BACKGROUND
multi-cellular organisms such as plants and animals use cell surface receptors to sense and transduce chemical signals for cell-to-cell communications.
one of the most important groups of cell surface receptors, the receptor-like protein kinases , has unique structural features that make them particularly suitable for cell-to-cell signaling.
a typical rlk contains an extracellular receptor domain to perceive a specific signal, a single-pass transmembrane domain to anchor the protein within the membrane, and a cytoplasmic kinase domain to transduce the signal downstream via autophosphorylation followed by further phosphorylation of specific substrates.
plant receptor kinases were originally named "receptor-like" protein kinases since ligands for these receptors were largely unknown at the time when the first rlk was identified in maize  <cit> .
since then, a small number of rlks have been functionally characterized in plants and a few specific ligands have been identified.
they play essential roles in plant growth, development, pathogen resistance and cell death  <cit> .
in the model plant arabidopsis, both transmembrane rlks and receptor-like cytoplasmic kinases  belong to a large, monophyletic gene superfamily of at least  <dig> members, representing nearly  <dig> % of the protein coding sequences within the entire genome  <cit> .
about two thirds of genes in this superfamily encode proteins with a typical n-terminal signal peptide and a hydrophobic transmembrane domain, which are consistent structural features of transmembrane rlks.
based on their structural and sequence similarities, the rlks are further grouped into more than  <dig> subfamilies.
leucine-rich repeat -rlks represent the largest subfamily in the arabidopsis genome with at least  <dig> members  <cit> .
despite the identification of a large number of lrr-rlks in arabidopsis, biological functions have been defined for only about  <dig> proteins , which play crucial roles in a variety of different physiological processes.
for instance, erecta  regulates organ shape and inflorescence architecture  <cit> ; clavata <dig>  determines the balance between undifferentiated and differentiated shoot and floral meristem cells  <cit> ; brassinosteroid-insensitive  <dig>  and bri1-associated receptor kinase  <dig>  are a pair of rlks involved in brassinosteroid  signaling  <cit> ; haesa controls floral organ abscission  <cit> ; flagellin-sensitive  <dig>  contributes to plant defense/pathogen-recognition  <cit> ; vascular highway  <dig>  influences leaf cell patterning  <cit> ; and excess microspocytes  <dig> , somatic embryogenesis receptor kinase  <dig>  and serk <dig> play important roles in microsporogenesis and male sterility  <cit> .
other lrr-rlks of known function include receptor-like protein kinase  <dig> , involved in abscisic acid early signaling  <cit> ; toad <dig> and its redundant homologue rpk <dig>  both required in arabidopsis embryonic pattern formation  <cit> ; pxy, responsible for maintaining vascular tissue polarity  <cit> ; and gassho <dig>  and gassho <dig>  which are essential for the normal development of epidermal surface of arabidopsis embryos  <cit> .
recently, two lrr-rlks, bir <dig> and sobir <dig>  were identified to regulate cell death and innate immunity in arabidopsis  <cit> .
interestingly, several rlks were found to possess dual or multiple roles during plant growth and development.
for example, erecta is involved in both plant development and pathogen defense responses  <cit> .
bak <dig> and bak1-like  <dig>  regulate br-dependent cell growth, and play an important role in cell-death control under various biotic and abiotic stresses.
when plants are attacked by bacterial pathogens, bak <dig> also can be recruited to the fls <dig> complex and regulates the innate immunity response  <cit> .
reverse genetics has been used as a routine and effective approach to dissect the biological functions of genes.
isolated complementary dna  sequences are valuable resources in many processes in determining the functions of their corresponding genes.
for example, the cdna sequences can be used for ectopic expression, complementary experiments for gene knock out lines, site-directed mutagenesis, dominant negative analysis, gene silencing and rna interference, subcellular localization of epitope-tagged fusion proteins, and protein-protein interaction analysis.
epitope-tagged fusion proteins can also facilitate the proteomic studies of interesting genes.
for example, in vivo phosphorylation sites of bri <dig> and bak <dig> were identified by immunoprecipitation of epitope-tagged bri1/bak <dig> from arabidopsis followed by liquid chromatography-tandem mass spectrometry  and the functions of the identified phosphorylation sites were determined in planta  <cit> .
in this paper, the full-length cdna cloning of the entire arabidopsis lrr-rlk subfamily genes is reported.
a total of  <dig> cdna sequences have been successfully amplified by rt-pcr and cloned into a gatewayr donor vector pdonr/zeor.
sequence analysis indicated that  <dig> cdnas are identical to the predicted or earlier submitted cdna sequences in the arabidopsis information resource  database, whereas  <dig> other genes showed altered cdna sequences distinct from those presented in the database, which is likely due to alternative splicing of pre-mrna.
one hundred eighty cdna sequences with 100% sequence accuracy were further transferred, by in vitro dna recombination, into two different destination vectors with either flag or gfp as the c-terminal fusion tags.
preliminary results indicated that most of the gene products can be detected by western hybridization analysis using anti-flag or anti-gfp antibodies.
the results and resources generated by this study will be useful tools for future functional analyses of lrr-rlks.
RESULTS
construction of gatewayr-compatible binary vectors for plant transformation
to facilitate future functional analyses of all lrr-rlks, we generated  <dig> different gatewayr-compatible binary vectors for high through-put cloning of lrr-rlks .
the four vectors contain a gatewayr cassette for dna recombination with plasmid dna of entry clones to produce final expression constructs.
gfp or flag sequences were integrated at the 3' terminus of the gatewayr cassette for the production of epitope-tagged fusion proteins that will facilitate subsequent immunoprecipitation and coimmunoprecipitation analyses.
the first vector, named pb35gwg, contains a basta resistance gene for selecting transgenic plants and a c-terminal gfp tag.
the second vector, designated pk35gwg, uses a kanamycin resistance gene for selecting transgenic plants, also with a c-terminal gfp tag.
the third vector, termed pb35gwf, uses the basta gene for transgenic selection and flag as the c-terminal fusion tag.
the fourth vector, labeled pk35gwf, contains a kanamycin resistance gene as the selectable marker and again has a c-terminal flag tag.
all vectors use the camv  <dig> s promoter with dual enhancers to drive expression of the gene of interest.
detailed sequence information of the junction region of the gatewayr cassette and the gfp or flag eptiope tags is also shown .
to examine whether the newly-constructed gatewayr-compatible vectors are reliable in generating lrr-rlk overexpressed transgenic plants, a functionally characterized gene, bak <dig>  was used for the test.
previous studies have shown that bak <dig> is involved in the br signal transduction pathway  <cit> .
overexpession of bak <dig> can suppress the dwarf phenotype of the bri <dig> weak allele, bri1- <dig>  to wildtype  <cit> .
to clone bak <dig> into the destination vectors, attb <dig> and attb <dig> flanked bak <dig> was pcr-amplified and gel purified as described in experimental procedures.
after bp and lr clonase reactions, bak <dig> was transferred into the destination vectors and introduced into bri1- <dig> mutant plants.
obtained transgenic plants showed a typical bri1- <dig> suppression phenotype .
western hybridization analysis using anti-flag or anti-gfp antibodies also indicated that both bak1-flag and bak1-gfp were truly overexpressed in the transgenic plants .
the results suggest that the generated destination vectors are fully functional and can be used for cloning and overexpression of all lrr-rlks in arabidopsis plants for future functional analyses.
gatewayr cloning of lrr-rlks
a three-step protocol was used to efficiently produce attb1- and attb2-flanked lrr-rlk orf fragments :  the reverse transcriptase reaction to generate single-stranded cdna;  the first round of pcr with gene-specific primers to amplify target orf flanked with partial attb <dig> and attb <dig> adaptor sequences; and  the second round of pcr with universal attb <dig> and attb <dig> adaptor primers to integrate complete attb <dig> and attb <dig> sites into the orf amplicons.
two hundred twenty three predicted lrr-rlks distributed on all five chromosomes of the arabidopsis genome with orf sizes ranging from  <dig> bp - <dig>  bp are presented in tair <dig>  the coding sequences of  <dig> lrr-rlks are larger than  <dig>  bp.
superscript iii was used to produce long cdnas with full-length orfs and a proof-reading polymerase  was employed to amplify the predicted orfs with high fidelity.
two rounds of pcr can produce enough dna for gatewayr cloning even for some genes with relatively low expression.
pcr products were obtained for  <dig> of the  <dig> predicted lrr-rlks genes, while  <dig> genes were never amplified by rt-pcr .
all pcr products were agarose gel purified and introduced into pdonr/zeor to produce the entry clones.
plasmid dna from entry clones was then used for lr clonase-mediated in vitro dna recombination with appropriate destination vectors to yield flag and gfp epitope tagged constructs.
sequence analysis of the isolated lrr-rlks
a total of  <dig> cdna sequences were successfully cloned into the donor vector and are summarized in table s <dig> in additional file  <dig>  among them,  <dig>  of the clones contain cdna sequences identical to those predicted in tair <dig> .
the other  <dig> isolated sequences  display gene structures that are different from their corresponding predictions in tair <dig> .
based on their structural differences, they can be divided into two groups:  one complete orf exists from the predicted start codon to the predicted stop codon despite the coding sequences being different from that predicted ;  no continuous orf exists from the predicted start codon to the predicted stop codon because of the different coding sequences .
the other  <dig> lrr-rlks  were not isolated successfully because of possible wrong annotation, specific and/or low expression, and bactericidal effect.
the first group includes  <dig> genes .
the detailed sequence differences are summarized in table s <dig> and the alignments among isolated cdnas, predicted orfs and the corresponding genomic dna sequences are shown in additional file  <dig>  the isolated sequence of at1g <dig> is  <dig> bp shorter than the prediction.
one clone  with the same sequence as the prediction was found in database, indicating that this gene has transcripts with alternative splicing .
the isolated sequence of at4g <dig> is  <dig> bp longer than the prediction, and both cs at position  <dig>  and  <dig>  in the isolated sequence are not found in the arabidopsis genome .
the isolated sequence of at5g <dig> displays two unpredicted exons and shows one unpredicted intron in predicted sequence .
an unpredicted exon is found in the isolated sequence of at5g <dig> .
two predicted introns in gene at3g <dig> are eliminated in the isolated sequence and have become a part of the first exon .
the first predicted exons of at3g <dig> and at4g <dig> have one unpredicted extra intron each .
the predicted 5th exon of at5g <dig> contains one extra unpredicted intron, and the intron/exon boundary is also different from that predicted .
two predicted exons disappear and one unpredicted intron is shown in the predicted 10th exon of at1g <dig>  and a different intron/exon boundary is also observed .
the isolated sequence of at5g <dig> is  <dig> bp shorter than the prediction because of the different intron/exon boundaries; and a riken clone  without a continuous orf from the predicted start codon to the predicted stop codon is available in the database .
isolated sequences of the other  <dig> genes, at1g <dig>  at1g <dig>  at1g <dig>  at1g <dig>  at1g <dig>  at1g <dig>  at2g <dig>  at3g <dig>  at4g <dig>  at4g <dig>  at5g <dig>  at5g <dig> and at5g <dig>  show different intron/exon boundaries compared with the predicted sequences, resulting in different mrna sequences .
the second group contains  <dig> genes .
the detailed sequence differences are summarized in table s <dig> in additional file  <dig> and the alignments among isolated cdnas, predicted orfs and the corresponding genomic dna sequences are shown in additional file  <dig>  unlike the genes in the first group, the isolated sequences in this group do not display continuous orfs from the predicted start codon to the predicted stop codon that were used to design the forward and reverse pcr primers for gatewayr cloning.
the isolated sequences of genes at1g <dig>  at1g <dig>  at1g <dig>  at1g <dig>  at3g <dig> and at5g <dig> exhibit different intron/exon boundaries compared to the predicted orf sequences .
different intron/exon boundaries are also found in the isolated orf sequences of at1g <dig>  at1g <dig>  at5g <dig>  at1g <dig> and at1g <dig>  with other structural differences .
the predicted 6th intron disappears in the isolated orf sequence of at1g <dig> .
the predicted intron  <dig>  exon  <dig> and exon  <dig> are merged into exon  <dig> and the predicted exon  <dig> is split into exon  <dig> and exon  <dig> in the isolated sequence of at1g <dig> .
the third predicted exon does not exist in the experimentally derived sequence of at5g <dig>  and the other six predicted exons are merged into two exons .
the first two exons and the first intron in the prediction of at1g <dig> merge into the first exon in the isolated sequence .
the predicted exon  <dig> is split into exon  <dig> and exon  <dig> in the isolated sequence of at1g <dig>  in database, a previously isolated sequence  is different from both the prediction of at1g <dig> in tair <dig> and the sequence from this report, losing the sequence from exon  <dig> to exon  <dig> and part of exon  <dig>  resulting in a much smaller protein with  <dig> aa compared to the predicted protein of  <dig>  aa .
two predicted introns in at2g <dig> do not exist in the isolated sequence .
at1g <dig> displays an unpredicted intron .
one extra unpredicted intron is shown in at4g <dig>  and the isolated sequence is different from both the existing sequence  and the tair prediction .
although the isolated sequences of genes at4g <dig> and at5g <dig> are the same as the current predictions in tair <dig>  the previously reported coding sequences of them are different .
the predicted exon  <dig> of at4g <dig> is split into exon  <dig> and exon  <dig> in sequence ak <dig>  .
gene at5g <dig> has a new annotation in tair <dig>  the isolated sequence contains the same orf as the current prediction, but the first two predicted exons in tair <dig> are arranged as three exons.
the existing sequence ak <dig>  shows a different intron/exon boundary between exon  <dig> and intron  <dig>  resulting in a smaller orf of  <dig> amino acids .
detection of alternative splicing of lrr-rlks
potentially alternatively spliced variants of  <dig> lrr-rlks were examined by rt-pcr with variant-specific primers according to the predicted mrna sequences and previous reports .
isolated cdna sequences from this study were not examined because they were identified by rt-pcr during the cloning procedure.
isolated cdna of at4g <dig> in this report showed a structure with slight difference from the prediction in database, which made it difficult to examine the sequence difference with variant-specific primers.
this gene was not included in the rt-pcr experiment.
from inflorescence,  <dig> variants of  <dig> lrr-rlks were confirmed by rt-pcr with expected size of products .
no rt-pcr products were obtained from at1g <dig>  at1g <dig>  at3g <dig>  at3g <dig> and at4g <dig>  the previously reported cdna sequence  of at1g <dig> was not amplified from this study, but the predicted variant sequence  of it was confirmed by rt-pcr.
both the previously reported cdna sequence  and the predicted mrna sequence  of at5g <dig> were confirmed in this study.
from leaf, rt-pcr fragments of  <dig> variants of  <dig> lrr-rlks were obtained .
no rt-pcr products with expected sizes were obtained for the same genes as in inflorescence except at1g <dig> that produced a larger fragment than predicted.
no rt-pcr product of previously reported cdna  of at4g <dig> was recovered.
together, a total of  <dig> lrr-rlks were confirmed with alternative splicing of pre-mrna, including four previously reported cdna variants of at1g <dig> , at4g <dig> , at5g <dig>  and at5g <dig> .
lrr-rlks phylogenetic analysis
sequence analyses of isolated lrr-rlks reported in this paper demonstrate that some of them encode protein sequences distinct from the predictions.
this sequence variation and the improved annotation of arabidopsis genome makes it necessary to examine the previously created phylogenies of this superfamily.
the previous report suggested  <dig> subfamilies because the sequences clearly fell into distinct clades  <cit> .
studies in this report based on the alignment of the full-length amino acid sequences result in a similar phylogenetic tree to the previous report  <cit>  with minor adjustments .
at1g <dig>  a member of the previously assigned subfamily lrr x, fell into the lrr vii subfamily;  two members  of the previously assigned subfamily lrr xii, one previously ungrouped gene , and one member  of the subfamily lrr xiv fell into the lrr xi subfamily.
epitope-tagged proteins of lrr-rlks in transgenic arabidopsis plants
the expression of lrr-rlks cloned in the destination vectors pb35gwf and pk35gwg and transformed into arabidopsis ecotype 'columbia- <dig> ' was verified by western hybridization analysis with αflag and αgfp antibodies respectively .
immunoprecipitated membrane protein was prepared and separated by sds-page for the detection of flag-tagged fusion proteins while total protein could be used directly to detect signals of gfp-tagged fusion proteins.
the flag- or gfp-tagged lrr-rlks could be detected in most of the examined transgenic lines usually as one distinct and specific protein band.
discussion
experimentally derived sequences help to verify and expand the predicted genome annotation
the tair annotation release tair <dig>  contains  <dig>  genes, including  <dig>  putative protein coding genes.
among all the putative protein coding genes,  <dig>  genes have not been experimentally supported by identified transcripts.
among the  <dig> predicted lrr-rlk genes,  <dig> of them have no est support.
est support for  <dig> of them is now provided .
a total of  <dig> lrr-rlk genes have no isolated full length coding sequence in the existing database.
from this study,  <dig> new lrr-rlk cdnas with full length coding sequence were provided .
the resources generated in this study will provide useful tools for future functional analyses of this important protein family.
at the same time, phylogenetic analysis can guide researchers to create double, even higher level, mutants to overcome functional redundancy of genes in one subfamily.
for example, elegant genetics studies in subfamily lrr ii revealed redundant functions of serk genes in brassinosteroid signal transduction  <cit> , male sporogenesis  <cit> , pathogen response  <cit>  and cell death  <cit> .
phylogenetic analysis in this study indicated that several subfamilies, such as lrr xii whose members fell into two different subfamilies based on the phylogeny of full length amino acid sequence, could be rearranged to aim future functional analysis of their gene members.
the sequence data generated from this project will also greatly improve genome annotation  <cit> .
in a previous study,  <dig>  full-length gene transcripts from arabidopsis were used to re-annotate its genome.
the results indicated that the gene structures of approximately 35% of the examined genes could be improved according to the isolated full-length cdna sequences  <cit> .
when examing existing est and full-length cdna sequences for all of the predicted lrr-rlks, one full-length cdna, clone rafl25-47-f <dig> , was identified that covered two predicted loci, at1g <dig> and at1g <dig>  with a full and complete orf of  <dig> aa.
the loci should be merged into one according to this data.
as described above, from this study a total of  <dig> genes were identified with different variant transcripts compared to the predictions .
all the data are useful for the improvement of arabidopsis lrr-rlk annotation.
gene functions and alternatively spliced transcripts
the tair <dig> release showed that  <dig>  of the annotated  <dig>  protein coding genes  have alternatively spliced transcripts.
in this report, sequence analyses show that a total of  <dig> lrr-rlk genes have different sequences from the tair <dig> predictions.
this includes two possibilities:  the prediction was not correct;  both the predicted and the isolated sequences exist in plant, which suggests some lrr-rlk genes have alternatively spliced transcripts, possibly in the same tissue, or in different tissues, or under different growth conditions.
the sequence analysis of isolated lrr-rlks in this report revealed different forms of the cds compared to tair <dig> predictions or the existing sequences in the database, including alternative intron donor and/or acceptor sites , unpredicted introns , unpredicted exons , unspliced introns  and different combinations of the aforementioned changes.
they form a continuous orf or several discontinuous orfs.
the presence of the observed alternative splicing was further confirmed by rt-pcr .
it is already known that alternative splicing can significantly increase the complexity of the transcriptome and proteome by synthesizing multiple transcripts and proteins from one gene.
several previous reports showed that approximately 20% of arabidopsis genes are alternatively spliced and some alternatively spliced transcripts have different functions  <cit> .
serine/arginine-rich  proteins form a conserved family of splicing regulators in eukaryotes.
the pre-mrnas of arabidopsis sr genes are extensively alternatively spliced, and about  <dig> transcripts are produced from  <dig> genes.
the transcriptome complexity of sr genes is increased by six-fold.
abiotic stresses regulate the alternative splicing of the pre-mrnas of sr genes to produce different isoforms of sr proteins that are likely to have altered functions in pre-mrna splicing  <cit> .
six mrna variants were generated by alternative splicing in the pre-mrna of a homologue of sr protein, atsr45a.
the transcript abundance and the splicing patterns of atsr45a were altered under various types of stress  <cit> .
the u <dig> small nuclear ribonucleoprotein particle  70k protein  interacts with splicing factors and is involved in basic and alternative splicing of pre-mrna  <cit> .
in arabidopsis, two distinct transcripts are produced by alternative splicing of the pre-mrna of the u <dig> snrna 70k gene.
only the short transcript encodes a full-length functional u1-70k, whereas the long transcript codes for a truncated u1-70k  <cit> .
cop <dig> is a negative regulator of arabidopsis light-dependent development.
cop1b is generated by alternative splicing, resulting in a 60-amino acid deletion in the wd- <dig> repeat domain relative to the full-length cop <dig>  which functions as a dominant negative regulator of cop <dig> function  <cit> .
the maize mik gene codes for a gck-like map4k that can be activated by interaction with maize atypical receptor kinase   <cit> .
four different mature mrnas of mik are generated by alternative splicing, and the resulting polypeptides display different kinase activity and are differentially activated by interaction with the mark receptor  <cit> .
recent studies further demonstrated that alternative splicing affected regions frequently code for intrinsically disordered regions of the corresponding protein products and the association of alternative splicing and intrinsic disorder results in various isoforms to increase the functional and regulatory diversity of the gene  <cit> .
lrr-rlks are critical proteins involved in many aspects of plant growth, development and stress responses.
it is noticed that six genes  produce rt-pcr fragments with different sizes in inflorescence and leaf , which indicates that different forms of lrr-rlk protein may be required for distinct tissue development and function.
some of the alternatively spliced transcripts of lrr-rlks will generate truncated versions of the predicted proteins.
the truncated proteins may be involved in the functional regulation of these genes in different developmental stages and different growth conditions/stresses.
future functional analyses of the alternatively spliced lrr-rlks, revealed from this study, would eventually elucidate the biological meaning of the process.
gene function and phosphorylation sites analysis
clones reported in this paper are not only a resource for gene annotation, but also will be very useful for gene function analysis.
genes in entry clones can be transferred freely to any gatewayr-compatible destination vectors and introduced into arabidopsis.
they can be used for overexpression in arabidopsis to dissect the resulting phenotypes that will indicate the possible related pathways and functions of the target genes.
they can be used to generate different epitope-tagged fusion proteins.
for example, as described above, gfp- and flag-tagged fusion protein can be produced in transgenic plants with different antibiotic resistances.
the subcellular localizations of the interesting genes can be determined with the help of confocal microscopy.
the homodimerization or heterodimerization between lrr-rlks can be detected and confirmed by coexpression in planta and coimmunoprecipitation analysis.
the transgenic plants can also be used to isolate protein complexes for each lrr-rlks, which can help to dissect the complicated signaling pathways that the genes participate in.
the cloned genes could be mutated directly by site-directed mutagenesis in entry clones to create kinase-inactive copies.
overexpression of kinase-inactive genes in arabidopsis will be useful to dissect the functions of the genes by dominant negative effects, especially in the case that functional redundancy is a problem during analysis of them.
in order to clearly understand lrr-rlk function, it is necessary to characterize cytoplasmic kinase domain phosphorylation and examine the role of receptor oligomerization in initiating signaling pathways.
the primary goal of this study was to generate resources for our current arabidopsis  <dig> project that is focused on mapping lrr-rlk phosphorylation sites, assessing the functions of the identified sites in plant growth and development, and examining the in vivo interactions of numerous lrr-rlks.
a prototype for this approach has been developed for the bri <dig> and bak <dig>  two lrr-rlks involved in br signaling.
for example, immunoprecipitated bri1-flag protein was analyzed by liquid chromatography-tandem mass spectrometry  and multiple in vivo phosphorylated ser and thr residues of bri <dig> were identified.
t- <dig> and s- <dig> are highly conserved activation loop residues that were shown to be essential for kinase function in vitro and bri <dig> signaling in planta  <cit> .
the interaction of bri <dig> and bak <dig> was studied in detail both in vitro and in vivo, and a novel mechanism of sequential transphosphorylation was developed, which helps explain the role of the bak <dig> co-receptor in regulating br signaling through bri <dig>  <cit> .
this approach, utilizing the resources developed here, is being expanded to examine the mechanisms of action of numerous lrr-rlks across this important family of regulatory proteins.
CONCLUSIONS
this study generated four gatewayr-compatible destination vectors for plant transformation and they were proved functional by overexpressing bak <dig> to suppress bri1- <dig> mutant plant phenotype.
complementary dna sequences of  <dig> arabidopsis lrr-rlks were cloned into the gatewayr donor vector pdonr/zeor and analyzed by dna sequencing.
a total of  <dig> isolated lrr-rlks showed distinct sequences from the database prediction or previously reported sequences.
alternative rna splicing was observed in some of them, which was thought involved in the regulation of gene functions and plant development.
experimental evidences for the annotation of these lrr-rlks were provided in is study.
the generated cdna clones, expression constructs and transgenic plants are useful resources for scientific communities and will accelerate the research in this field.

 multi-cellular organisms such as plants and animals use cell surface receptors to sense and transduce chemical signals for cell-to-cell communications. one of the most important groups of cell surface receptors, the receptor-like protein kinases, has unique structural features that make them particularly suitable for cell-to-cell signaling. a typical rlk contains an extracellular receptor domain to perceive a specific signal, a single-pass transmembrane domain to anchor the protein within the membrane, and a cytoplasmic kinase domain to transduce the signal downstream via autophosphorylation followed by further phosphorylation of specific substrates. plant receptor kinases were originally named "receptor-like" protein kinases since ligands for these receptors were largely unknown at the time when the first rlk was identified in maize. since then, a small number of rlks have been functionally characterized in plants and a few specific ligands have been identified. they play essential roles in plant growth, development, pathogen resistance and cell death. in the model plant arabidopsis, both transmembrane rlks and receptor-like cytoplasmic kinases belong to a large, monophyletic gene superfamily of at least members, representing nearly % of the protein coding sequences within the entire genome. about two thirds of genes in this superfamily encode proteins with a typical n-terminal signal peptide and a hydrophobic transmembrane domain, which are consistent structural features of transmembrane rlks. based on their structural and sequence similarities, the rlks are further grouped into more than subfamilies. leucine-rich repeat -rlks represent the largest subfamily in the arabidopsis genome with at least members. despite the identification of a large number of lrr-rlks in arabidopsis, biological functions have been defined for only about proteins, which play crucial roles in a variety of different physiological processes. for instance, erecta regulates organ shape and inflorescence architecture; clavata determines the balance between undifferentiated and differentiated shoot and floral meristem cells; brassinosteroid-insensitive and bri1-associated receptor kinase are a pair of rlks involved in brassinosteroid signaling; haesa controls floral organ abscission; flagellin-sensitive contributes to plant defense/pathogen-recognition; vascular highway influences leaf cell patterning; and excess microspocytes, somatic embryogenesis receptor kinase and serk play important roles in microsporogenesis and male sterility. other lrr-rlks of known function include receptor-like protein kinase, involved in abscisic acid early signaling; toad and its redundant homologue rpk both required in arabidopsis embryonic pattern formation; pxy, responsible for maintaining vascular tissue polarity; and gassho and gassho which are essential for the normal development of epidermal surface of arabidopsis embryos. recently, two lrr-rlks, bir and sobir were identified to regulate cell death and innate immunity in arabidopsis. interestingly, several rlks were found to possess dual or multiple roles during plant growth and development. for example, erecta is involved in both plant development and pathogen defense responses. bak and bak1-like regulate br-dependent cell growth, and play an important role in cell-death control under various biotic and abiotic stresses. when plants are attacked by bacterial pathogens, bak also can be recruited to the fls complex and regulates the innate immunity response. reverse genetics has been used as a routine and effective approach to dissect the biological functions of genes. isolated complementary dna sequences are valuable resources in many processes in determining the functions of their corresponding genes. for example, the cdna sequences can be used for ectopic expression, complementary experiments for gene knock out lines, site-directed mutagenesis, dominant negative analysis, gene silencing and rna interference, subcellular localization of epitope-tagged fusion proteins, and protein-protein interaction analysis. epitope-tagged fusion proteins can also facilitate the proteomic studies of interesting genes. for example, in vivo phosphorylation sites of bri and bak were identified by immunoprecipitation of epitope-tagged bri1/bak from arabidopsis followed by liquid chromatography-tandem mass spectrometry and the functions of the identified phosphorylation sites were determined in planta. in this paper, the full-length cdna cloning of the entire arabidopsis lrr-rlk subfamily genes is reported. a total of cdna sequences have been successfully amplified by rt-pcr and cloned into a gatewayr donor vector pdonr/zeor. sequence analysis indicated that cdnas are identical to the predicted or earlier submitted cdna sequences in the arabidopsis information resource database, whereas other genes showed altered cdna sequences distinct from those presented in the database, which is likely due to alternative splicing of pre-mrna. one hundred eighty cdna sequences with 100% sequence accuracy were further transferred, by in vitro dna recombination, into two different destination vectors with either flag or gfp as the c-terminal fusion tags. preliminary results indicated that most of the gene products can be detected by western hybridization analysis using anti-flag or anti-gfp antibodies. the results and resources generated by this study will be useful tools for future functional analyses of lrr-rlks.  construction of gatewayr-compatible binary vectors for plant transformation to facilitate future functional analyses of all lrr-rlks, we generated different gatewayr-compatible binary vectors for high through-put cloning of lrr-rlks. the four vectors contain a gatewayr cassette for dna recombination with plasmid dna of entry clones to produce final expression constructs. gfp or flag sequences were integrated at the 3' terminus of the gatewayr cassette for the production of epitope-tagged fusion proteins that will facilitate subsequent immunoprecipitation and coimmunoprecipitation analyses. the first vector, named pb35gwg, contains a basta resistance gene for selecting transgenic plants and a c-terminal gfp tag. the second vector, designated pk35gwg, uses a kanamycin resistance gene for selecting transgenic plants, also with a c-terminal gfp tag. the third vector, termed pb35gwf, uses the basta gene for transgenic selection and flag as the c-terminal fusion tag. the fourth vector, labeled pk35gwf, contains a kanamycin resistance gene as the selectable marker and again has a c-terminal flag tag. all vectors use the camv s promoter with dual enhancers to drive expression of the gene of interest. detailed sequence information of the junction region of the gatewayr cassette and the gfp or flag eptiope tags is also shown. to examine whether the newly-constructed gatewayr-compatible vectors are reliable in generating lrr-rlk overexpressed transgenic plants, a functionally characterized gene, bak was used for the test. previous studies have shown that bak is involved in the br signal transduction pathway. overexpession of bak can suppress the dwarf phenotype of the bri weak allele, bri1- to wildtype. to clone bak into the destination vectors, attb and attb flanked bak was pcr-amplified and gel purified as described in experimental procedures. after bp and lr clonase reactions, bak was transferred into the destination vectors and introduced into bri1- mutant plants. obtained transgenic plants showed a typical bri1- suppression phenotype. western hybridization analysis using anti-flag or anti-gfp antibodies also indicated that both bak1-flag and bak1-gfp were truly overexpressed in the transgenic plants. the results suggest that the generated destination vectors are fully functional and can be used for cloning and overexpression of all lrr-rlks in arabidopsis plants for future functional analyses. gatewayr cloning of lrr-rlks a three-step protocol was used to efficiently produce attb1- and attb2-flanked lrr-rlk orf fragments: the reverse transcriptase reaction to generate single-stranded cdna; the first round of pcr with gene-specific primers to amplify target orf flanked with partial attb and attb adaptor sequences; and the second round of pcr with universal attb and attb adaptor primers to integrate complete attb and attb sites into the orf amplicons. two hundred twenty three predicted lrr-rlks distributed on all five chromosomes of the arabidopsis genome with orf sizes ranging from bp - bp are presented in tair the coding sequences of lrr-rlks are larger than bp. superscript iii was used to produce long cdnas with full-length orfs and a proof-reading polymerase was employed to amplify the predicted orfs with high fidelity. two rounds of pcr can produce enough dna for gatewayr cloning even for some genes with relatively low expression. pcr products were obtained for of the predicted lrr-rlks genes, while genes were never amplified by rt-pcr. all pcr products were agarose gel purified and introduced into pdonr/zeor to produce the entry clones. plasmid dna from entry clones was then used for lr clonase-mediated in vitro dna recombination with appropriate destination vectors to yield flag and gfp epitope tagged constructs. sequence analysis of the isolated lrr-rlks a total of cdna sequences were successfully cloned into the donor vector and are summarized in table s in additional file among them, of the clones contain cdna sequences identical to those predicted in tair. the other isolated sequences display gene structures that are different from their corresponding predictions in tair. based on their structural differences, they can be divided into two groups: one complete orf exists from the predicted start codon to the predicted stop codon despite the coding sequences being different from that predicted; no continuous orf exists from the predicted start codon to the predicted stop codon because of the different coding sequences. the other lrr-rlks were not isolated successfully because of possible wrong annotation, specific and/or low expression, and bactericidal effect. the first group includes genes. the detailed sequence differences are summarized in table s and the alignments among isolated cdnas, predicted orfs and the corresponding genomic dna sequences are shown in additional file the isolated sequence of at1g is bp shorter than the prediction. one clone with the same sequence as the prediction was found in database, indicating that this gene has transcripts with alternative splicing. the isolated sequence of at4g is bp longer than the prediction, and both cs at position and in the isolated sequence are not found in the arabidopsis genome. the isolated sequence of at5g displays two unpredicted exons and shows one unpredicted intron in predicted sequence. an unpredicted exon is found in the isolated sequence of at5g. two predicted introns in gene at3g are eliminated in the isolated sequence and have become a part of the first exon. the first predicted exons of at3g and at4g have one unpredicted extra intron each. the predicted 5th exon of at5g contains one extra unpredicted intron, and the intron/exon boundary is also different from that predicted. two predicted exons disappear and one unpredicted intron is shown in the predicted 10th exon of at1g and a different intron/exon boundary is also observed. the isolated sequence of at5g is bp shorter than the prediction because of the different intron/exon boundaries; and a riken clone without a continuous orf from the predicted start codon to the predicted stop codon is available in the database. isolated sequences of the other genes, at1g at1g at1g at1g at1g at1g at2g at3g at4g at4g at5g at5g and at5g show different intron/exon boundaries compared with the predicted sequences, resulting in different mrna sequences. the second group contains genes. the detailed sequence differences are summarized in table s in additional file and the alignments among isolated cdnas, predicted orfs and the corresponding genomic dna sequences are shown in additional file unlike the genes in the first group, the isolated sequences in this group do not display continuous orfs from the predicted start codon to the predicted stop codon that were used to design the forward and reverse pcr primers for gatewayr cloning. the isolated sequences of genes at1g at1g at1g at1g at3g and at5g exhibit different intron/exon boundaries compared to the predicted orf sequences. different intron/exon boundaries are also found in the isolated orf sequences of at1g at1g at5g at1g and at1g with other structural differences. the predicted 6th intron disappears in the isolated orf sequence of at1g. the predicted intron exon and exon are merged into exon and the predicted exon is split into exon and exon in the isolated sequence of at1g. the third predicted exon does not exist in the experimentally derived sequence of at5g and the other six predicted exons are merged into two exons. the first two exons and the first intron in the prediction of at1g merge into the first exon in the isolated sequence. the predicted exon is split into exon and exon in the isolated sequence of at1g in database, a previously isolated sequence is different from both the prediction of at1g in tair and the sequence from this report, losing the sequence from exon to exon and part of exon resulting in a much smaller protein with aa compared to the predicted protein of aa. two predicted introns in at2g do not exist in the isolated sequence. at1g displays an unpredicted intron. one extra unpredicted intron is shown in at4g and the isolated sequence is different from both the existing sequence and the tair prediction. although the isolated sequences of genes at4g and at5g are the same as the current predictions in tair the previously reported coding sequences of them are different. the predicted exon of at4g is split into exon and exon in sequence ak. gene at5g has a new annotation in tair the isolated sequence contains the same orf as the current prediction, but the first two predicted exons in tair are arranged as three exons. the existing sequence ak shows a different intron/exon boundary between exon and intron resulting in a smaller orf of amino acids. detection of alternative splicing of lrr-rlks potentially alternatively spliced variants of lrr-rlks were examined by rt-pcr with variant-specific primers according to the predicted mrna sequences and previous reports. isolated cdna sequences from this study were not examined because they were identified by rt-pcr during the cloning procedure. isolated cdna of at4g in this report showed a structure with slight difference from the prediction in database, which made it difficult to examine the sequence difference with variant-specific primers. this gene was not included in the rt-pcr experiment. from inflorescence, variants of lrr-rlks were confirmed by rt-pcr with expected size of products. no rt-pcr products were obtained from at1g at1g at3g at3g and at4g the previously reported cdna sequence of at1g was not amplified from this study, but the predicted variant sequence of it was confirmed by rt-pcr. both the previously reported cdna sequence and the predicted mrna sequence of at5g were confirmed in this study. from leaf, rt-pcr fragments of variants of lrr-rlks were obtained. no rt-pcr products with expected sizes were obtained for the same genes as in inflorescence except at1g that produced a larger fragment than predicted. no rt-pcr product of previously reported cdna of at4g was recovered. together, a total of lrr-rlks were confirmed with alternative splicing of pre-mrna, including four previously reported cdna variants of at1g, at4g, at5g and at5g. lrr-rlks phylogenetic analysis sequence analyses of isolated lrr-rlks reported in this paper demonstrate that some of them encode protein sequences distinct from the predictions. this sequence variation and the improved annotation of arabidopsis genome makes it necessary to examine the previously created phylogenies of this superfamily. the previous report suggested subfamilies because the sequences clearly fell into distinct clades. studies in this report based on the alignment of the full-length amino acid sequences result in a similar phylogenetic tree to the previous report with minor adjustments. at1g a member of the previously assigned subfamily lrr x, fell into the lrr vii subfamily; two members of the previously assigned subfamily lrr xii, one previously ungrouped gene, and one member of the subfamily lrr xiv fell into the lrr xi subfamily. epitope-tagged proteins of lrr-rlks in transgenic arabidopsis plants the expression of lrr-rlks cloned in the destination vectors pb35gwf and pk35gwg and transformed into arabidopsis ecotype'columbia-' was verified by western hybridization analysis with αflag and αgfp antibodies respectively. immunoprecipitated membrane protein was prepared and separated by sds-page for the detection of flag-tagged fusion proteins while total protein could be used directly to detect signals of gfp-tagged fusion proteins. the flag- or gfp-tagged lrr-rlks could be detected in most of the examined transgenic lines usually as one distinct and specific protein band. discussion experimentally derived sequences help to verify and expand the predicted genome annotation the tair annotation release tair contains genes, including putative protein coding genes. among all the putative protein coding genes, genes have not been experimentally supported by identified transcripts. among the predicted lrr-rlk genes, of them have no est support. est support for of them is now provided. a total of lrr-rlk genes have no isolated full length coding sequence in the existing database. from this study, new lrr-rlk cdnas with full length coding sequence were provided. the resources generated in this study will provide useful tools for future functional analyses of this important protein family. at the same time, phylogenetic analysis can guide researchers to create double, even higher level, mutants to overcome functional redundancy of genes in one subfamily. for example, elegant genetics studies in subfamily lrr ii revealed redundant functions of serk genes in brassinosteroid signal transduction, male sporogenesis, pathogen response and cell death. phylogenetic analysis in this study indicated that several subfamilies, such as lrr xii whose members fell into two different subfamilies based on the phylogeny of full length amino acid sequence, could be rearranged to aim future functional analysis of their gene members. the sequence data generated from this project will also greatly improve genome annotation. in a previous study, full-length gene transcripts from arabidopsis were used to re-annotate its genome. the results indicated that the gene structures of approximately 35% of the examined genes could be improved according to the isolated full-length cdna sequences. when examing existing est and full-length cdna sequences for all of the predicted lrr-rlks, one full-length cdna, clone rafl25-47-f, was identified that covered two predicted loci, at1g and at1g with a full and complete orf of aa. the loci should be merged into one according to this data. as described above, from this study a total of genes were identified with different variant transcripts compared to the predictions. all the data are useful for the improvement of arabidopsis lrr-rlk annotation. gene functions and alternatively spliced transcripts the tair release showed that of the annotated protein coding genes have alternatively spliced transcripts. in this report, sequence analyses show that a total of lrr-rlk genes have different sequences from the tair predictions. this includes two possibilities: the prediction was not correct; both the predicted and the isolated sequences exist in plant, which suggests some lrr-rlk genes have alternatively spliced transcripts, possibly in the same tissue, or in different tissues, or under different growth conditions. the sequence analysis of isolated lrr-rlks in this report revealed different forms of the cds compared to tair predictions or the existing sequences in the database, including alternative intron donor and/or acceptor sites, unpredicted introns, unpredicted exons, unspliced introns and different combinations of the aforementioned changes. they form a continuous orf or several discontinuous orfs. the presence of the observed alternative splicing was further confirmed by rt-pcr. it is already known that alternative splicing can significantly increase the complexity of the transcriptome and proteome by synthesizing multiple transcripts and proteins from one gene. several previous reports showed that approximately 20% of arabidopsis genes are alternatively spliced and some alternatively spliced transcripts have different functions. serine/arginine-rich proteins form a conserved family of splicing regulators in eukaryotes. the pre-mrnas of arabidopsis sr genes are extensively alternatively spliced, and about transcripts are produced from genes. the transcriptome complexity of sr genes is increased by six-fold. abiotic stresses regulate the alternative splicing of the pre-mrnas of sr genes to produce different isoforms of sr proteins that are likely to have altered functions in pre-mrna splicing. six mrna variants were generated by alternative splicing in the pre-mrna of a homologue of sr protein, atsr45a. the transcript abundance and the splicing patterns of atsr45a were altered under various types of stress. the u small nuclear ribonucleoprotein particle 70k protein interacts with splicing factors and is involved in basic and alternative splicing of pre-mrna. in arabidopsis, two distinct transcripts are produced by alternative splicing of the pre-mrna of the u snrna 70k gene. only the short transcript encodes a full-length functional u1-70k, whereas the long transcript codes for a truncated u1-70k. cop is a negative regulator of arabidopsis light-dependent development. cop1b is generated by alternative splicing, resulting in a 60-amino acid deletion in the wd- repeat domain relative to the full-length cop which functions as a dominant negative regulator of cop function. the maize mik gene codes for a gck-like map4k that can be activated by interaction with maize atypical receptor kinase. four different mature mrnas of mik are generated by alternative splicing, and the resulting polypeptides display different kinase activity and are differentially activated by interaction with the mark receptor. recent studies further demonstrated that alternative splicing affected regions frequently code for intrinsically disordered regions of the corresponding protein products and the association of alternative splicing and intrinsic disorder results in various isoforms to increase the functional and regulatory diversity of the gene. lrr-rlks are critical proteins involved in many aspects of plant growth, development and stress responses. it is noticed that six genes produce rt-pcr fragments with different sizes in inflorescence and leaf, which indicates that different forms of lrr-rlk protein may be required for distinct tissue development and function. some of the alternatively spliced transcripts of lrr-rlks will generate truncated versions of the predicted proteins. the truncated proteins may be involved in the functional regulation of these genes in different developmental stages and different growth conditions/stresses. future functional analyses of the alternatively spliced lrr-rlks, revealed from this study, would eventually elucidate the biological meaning of the process. gene function and phosphorylation sites analysis clones reported in this paper are not only a resource for gene annotation, but also will be very useful for gene function analysis. genes in entry clones can be transferred freely to any gatewayr-compatible destination vectors and introduced into arabidopsis. they can be used for overexpression in arabidopsis to dissect the resulting phenotypes that will indicate the possible related pathways and functions of the target genes. they can be used to generate different epitope-tagged fusion proteins. for example, as described above, gfp- and flag-tagged fusion protein can be produced in transgenic plants with different antibiotic resistances. the subcellular localizations of the interesting genes can be determined with the help of confocal microscopy. the homodimerization or heterodimerization between lrr-rlks can be detected and confirmed by coexpression in planta and coimmunoprecipitation analysis. the transgenic plants can also be used to isolate protein complexes for each lrr-rlks, which can help to dissect the complicated signaling pathways that the genes participate in. the cloned genes could be mutated directly by site-directed mutagenesis in entry clones to create kinase-inactive copies. overexpression of kinase-inactive genes in arabidopsis will be useful to dissect the functions of the genes by dominant negative effects, especially in the case that functional redundancy is a problem during analysis of them. in order to clearly understand lrr-rlk function, it is necessary to characterize cytoplasmic kinase domain phosphorylation and examine the role of receptor oligomerization in initiating signaling pathways. the primary goal of this study was to generate resources for our current arabidopsis project that is focused on mapping lrr-rlk phosphorylation sites, assessing the functions of the identified sites in plant growth and development, and examining the in vivo interactions of numerous lrr-rlks. a prototype for this approach has been developed for the bri and bak two lrr-rlks involved in br signaling. for example, immunoprecipitated bri1-flag protein was analyzed by liquid chromatography-tandem mass spectrometry and multiple in vivo phosphorylated ser and thr residues of bri were identified. t- and s- are highly conserved activation loop residues that were shown to be essential for kinase function in vitro and bri signaling in planta. the interaction of bri and bak was studied in detail both in vitro and in vivo, and a novel mechanism of sequential transphosphorylation was developed, which helps explain the role of the bak co-receptor in regulating br signaling through bri. this approach, utilizing the resources developed here, is being expanded to examine the mechanisms of action of numerous lrr-rlks across this important family of regulatory proteins.  this study generated four gatewayr-compatible destination vectors for plant transformation and they were proved functional by overexpressing bak to suppress bri1- mutant plant phenotype. complementary dna sequences of arabidopsis lrr-rlks were cloned into the gatewayr donor vector pdonr/zeor and analyzed by dna sequencing. a total of isolated lrr-rlks showed distinct sequences from the database prediction or previously reported sequences. alternative rna splicing was observed in some of them, which was thought involved in the regulation of gene functions and plant development. experimental evidences for the annotation of these lrr-rlks were provided in is study. the generated cdna clones, expression constructs and transgenic plants are useful resources for scientific communities and will accelerate the research in this field. 
transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. as a resource for the in-depth analysis of this important protein family, the complementary dna sequences of lrr-rlks were cloned into the gatewayr donor vector pdonr/zeor and analyzed by dna sequencing. most of the arabidopsis lrr-rlk genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. the generated resources, including cdna entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.
 transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. in arabidopsis thaliana, there are at least leucine-rich repeat receptor-like kinases, representing one of the largest protein families. although functional roles for a handful of lrr-rlks have been revealed, the functions of the majority of members in this protein family have not been elucidated.  as a resource for the in-depth analysis of this important protein family, the complementary dna sequences of lrr-rlks were cloned into the gatewayr donor vector pdonr/zeor and analyzed by dna sequencing. among them, clones showed sequences identical to the predictions in the arabidopsis sequence resource, tair the other cdnas showed gene structures distinct from the predictions of tair which was mainly caused by alternative splicing of pre-mrna. most of the genes have been further cloned into gatewayr destination vectors with gfp or flag epitope tags and have been transformed into arabidopsis for in planta functional analysis. all clones from this study have been submitted to the arabidopsis biological resource center at ohio state university for full accessibility by the arabidopsis research community.  most of the arabidopsis lrr-rlk genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. the generated resources, including cdna entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family. 