKF1 P   Y (21-40; 591-608;   1021   At1g29750.2   LRR   474
  Fam-6     621-645;       VIII-2
      868-887)
  No   LRR   Y (427-444; 457-476)   853   At2g24230   LRR VII   337
  Fam-7
  No   LRR   Y (437-459; 532-551)   785   At5g58150   LRR VII   338
  Fam-7
  No   CRK13   Y (6-24; 226-243;   610   At4g23210.1   DUF26   422
  Fam-8     302-320)
  No   CRK13   Y (6-24; 226-243;   524   At4g23210.2   DUF26   422
  Fam-8     302-320)
  No   CRK11   Y (6-23; 290-308;   667   At4g23190   DUF26   401
  Fam-8     395-412;
      616-633)
  No   PK   N   609   At1g66920   LRK10L-2   133
  Fam-9
  No   PK   Y (19-41; 573-595)   692   At1g80870   RLCK XI   280
  Fam-10
  No   PK   Y (37-61)   458   At1g54820   Extensin   80
  Fam-11
  No   PK   N   270   At3g21450   RLCK IX   573
  Fam-11
  No   PK   Y (300-319)   674   At3g24660   LRR III   332
  Fam-12
 
  *The sequences associated with the AGI accession numbers are incorporated herein by reference.
[00002] [TABLE-US-00002]
  TABLE 2
 
  Phenotypes for all DN-RLK mutants generated grown on soil and on other
growth media in the T2 and T3 homozygous generations.
            Confirmed
    DN-RLKT2   PreliminaryT3   Phenotypes
  RLK   Construct   Transgenic   Phenotypes   Homozygous   (various growth
  Subfamily   (AGI)   Lines   (soil grown)   Lines   media)
 
  1.Other-9   At4g20790   14   none   4   shorter
            roots/less
            lateral roots*
            on MS
  1.Other-   At5g39390   17   None   2   none
  10
  1.Other-   At5g45800   18   senescent   3   none
  11       leaves with
        more
        serrations/
        stunted plant
        height/long
        skinny
        cauline
        leaves
  1.Other-   At5g10020   7   none   2   longer roots on
  12           MS/longer
            roots on-
            sucrose media
  1.Other-   At2g46850   3   none   2   none
  13
  1.1-2   At3g21630   18   short stem/   2   none
        larger leaves
  1.1-4   At3g14350   13   larger leaves   4   more lateral
            roots*/larger
            epidermal
            cells/
            increased
            cellulose
            content
  1.1-6   At5g06820   12   short stem/   2   short stem/
        narrow leaves     narrow leaves
  1.1-7   At4g03390   3   none   1   none
  1.2-28   At5g01020   12   none   2   none
  1.2-29   At2g20300   16   none   8   none
  1.2-31   At2g28250   6   longer   3   longer
        flowering     flowering time
        time
  1.3-2   At1g49730   6   none   3   none
  1.3-4   At5g59700   3   none   3   short roots on
            MS, short roots
            on-sucrose
            media
  1.3-5   At2g21480   13   short stem   2   nd
  1.3-9   At5g49760   5   small leaves/   4   short hypocotyl
        short stem     on-sucrose
            media in dark
  1.5-1   At2g11520   4   none   2   short roots on
            MS
  1.5-2   At1g16260   10   none   3   none
  1.5-3   At1g16130   6   none   3   short roots on-
            sucrose media
  1.5-5   At1g16110   8   small round   3   nd
        leaves/short
        petiole
  1.5-11   At2g23450   20   senescent   5   short roots on
        leave     MS, short roots
            on-sucrose & -
            nitrogen media
            and under low
            light
  1.5-13   At1g18390   20   none   3   nd
  1.5-14   At5g38210   2   none   1   nd
  1.6-2   At1g55200   20   none   2   nd
  1.6-13   At1g26150   5   none   2   nd
  1.6-14   At1g66150   19   apically   2   variable
        dominant
  1.7-10   At1g70520   3   late   2   late flowering/
        flowering/     short stem
        short stem
  1.7-13   At4g23140   11   none   1   none
  1.7-14   At4g23150   5   none   2   none
  1.7-19   At4g23290   20   none   4   short roots on
            MS, short roots
            on-sucrose
            media/more
            lateral roots
            on MS*
  1.7-21   At4g11480   1   none   1   none
  1.7-25   At4g04570   8   none   6   longer roots on
            MS, -nitrogen &
            sorbitol/more
            lateral roots
            on MS*
  1.7-29   At1g61380   5   none   3   longer roots/
            more lateral
            roots on MS*
  1.7-31   At1g11410   7   none   2   nd
  1.7-34   At1g61610   1   none   1   nd
  1.9-1   At5g38990   1   late   1   late flowering/
        flowering/     large leaves/
        large leaves/     more leaves/
        more leaves/     thick stem
        thick stem
  1.9-7   At1g34300   9   late   2   late flowering/
        flowering/     large leaves/
        large leaves/     more leaves/
        more leaves/     thick stem
        thick stem
  1.9-8   At4g32300   1   late   1   nd
        flowering/
        more leaves
  1.10-1   At1g21590   1   none   1   long roots
            MS/branched
            root
            hairs*/short
            roots on-
            sucrose media
  1.11-3   At5g03140   6   none   4   long roots MS/
            short roots on-
            sucrose media
  1.11-5   At2g37710   6   none   1   short roots on
            MS
  1.11-10   At5g60300   4   none   3   nd
  1.11-11   At5g01540   10   none   3   nd
  1.12-3   At1g69270   15   none   3   longer roots on
            MS
  1.12-5   At3g28450   5   none   4   short roots on
            MS and -sucrose
            media/bulbous
            root hairs*
  1.12-6   At3g51740   7   none   7   longer roots on
            MS, -sucrose
            and 6% sucrose
  1.12-8   At1g12460   8   none   5   none
  1.12-12   At5g56040   18   none   8   none
  1.12-13   At1g73080   11   none   4   longer roots on
            MS, -sucrose
            and -nitrogen
            media
  1.12-19   At5g65700   10   none   3   longer roots on
            MS
  1.12-21   At3g47570   15   none   4   none
  1.12-23   At5g01890   7   none   4   bulbous root
            hairs*
  1.12-26   At3g25560   16   none   2   short hypocotyl
            on-sucrose
            media in dark
  1.12-27   At1g71830   7   none   3   longer roots on
            MS
  1.12-28   At5g10290   12   none   2   longer roots on
            MS/branching
            root hairs*
  1.12-29   At5g63710   15   none   8   none
  1.12-30   At5g62710   16   large leaves/   8   root growth
        thick stem/     effected on MS,
        longer stems     short hypocotyl
            on-sucrose
            media in dark
  1.13-2   At5g67200   4   none   2   root hair
            phenotype*/
            reduction in
            pavement cell
            lobe number/
            longer roots on
            MS
  1.13-3   At3g08680   8   none   6   none
  1.13-4   At3g02880   15   none   3   long roots on
            MS
  1.13-5   At4g23740   7   none   2   wavy root hair
            phenotype*
  1.13-9   At3g57830   5   none   3   short roots on-
            sucrose media
  1.14-5   At1g78940   6   late   3   long roots on
        flowering/     MS
        long
        petioles/
        dark green
        leaves
  1.14-7   At5g26150   6   none   3   none
  1.14-10   At2g45910   5   none   2   root hair
            phenotype*
  1.15-3   At5g58540   15   none   5   none
  1.15-4   At3g03770   4   none   3   none
  1.15-5   At4g39270   5   none   5   short roots on-
            sucrose media
  1.15-6   At5g41180   3   none   1   nd
  No Fam-6   At1g29750   1   short thick   1   nd
        stem
  No Fam-7   At2g24230   12   none   6   none
  No Fam-9   At1g66920   8   none   3   nd
 
  *Root hair phenotypes examined by Ornusa Khamsuk
[00003] [TABLE-US-00003]
  TABLE 3
 
  DN-RLK constructs generated for this project from original cDNA
  and confirmed using DNA sequencing.
      DN-RLK
    RLK   Constructs
    Subfamily   (AGI)
   
    1.Other-9   At4g20790
    1.Other-10   At5g39390
    1.Other-13   At2g46850
    1.1-2   At3g21630
    1.1-6   At5g06820
    1.3-4   At5g59700
    1.3-5   At2g21480
    1.5-2   At1g16260
    1.5-13   At1g18390
    1.6-13   At1g26150
    1.7-14   At4g23150
    1.7-21   At4g11480
    1.7-31   At1g11410
    1.7-34   At1g61610
    1.14-7   At5g26150
    1.15-3   At5g58540
    No Fam-9   At1g66920
   
[0038] As used herein, the terms “host cells” and “recombinant host cells” are used interchangeably and refer to cells (for example, an Arabidposis sp., or other plant cell) into which the compositions of the presently disclosed subject matter, for example, an expression vector comprising a dominant negative RLK can be introduced. Furthermore, the terms refer not only to the particular plant cell into which an expression construct is initially introduced, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny might not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
[0039] As used herein, the terms “complementarity” and “complementary” refer to a nucleic acid that can form one or more hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interactions. In reference to the nucleic molecules of the presently disclosed subject matter, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, in some embodiments, ribonuclease activity. Determination of binding free energies for nucleic acid molecules is well known in the art. See e.g., Freier et al., 1986; Turner et al., 1987.
[0040] A “dominant negative RLK” refers to a polypeptide variant of a native RLK sequence whose expression interferes with or otherwise counteracts native RLK activity. Dominant negative RLK mutants can include a fragment of a RLK polypeptide sequence with at least one mutation. Exemplary mutations include, e.g., RLK polypeptide lacking a functional domain. In other embodiment, the RLK comprises a transmembrane domain but lacks either a kinase domain or a ligand binding domain. In some embodiments, the dominant negative RLK comprise a polypeptide at least 50%, 60%, 70%, 80%, or 90% identical to a wild-type RLK.
[0041] As used herein, the phrase “percent complementarity” refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). The terms “100% complementary”, “fully complementary”, and “perfectly complementary” indicate that all of the contiguous residues of a nucleic acid sequence can hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
[0042] As used herein, the term “gene” refers to a nucleic acid sequence that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. The term “gene” also refers broadly to any segment of DNA associated with a biological function. As such, the term “gene” encompasses sequences including but not limited to a coding sequence, a promoter region, a transcriptional regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation from one or more existing sequences.
[0043] As is understood in the art, a gene typically comprises a coding strand and a non-coding strand. As used herein, the terms “coding strand” and “sense strand” are used interchangeably, and refer to a nucleic acid sequence that has the same sequence of nucleotides as an mRNA from which the gene product is translated. As is also understood in the art, when the coding strand and/or sense strand is used to refer to a DNA molecule, the coding/sense strand includes thymidine residues instead of the uridine residues found in the corresponding mRNA. Additionally, when used to refer to a DNA molecule, the coding/sense strand can also include additional elements not found in the mRNA including, but not limited to promoters, enhancers, and introns. Similarly, the terms “template strand” and “antisense strand” are used interchangeably and refer to a nucleic acid sequence that is complementary to the coding/sense strand.
[0044] The phrase “gene expression” generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence and exhibits a biological activity in a cell. As such, gene expression involves the processes of transcription and translation, but also involves post-transcriptional and post-translational processes that can influence a biological activity of a gene or gene product. These processes include, but are not limited to RNA syntheses, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of polypeptides. Additionally, processes that affect protein-protein interactions within the cell can also affect gene expression as defined herein.
[0045] However, in the case of genes that do not encode protein products, for example nucleic acid sequences that encode RNAs or precursors thereof that induce RNAi, the term “gene expression” refers to the processes by which the RNA is produced from the nucleic acid sequence. Typically, this process is referred to as transcription, although unlike the transcription of protein-coding genes, the transcription products of an RNAi-inducing RNA (or a precursor thereof are not translated to produce a protein. Nonetheless, the production of a mature RNAi-inducing RNA from an RNAi-inducing RNA precursor nucleic acid sequence is encompassed by the term “gene expression” as that term is used herein.
[0046] The terms “heterologous gene”, “heterologous DNA sequence”, “heterologous nucleotide sequence”, “exogenous nucleic acid molecule”, “exogenous DNA segment”, and “transgene” as used herein refer to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native transcriptional regulatory sequences. The terms also include non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid wherein the element is not ordinarily found.
[0047] As used herein, the term “isolated” refers to a molecule substantially free of other nucleic acids, proteins, lipids, carbohydrates, and/or other materials with which it is normally associated, such association being either in cellular material or in a synthesis medium. Thus, the term “isolated nucleic acid” refers to a ribonucleic acid molecule or a deoxyribonucleic acid molecule (for example, a genomic DNA, cDNA, mRNA, RNAi-inducing RNA or a precursor thereof, etc.) of natural or synthetic origin or some combination thereof, which (1) is not associated with the cell in which the “isolated nucleic acid” is found in nature, or (2) is operatively linked to a polynucleotide to which it is not linked in nature. Similarly, the term “isolated polypeptide” refers to a polypeptide, in some embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
[0048] The term “isolated”, when used in the context of an “isolated cell”, refers to a cell that has been removed from its natural environment, for example, as a part of an organ, tissue, or organism.
[0049] As used herein, the term “modulate” refers to an increase, decrease, or other alteration of any, or all, chemical and biological activities or properties of a biochemical entity, e.g., a wild type or mutant nucleic acid molecule. For example, the term “modulate” can refer to a change in the expression level of a gene or a level of an RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits; or to an activity of one or more proteins or protein subunits that is upregulated or downregulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit” or “suppress”, but the use of the word “modulate” is not limited to this definition.
[0050] The term “naturally occurring”, as applied to an object, refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including bacteria) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. It must be understood, however, that any manipulation by the hand of man can render a “naturally occurring” object an “isolated” object as that term is used herein.
[0051] As used herein, the terms “nucleic acid”, “nucleic acid molecule” and polynucleotide refer to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), or analogs of naturally occurring nucleotides (e.g., alpha-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term “nucleic acid” also includes so-called “peptide nucleic acids”, which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
[0052] The terms “operably linked” and “operatively linked” are used interchangeably. When describing the relationship between two nucleic acid regions, each term refers to a juxtaposition wherein the regions are in a relationship permitting them to function in their intended manner. For example, a control sequence “operably linked” to a coding sequence can be ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules (e.g., inducers and polymerases) are bound to the control or regulatory sequence(s). Thus, in some embodiments, the phrase “operably linked” refers to a promoter connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that promoter. Techniques for operably linking a promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the promoter.
[0053] Thus, the term “operably linked” can refer to a promoter region that is connected to a nucleotide sequence in such a way that the transcription of that nucleotide sequence is controlled and regulated by that promoter region. Similarly, a nucleotide sequence is said to be under the “transcriptional control” of a promoter to which it is operably linked. Techniques for operably linking a promoter region to a nucleotide sequence are known in the art. In some embodiments, a nucleotide sequence comprises a coding sequence and/or an open reading frame. The term “operably linked” can also refer to a transcription termination sequence that is connected to a nucleotide sequence in such a way that termination of transcription of that nucleotide sequence is controlled by that transcription termination sequence.
[0054] The term “operably linked” can also refer to a transcription termination sequence that is connected to a nucleotide sequence in such a way that termination of transcription of that nucleotide sequence is controlled by that transcription termination sequence.
[0055] In some embodiments, more than one of these elements can be operably linked in a single molecule. Thus, in some embodiments multiple terminators, coding sequences, and promoters can be operably linked together. Techniques are known to one of ordinary skill in the art that would allow for the generation of nucleic acid molecules that comprise different combinations of coding sequences and/or regulatory elements that would function to allow for the expression of one or more nucleic acid sequences in a cell.
[0056] The phrases “percent identity” and “percent identical,” in the context of two nucleic acid or protein sequences, refer to two or more sequences or subsequences that have in some embodiments at least 60%, in some embodiments at least 70%, in some embodiments at least 80%, in some embodiments at least 85%, in some embodiments at least 90%, in some embodiments at least 95%, in some embodiments at least 98%, and in some embodiments at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The percent identity exists in some embodiments over a region of the sequences that is at least about 50 residues in length, in some embodiments over a region of at least about 100 residues, and in some embodiments the percent identity exists over at least about 150 residues. In some embodiments, the percent identity exists over the entire length of a given region, such as a coding region.
[0057] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0058] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm described in Smith & Waterman, 1981, by the homology alignment algorithm described in Needleman & Wunsch, 1970, by the search for similarity method described in Pearson & Lipman, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG WISCONSIN PACKAGE, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, Ausubel et al., 1989.
[0059] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information via the World Wide Web. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff, 1992.
[0060] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See e.g., Karlin & Altschul 1993. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in some embodiments less than about 0.1, in some embodiments less than about 0.01, and in some embodiments less than about 0.001.
[0061] As used herein, the terms “polypeptide”, “protein”, and “peptide”, which are used interchangeably herein, refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size or function. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably herein when referring to a gene product. The term “polypeptide” encompasses proteins of all functions, including enzymes. Thus, exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
[0062] The terms “polypeptide fragment” or “fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, 8, or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40, or 50 amino acids long, at least 75 amino acids long, or at least 100, 150, 200, 300, 500, or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. Further, fragments can include a sub-fragment of a specific region, which sub-fragment retains a function of the region from which it is derived.
[0063] As used herein, the term “primer” refers to a sequence comprising in some embodiments two or more deoxyribonucleotides or ribonucleotides, in some embodiments more than three, in some embodiments more than eight, and in some embodiments at least about 20 nucleotides of an exonic or intronic region. Such oligonucleotides are in some embodiments between ten and thirty bases in length.
[0064] The term “promoter” or “promoter region” each refers to a nucleotide sequence within a gene that is positioned 5′ to a coding sequence and functions to direct transcription of the coding sequence. The promoter region comprises a transcriptional start site, and can additionally include one or more transcriptional regulatory elements. In some embodiments, a method of the presently disclosed subject matter employs a RNA polymerase III promoter.
[0065] A “minimal promoter” is a nucleotide sequence that has the minimal elements required to enable basal level transcription to occur. As such, minimal promoters are not complete promoters but rather are subsequences of promoters that are capable of directing a basal level of transcription of a reporter construct in an experimental system. Minimal promoters are often augmented with one or more transcriptional regulatory elements to influence the transcription of an operatively linked gene. For example, cell-type-specific or tissue-specific transcriptional regulatory elements can be added to minimal promoters to create recombinant promoters that direct transcription of an operatively linked nucleotide sequence in a cell-type-specific or tissue-specific manner.
[0066] Different promoters have different combinations of transcriptional regulatory elements. Whether or not a gene is expressed in a cell is dependent on a combination of the particular transcriptional regulatory elements that make up the gene's promoter and the different transcription factors that are present within the nucleus of the cell. As such, promoters are often classified as “constitutive”, “tissue-specific”, “cell-type-specific”, or “inducible”, depending on their functional activities in vivo or in vitro. For example, a constitutive promoter is one that is capable of directing transcription of a gene in a variety of cell types (in some embodiments, in all cell types) of an organism. “Tissue-specific” or “cell-type-specific” promoters, on the other hand, direct transcription in some tissues or cell types of an organism but are inactive in some or all others tissues or cell types. Exemplary tissue-specific promoters include those promoters described in more detail hereinbelow, as well as other tissue-specific and cell-type specific promoters known to those of skill in the art. In some embodiments, a tissue-specific promoter is a seed-specific promoter, leaf specific, root specific promoter.
[0067] When used in the context of a promoter, the term “linked” as used herein refers to a physical proximity of promoter elements such that they function together to direct transcription of an operatively linked nucleotide sequence
[0068] The term “transcriptional regulatory sequence” or “transcriptional regulatory element”, as used herein, each refers to a nucleotide sequence within the promoter region that enables responsiveness to a regulatory transcription factor. Responsiveness can encompass a decrease or an increase in transcriptional output and is mediated by binding of the transcription factor to the DNA molecule comprising the transcriptional regulatory element. In some embodiments, a transcriptional regulatory sequence is a transcription termination sequence, alternatively referred to herein as a transcription termination signal.
[0069] The term “transcription factor” generally refers to a protein that modulates gene expression by interaction with the transcriptional regulatory element and cellular components for transcription, including RNA Polymerase, Transcription Associated Factors (TAFs), chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription.
[0070] The term “purified” refers to an object species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
[0071] A “reference sequence” is a defined sequence used as a basis for a sequence comparison. A reference sequence can be a subset of a larger sequence, for example, as a segment of a full-length nucleotide, or amino acid sequence, or can comprise a complete sequence. Generally, when used to refer to a nucleotide sequence, a reference sequence is at least 200, 300, or 400 nucleotides in length, frequently at least 600 nucleotides in length, and often at least 800 nucleotides in length. Because two proteins can each (1) comprise a sequence (i.e., a portion of the complete protein sequence) that is similar between the two proteins, and (2) can further comprise a sequence that is divergent between the two proteins, sequence comparisons between two (or more) proteins are typically performed by comparing sequences of the two proteins over a “comparison window” (defined hereinabove) to identify and compare local regions of sequence similarity.
[0072] The term “regulatory sequence” is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators, promoters, and termination sequences, which are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operatively linked. Exemplary regulatory sequences are described in Goeddel, 1990, and include, for example, the early and late promoters of simian virus 40 (SV40), adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The nature and use of such control sequences can differ depending upon the host organism. In prokaryotes, such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences. The term “regulatory sequence” is intended to include, at a minimum, components the presence of which can influence expression, and can also include additional components the presence of which is advantageous, for example, leader sequences and fusion partner sequences.
[0073] In some embodiments, transcription of a polynucleotide sequence is under the control of a promoter sequence (or other regulatory sequence) that controls the expression of the polynucleotide in a cell-type in which expression is intended. It will also be understood that the polynucleotide can be under the control of regulatory sequences that are the same or different from those sequences which control expression of the naturally occurring form of the polynucleotide. As used herein, the phrase “functional derivative” refers to a subsequence of a promoter or other regulatory element that has substantially the same activity as the full length sequence from which it was derived. As such, a “functional derivative” of a seed-specific promoter can itself function as a seed-specific promoter.
[0074] Termination of transcription of a polynucleotide sequence is typically regulated by an operatively linked transcription termination sequence (for example, an RNA polymerase III termination sequence). In certain instances, transcriptional terminators are also responsible for correct mRNA polyadenylation. The 3′ non-transcribed regulatory DNA sequence includes in some embodiments about 50 to about 1,000, and in some embodiments about 100 to about 1,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences. Appropriate transcriptional terminators and those that are known to function in plants include the cauliflower mosaic virus (CaMV) 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3′end of the protease inhibitor I or II genes from potato or tomato, although other 3′ elements known to those of skill in the art can also be employed. Alternatively, a gamma coixin, oleosin 3, or other terminator from the genus Coix can be used.
[0075] As used herein, the term “RNA” refers to a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a beta-D-ribofuranose moiety. The terms encompass double stranded RNA, single stranded RNA, RNAs with both double stranded and single stranded regions, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA, or analog RNA, that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of an RNA molecule or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the presently disclosed subject matter can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of a naturally occurring RNA.
[0076] As used herein, the phrase “double stranded RNA” refers to an RNA molecule at least a part of which is in Watson-Crick base pairing forming a duplex. As such, the term is to be understood to encompass an RNA molecule that is either fully or only partially double stranded. Exemplary double stranded RNAs include, but are not limited to molecules comprising at least two distinct RNA strands that are either partially or fully duplexed by intermolecular hybridization. Additionally, the term is intended to include a single RNA molecule that by intramolecular hybridization can form a double stranded region (for example, a hairpin). Thus, as used herein the phrases “intermolecular hybridization” and “intramolecular hybridization” refer to double stranded molecules for which the nucleotides involved in the duplex formation are present on different molecules or the same molecule, respectively.
[0077] As used herein, the phrase “double stranded region” refers to any region of a nucleic acid molecule that is in a double stranded conformation via hydrogen bonding between the nucleotides including, but not limited to hydrogen bonding between cytosine and guanosine, adenosine and thymidine, adenosine and uracil, and any other nucleic acid duplex as would be understood by one of ordinary skill in the art. The length of the double stranded region can vary from about 15 consecutive basepairs to several thousand basepairs. In some embodiments, the double stranded region is at least 15 basepairs, in some embodiments between 15 and 50 basepairs, in some embodiments between 50 and 100 basepairs, in some embodiments between 100 and 500 basepairs, in some embodiments between 500 and 1000 basepairs, and in some embodiments is at least 1000 basepairs. As describe hereinabove, the formation of the double stranded region results from the hybridization of complementary RNA strands (for example, a sense strand and an antisense strand), either via an intermolecular hybridization (i.e., involving 2 or more distinct RNA molecules) or via an intramolecular hybridization, the latter of which can occur when a single RNA molecule contains self-complementary regions that are capable of hybridizing to each other on the same RNA molecule. These self-complementary regions are typically separated by a stretch of nucleotides such that the intramolecular hybridization event forms what is referred to in the art as a “hairpin” or a “stem-loop structure”. In some embodiments, the stretch of nucleotides between the self-complementary regions comprises an intron that is excised from the nucleic acid molecule by RNA processing in the cell.
[0078] As used herein, “significance” or “significant” relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is “significant” or has “significance”, statistical manipulations of the data can be performed to calculate a probability, expressed as a “P-value”. Those P-values that fall below a user-defined cutoff point are regarded as significant. In some embodiments, a P-value less than or equal to 0.05, in some embodiments less than 0.01, in some embodiments less than 0.005, and in some embodiments less than 0.001, are regarded as significant.
[0079] An exemplary nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic in some embodiments at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the presently disclosed subject matter. In one example, probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of a given gene. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical synthesis, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA).
[0080] As used herein, the term “transcription” refers to a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene. The process includes, but is not limited to, the following steps: (a) the transcription initiation; (b) transcript elongation; (c) transcript splicing; (d) transcript capping; (e) transcript termination; (f) transcript polyadenylation; (g) nuclear export of the transcript; (h) transcript editing; and (i) stabilizing the transcript.
[0081] The term “transfection” refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell, which in certain instances involves nucleic acid-mediated gene transfer. The term “transformation” refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. For example, a transformed cell can express a recombinant form of a polypeptide of the presently disclosed subject matter.
[0082] The transformation of a cell with an exogenous nucleic acid (for example, an expression vector) can be characterized as transient or stable. As used herein, the term “stable” refers to a state of persistence that is of a longer duration than that which would be understood in the art as “transient”. These terms can be used both in the context of the transformation of cells (for example, a stable transformation), or for the expression of a transgene (for example, the stable expression of a vector-encoded nucleic acid sequence comprising a trigger sequence) in a transgenic cell. In some embodiments, a stable transformation results in the incorporation of the exogenous nucleic acid molecule (for example, an expression vector) into the genome of the transformed cell. As a result, when the cell divides, the vector DNA is replicated along with plant genome so that progeny cells also contain the exogenous DNA in their genomes.
[0083] In some embodiments, the term “stable expression” relates to expression of a nucleic acid molecule (for example, a vector-encoded nucleic acid sequence comprising a trigger sequence) over time. Thus, stable expression requires that the cell into which the exogenous DNA is introduced express the encoded nucleic acid at a consistent level over time. Additionally, stable expression can occur over the course of generations. When the expressing cell divides, at least a fraction of the resulting daughter cells can also express the encoded nucleic acid, and at about the same level. It should be understood that it is not necessary that every cell derived from the cell into which the vector was originally introduced express the nucleic acid molecule of interest. Rather, particularly in the context of a whole plant, the term “stable expression” requires only that the nucleic acid molecule of interest be stably expressed in tissue(s) and/or location(s) of the plant in which expression is desired. In some embodiments, stable expression of an exogenous nucleic acid is achieved by the integration of the nucleic acid into the genome of the host cell.
[0084] The term “vector” refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector that can be used in accord with the presently disclosed subject matter is an Agrobacterium binary vector, i.e., a nucleic acid capable of integrating the nucleic acid sequence of interest into the host cell (for example, a plant cell) genome. Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector. However, the presently disclosed subject matter is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
[0085] The term “expression vector” as used herein refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to transcription termination sequences. It also typically comprises sequences required for proper translation of the nucleotide sequence. The construct comprising the nucleotide sequence of interest can be chimeric. The construct can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The nucleotide sequence of interest, including any additional sequences designed to effect proper expression of the nucleotide sequences, can also be referred to as an “expression cassette”.
[0086] Embodiments of the presently disclosed subject matter provide an expression cassette comprising one or more elements operably linked in an isolated nucleic acid. In some embodiments, the expression cassette comprises one or more operably linked promoters, coding sequences, and/or promoters.
[0087] Further encompassed within the presently disclosed subject matter are recombinant vectors comprising an expression cassette according to the embodiments of the presently disclosed subject matter. Also encompassed are plant cells comprising expression cassettes according to the present disclosure, and plants comprising these plant cells.
[0088] In some embodiments, the expression cassette is expressed in a specific location or tissue of a plant. In some embodiments, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof.
[0089] Embodiments of the presently disclosed subject matter also relate to an expression vector comprising an expression cassette as disclosed herein. In some embodiments, the expression vector comprises one or more elements including, but not limited to, a promoter sequence, an enhancer sequence, a selection marker sequence, a trigger sequence, an intron-containing hairpin transformation construct, an origin of replication, and combinations thereof.
[0090] The method comprises in some embodiments introducing into a plant cell an expression cassette comprising a nucleic acid molecule encoding a DN-RLK of the to obtain a transformed plant cell or tissue (also referred to herein as a “transgenic” plant cell or tissue), and culturing the transformed plant cell or tissue. The nucleic acid molecule can be under the regulation of a constitutive or inducible promoter, and in some embodiments can be under the regulation of a tissue—or cell type-specific promoter.
[0091] A plant or plant part comprising a cassette encoding a DN-RLK can be analyzed and selected using methods known to those skilled in the art including, but not limited to, Southern blotting, DNA sequencing, and/or PCR analysis using primers specific to the nucleic acid molecule, morphological changes and detecting amplicons produced therefrom.
[0092] Coding sequences intended for expression in transgenic plants can be first assembled in expression cassettes operably linked to a suitable promoter expressible in plants. The expression cassettes can also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not limited to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the transgene-encoded product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors disclosed below. The following is a description of various components of typical expression cassettes.
[0093] The selection of the promoter used in expression cassettes can determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters can express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves, flowers, or seeds, for example) and the selection can reflect the desired location for accumulation of the transgene. Alternatively, the selected promoter can drive expression of the gene under various inducing conditions. Promoters vary in their strength; i.e., their abilities to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non-limiting examples of promoters that can be used in expression cassettes.
[0094] Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower-Binet et al., 1991; maize-Christensen & Quail, 1989; and Arabidposis-Callis et al., 1990). The Arabidposis ubiquitin promoter is suitable for use with the nucleotide sequences of the presently disclosed subject matter. The ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors disclosed herein, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.
[0095] Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter can be used as a constitutive promoter. In particular, the promoter from the rice Actl gene has been cloned and characterized (McElroy et al., 1990). A 1.3 kilobase (kb) fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, expression vectors based on the Acti promoter have been constructed (McElroy et al., 1991). These incorporate the Actl-intron 1, Adhl 5′ flanking sequence (from the maize alcohol dehydrogenase gene) and Adhl-intron 1 and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5′ flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the beta-glucuronidase (GUS) reporter gene) also enhanced expression.
[0096] The promoter expression cassettes disclosed in McElroy et al., 1991, can be easily modified for gene expression. For example, promoter-containing fragments are removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al., 1993).
[0097] A promoter inducible by certain alcohols or ketones, such as ethanol, can also be used to confer inducible expression of a coding sequence of the presently disclosed subject matter. Such a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et a., 1998). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer. For the purposes of the presently disclosed subject matter, the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al., 1998) are replaced by a coding sequence of the presently disclosed subject matter to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods known in the art.
[0098] Induction of expression of a nucleic acid sequence of the presently disclosed subject matter using systems based on steroid hormones is also provided. For example, a glucocorticoid-mediated induction system can be used and gene expression is induced by application of a glucocorticoid, for example, a synthetic glucocorticoid, for example dexamethasone, at a concentration ranging in some embodiments from 0.1 mM to 1 mM, and in some embodiments from 10 mM to 100 mM.
[0099] Another pattern of gene expression is root expression. A suitable root promoter is the promoter of the maize metallothionein-like (MTL) gene disclosed in de Framond, 1991, and also in U.S. Pat. No. 5,466,785, each of which is incorporated herein by reference. This “MTL” promoter is transferred to a suitable vector such as pCGN 1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
[0100] Wound-inducible promoters can also be suitable for gene expression. Numerous such promoters have been disclosed (e.g. Xu et al., 1993; Logemann et al., 1989; Rohrmeier & Lehle, 1993; Firek et al., 1993; Warner et al., 1993) and all are suitable for use with the presently disclosed subject matter. Logemann et al. describe the 5′ upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA that is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similarly, Firek et al. and Warner et al. have disclosed a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to the presently disclosed subject matter, and used to express these genes at the sites of plant wounding.
[0101] A maize gene encoding phosphoenol carboxylase (PEPC) has been disclosed by Hudspeth and Grula, 1989. Using standard molecular biological techniques, the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
[0102] A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for termination of transcription and correct mRNA polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the octopine synthase terminator, and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator can be used.
[0103] Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of the presently disclosed subject matter to increase their expression in transgenic plants.
[0104] Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., 1987). In the same experimental system, the intron from the maize bronzel gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
[0105] A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV; the “W-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (see e.g., Gallie et al., 1987; Skuzeski et al., 1990). Other leader sequences known in the art include, but are not limited to, picornavirus leaders, for example, EMCV (encephalomyocarditis virus) leader (5′ noncoding region; see Elroy-Stein et al., 1989); potyvirus leaders, for example, from Tobacco Etch Virus (TEV; see Allison et al., 1986); Maize Dwarf Mosaic Virus (MDMV; see Kong & Steinbiss 1998); human immunoglobulin heavy-chain binding polypeptide (BiP) leader (Macejak & Sarnow, 1991); untranslated leader from the coat polypeptide mRNA of alfalfa mosaic virus (AMV; RNA 4; see Jobling & Gehrke, 1987); tobacco mosaic virus (TMV) leader (Gallie et al., 1989); and Maize Chlorotic Mottle Virus (MCMV) leader (Lommel et al., 1991). See also Della-Cioppa et al., 1987.
[0106] Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation art, and the genes pertinent to the presently disclosed subject matter can be used in conjunction with any such vectors. The selection of vector will depend upon the selected transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers might be employed. Selection markers used routinely in transformation include the nptil gene, which confers resistance to kanamycin and related antibiotics (Messing & Vieira, 1982; Bevan et al., 1983); the bargene, which confers resistance to the herbicide phosphinothricin (White et al., 1990; Spencer et al., 1990); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, 1984); the dhfr gene, which confers resistance to methotrexate (Bourouis & Jarry, 1983); the EPSP synthase gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642); and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).
[0107] Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as PBIN19 (Bevan, 1984). Below, the construction of two typical vectors suitable for Agrobacterium transformation is disclosed.
[0108] Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector, and consequently vectors lacking these sequences can be utilized in addition to other vectors that contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. polyethylene glycol (PEG) and electroporation), and microinjection. The choice of vector depends largely on the species being transformed.
[0109] Once a DN-RLK is obtained and has been cloned into an expression system, it is transformed into a plant cell. The expression cassettes of the presently disclosed subject matter can be introduced into the plant cell in a number of art-recognized ways. Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.
[0110] Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation-mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are disclosed in Paszkowski et al., 1984; Potrykus et al., 1985; and Klein et al., 1987. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
[0111] Agrobacterium-mediated transformation is a useful technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of a binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain which can depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally.
[0112] Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
[0113] Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050; 5,036,006; and 5,100,792; all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium, or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.
[0114] The following examples are provided to further illustrate but not limit the disclosure.

Examples

[0115] One of the major obstacles to studying the function of receptor-like kinases (RLKs) was that in many cases there are many genes in a subfamily and there was the potential for functional redundancy among subfamily members. This redundancy can explain why few RLK genes have been identified using forward genetics-based mutant screens as well as making it difficult to investigate RLK using gene knockout-based reverse genetics. The disclosure provides a novel approach to circumvent this functional redundancy. The approach uses the similarity of the extracellular domains among subfamily members as a way to disrupt the function of the entire subfamily group.
[0116] In plants the mechanisms for monitoring the nutrient status is critical for plant growth, development, and responses to the environment. Such mechanisms are presumably linked to nutrient uptake, mobilization and redistribution to regulate plant vegetative growth and reproductive development and growth. However, little was known about the molecular basis of nutrient sensing mechanisms in plants.
[0117] Bioinformatics of the Receptor-like Kinase Family in Arabidposis Sequence Annotation, Alignment, and Phylogenetic Analysis. Arabidposis receptor-like kinase gene information was taken from three databases: The Arabidposis Information Resource (TAIR) (www.arabidopsis.org), PlantsP (plantsp.genomics.purdue.edu) and Shiu and Bleecker's 2001 PNAS paper that totaled 651 putative RLKs. Alignment was made using sequences with and without the predicted kinase domain. Because of the interest in extracellular domain homology the methods concentrated on the kinase deletion alignment for further analysis.
[0118] Plants used in this project were Arabidposis thaliana ecotype Columbia-0 (Col-0). Before plating, seeds were surface sterilized. First, the seeds were washed in 95% ethanol for 10 minutes, which was removed then the sterilization solution was added (20% bleach, 0.05% tween-20 (Sigma) and double distilled water) and shaken for 10 minutes. The sterilization solution was removed and the seeds were washed three times with sterile distilled water. The seeds were then cold treated for 4 days at 4° C. after plating them on the plates. Four different growth media were prepared for these experiments. For the control conditions: one-half strength Murashige and Skoog (MS) salts (Sigma), 0.5% sucrose (Sigma), 0.8% phyto agar (Research Products International Corp.), 1× B5 (1,000× in double distilled water: 10% myo-inositol, 0.1% nicotinic acid and 0.1% pyroxidine HCl) and 1× Thiamin (2,000× in double distilled water: 0.2% thiamin HCl). For low nitrogen media: 10× MS micronutrient media (Sigma) was diluted to 0.5× and 10× MS macronutrient containing no nitrogen (40 mM CaCl2.2H2O, 30 mM MgSO4.7H2O and 12.5 mM KH2PO4) was also diluted to 0.5× and 100× Fe.EDTA (18.3 mM FeSO4 and 12.5 mM EDTA) was also added to a final concentration of 1×. All the other components of the control media were kept the same. For sucrose-less media all components of the control media were included except for the omission of sucrose. All media was brought to pH 5.8 with 1N KOH and autoclaved for 20 minutes. Plates were arranged vertically in the growth room and grown at 22° C. with 150 μM photons/m−1s−1 with a 16 h light, 8 h dark photoperiod.
[0119] The Invitrogen Gateway technology was used to expedite the generation of the different RLK mutations used in this study. Generally, a RIKEN cDNA clone (55 RIKEN clones) or wild type seedling cDNA (17 generated by, Table 2.3) was used as a template for polymerase chain reaction (PCR) amplification of the dominant negative. The PCR product was then gel eluted using the Qiagen QIAquick gel extraction kit using the manufacturer's protocol. Eluted DNA was subsequently ligated into Promega's pGEM-Teasy PCR vector. Positive colonies were picked and those with insertions of the DN-RLK into the pGEM vector were confirmed by DNA sequencing: using the T7 and S6 primer sites on the pGEM vector. Confirmed DN-RLK inserts were then restriction digested using the PCR introduced restriction sites (usually SaIl or NotI). The restriction digest was run on a 1% agarose (Invitrogen) gel and the digested insert was removed using the QIAquick kit. The fragment was then ligated into a TAP tagged entry vector that was made by taking the pENTR-1A vector (Invitrogen) and introducing a 6× His and T7 epitope DNA sequence into the EcoRV restriction site in the pENTR-1A vector. This vector was designated pENTR-TAP2. The 3′ ends of all PCR fragments were designed to go into frame with the TAP sequence. The pENTR-TAP2 vectors containing the desired fragments were then introduced into the final destination binary vector that contains the cauliflower mosaic virus (CaMV) 35S promoter, pGWB2 (Invitrogen, Nakagawa). This construct was introduced into Arabidposis (Col-0) via the floral dip method (Bechtold et al., 1993). Subsequent generations of the seeds were selected for using 50 μg/ml Kanamycin (Sigma) in MS media and then transferred to soil until seed set. This process was carried out for subsequent generations until T3 homozygous lines were found and these lines were used for all of the following experiments. For each construct a minimum of 5 independent lines was generated, but in a few cases less then this was achieved.
[0120] Dominant Negative (DN)-RLK plants were examined at all stages of growth for morphological phenotypes. Beginning in the T1 generation plants were examined when grown on soil and compared to wild type (Col-0) plants for changes in flowering time, leaf size and phyllotaxic aberrations. These phenotypes were recorded and examined in further generations. If the phenotype persisted until the homozygous lines were isolated these phenotypes would then be more carefully examined.
[0121] RNA was collected from 10-day old vertically grown seedlings using Qiagen's RNeasy Kit following the manufacture's protocol. Three micrograms of total RNA was used in a reverse transcriptase (Superscript II, Invitrogen) reaction in a 20 μl reaction volume. cDNA obtained from DN-RLK lines was then amplified using gene specific primes and compared to the wild type plants and actin 2 (ACT2) was used as an amplification control.
[0122] Examination of Carbon, Nitrogen and Light Requirements of DN-RLKs. Many DN-RLK lines did not show any apparent phenotypes when grown on soil under normal growing conditions, possible RLK functions were examined using nutritional and light screening methods. Because of the many DN-RLK lines that needed to be screened a vertical plate based growth system was used. For examining the responses to sucrose plates containing 0%, 0.5% (normal) and 3-6% sucrose plates were used and root growth examined. For nitrogen requirements DN-RLK lines were grown on 0 mM and 40 mM nitrogen plates and again root growth was examined. Sucrose and light requirements were also examined using 0% and 0.5% sucrose plates grown in the dark and hypocotyl lengths were examined.
[0123] The approached provided herein was used to identify potential nutrient sensing molecules from the superfamily, receptor-like kinases. As a proof of concept, Arabidposis dominant negative-RLK transgenic lines were screened on a MS agar medium lacking sucrose and identified four RLK genes that affect sucrose sensing. These results suggest that RLKs play an important role in the regulation of sugar status in plants most likely through its potential role in sensing sugar.
[0124] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
(57)

Claim

1. A method of identifying receptor-like kinases (RLKs) that modulate plant function and morphology comprising:
identifying a family of RLKs that comprise at least 50% sequence identity in the extracellular and transmembrane domains;
using a set of PCR primer pair, generating from a cDNA library of RLKs a plurality of RLKs lacking a functional kinase domain (DN-RLKs);
cloning the DN-RLKs into a plant species to obtain recombinant plants comprising at least one DN-RLK from the plurality of DN-RLKs;
expressing the DN-RLKs; and
identifying recombinant plants having morphological or functional traits different than a wild-type plant species.
2. The method of claim 1, wherein the family of RLKs has at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity between members of the family.
3. The method of claim 1, wherein the PCR primer pair comprise a first primer comprises a sequence corresponding to the extracellular domain end of the coding sequence and the second primer comprises a sequence that truncates the kinase domain or induces a mutation in the kinase domain that results in a domain lacks kinase activity.
4. The method of claim 1, wherein the plant species is Arabidposis.
5. A plant generated by the method of claim 1.
6. The recombinant plant of claim 5, wherein the plant comprises improved growth characteristics, pathogen resistance, plant height or metabolic activity compared to a wild-type plant.
7. A method of generating a transgene comprising a dominant-negative receptor-like kinases (RLKs) that modulate plant function and morphology comprising:
identifying a family of RLKs that comprise at least 50% sequence identity in the extracellular and transmembrane domains;
using a set of PCR primer pair, generating from a cDNA library of RLKs a plurality of RLKs lacking a functional kinase domain (DN-RLKs);
cloning at least one DN-RLK from the plurality of DN-RLKs into a vector.
8. A method for modulating plant height, organ shape, metabolism, growth characteristics or pathogen resistance comprising the step of expressing a transgene of claim 7 in a plant, wherein the transgene encodes a receptor-like kinase (RLK) protein lacking an active receptor domain or kinase domain and wherein expression of the transgene modulates plant height, organ shape, metabolism, growth characteristics or pathogen resistance.
9. The method of claim 1, 5, 7 or 8, wherein the plant species is a crop plant.
10. A method for enhancing the plant height, organ shape, metabolism, growth characteristics or pathogen resistance of a plant, comprising the steps of: (a) introducing a transgene of claim 7 into a plant, wherein the transgene encodes a receptor-like kinase protein lacking an active receptor domain or kinase domain and wherein expression of the transgene enhances the plant height, organ shape, metabolism, growth characteristics or pathogen resistance of the crop plant; and
(b) growing the transgenic plant under conditions in which the transgene is expressed to enhance the plant height, organ shape, metabolism, growth characteristics or pathogen resistance of the plant.
11. A library of dominant-negative RLK-encoding polynucleotides wherein the polynucleotide encodes a dominant-negative RLK lacking a receptor domain or kinase domain, the library obtained by the method of claim 7.
12. A method of making a library of dominant-negative RLK encoding polynucleotides comprising:
(a) identifying a family of RLKs having at least 50% identity to one another;
(b) mutating the RLKs having identity to disrupt function ligand binding function or kinase function; and
(c) cloning the mutant RLKs.
13. The method of claim 12, further comprising transforming plant cells with the mutant RLKs.
14. The method of claim 13, further comprising growing the mutant cells and identifying cells displaying a mutant phenotype.
15. A library of dominant negative plant cells comprising a transgene encoding a receptor-like kinase lacking a receptor domain or a kinase domain.
*****

Download Citation


Sign in to the Lens

Feedback