plants and animals respond to changes in their environment via cell surface receptors , which allow them to sense both external and internal signals and adapt accordingly . receptor - like protein kinases ( rlks ) are one of the most important groups of cell surface receptors . these proteins have special structural features that make them particularly suitable for cell - to - cell signaling . since the first rlk was identified in maize , many studies have functionally characterized rlks from various plants , including rice , poplar , soybean , and potato , and have shown that the rlks make up a superfamily in plants . a typical rlk usually includes three distinct parts : an extracellular n - terminal domain , a single transmembrane ( tm ) domain , and a c - terminal intracellular kinase domain . the rlks with a leucine - rich - repeat ( lrr ) n - terminal domain , the lrr - rlks , are the largest group of proteins in the rlk superfamily . lrr - rlk proteins in various organisms contain a consensus motif of 2030 amino acid residues   that is tandemly repeated to build the domain . the distinguishing feature of an lrr motif is an 11-amino acid consensus sequence , lxxlxlxxnxl , where x is any amino acid . the protein kinase ( pk ) domain of lrr - rlks usually consists of approximately 250300 amino acid residues   and has a cytoplasmic pk domain . lrr - rlks can be classified into three types depending on their cytoplasmic pk domain : ( 1 ) protein ser / thr kinases , ( 2 ) protein tyrosine kinases , and ( 3 ) protein histidine kinases . the ser / thr kinases have been well studied in plants . the ser / thr domain transduces signals downstream via autophosphorylation and then phosphorylates specific substrates . previous studies have shown that the lrr - rlk family has 216 members in arabidopsis thaliana , 234 members in solanum lycopersicum , 379 members in populus trichocarpa , and 309 members in oryza sativa . members of the lrr - rlk family have been shown to play critical and diverse roles in physiological processes such as secondary wall formation , embryogenesis , meristematic growth , maintaining vascular tissue polarity , germination speed , regulation of organ shape , pollen self - incompatibility , negative regulator - programmed cell death , signaling pathways , abscisic acid ( aba ) early signaling , brassinosteroid signaling , hormone regulation , pathogen defense , tolerance to oxidative stress , and tolerance to salt and heat stress . to date , most lrr - rlk genes have been isolated from model plants and herbs , rather than woody oil plants . tung oil tree ( vernicia fordii ) and wood oil tree ( vernicia montana ) are important industrial oil plants belonging to the euphorbiaceae family . the oil extracted from tung seeds is an excellent drying oil that is renewable , safe , and environmentally friendly . this oil is widely used in industrial products such as paints , plasticizers , resins , medicine , synthetic rubber , and printing ink , and as a raw material for biodiesel production . interestingly , the two different species of vernicia show different degrees of resistance to this disease ; v. fordii , which is the main oil - producing species , is susceptible to the disease , while wood oil tree ( v. montana ) is resistant . a previous study showed that many lrr - rlks are defense - related ; therefore , studies on the lrr - rlks of these two vernicia species may help to clarify why one species is more resistant than the other . in this study , we identified the lrr - rlks in two vernicia species and conducted multiple sequence alignments , phylogenetic analyses , and conserved motif analyses of the vflrr - rlk and vmlrr - rlk gene families . we selected several lrr - rlk genes for gene expression analyses in various tissues of v. fordii and v. montana . finally , we investigated the changes in expression of 22 vm / flrr - rlk genes during infection with fusarium oxysporum . these results will be useful for further studies on the functions of lrr - rlks in woody oil trees . samples of v. fordii and v. montana were collected from fuyang urban forest park , hangzhou city , zhejiang province , china , and then separated into roots , stems , leaves , flower buds , ovaries , and kernels . total rna was extracted separately from each sample using an rn38-easy spin plus plant rna kit ( aidlab biotech , beijing , china ) following the manufacturer 's instructions . the concentration of purified rna was determined by agarose gel electrophoresis and spectrophotometry ( nanodrop 5000 , thermo scientific , waltham , ma , usa ) . only rna samples with a 260/280 wavelength ratio between 2.0 and 2.2 and a 260/230 wavelength ratio greater than 1.8 were used for cdna synthesis . the cdna was synthesized using superscript iii rt ( invitrogen , carlsbad , ca , usa ) following the manufacturer 's instructions . all cdna synthesis reactions were performed at the same time so that the efficiency of reverse transcription was approximately equal among the samples . the cdnas were diluted 1  :  10 with nuclease - free water for rt - pcr and amplification . the members of the lrr - rlk superfamily in the two vernicia species were first identified from transcriptome data using look - up function of computer and using  lrr  as the key word ; then we sought the selected genes one by one according to their descriptions of annotations . for all of the obtained protein sequences , the presence of characteristic domains ( lrr , tm , and rlk domains ) was confirmed using the conserved domain database of ncbi ( http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi ) . the simple modular architecture research tool ( smart ) ( http://smart.embl-heidelberg.de/ )   was used as a secondary method to confirm the presence of the domain(s ) . arabidopsis lrr - rlk amino acid sequences with known functions were downloaded from the ncbi database . multiple sequence alignments of amino acid sequences of rlk domains and full - length amino acid sequences of vernicia lrr - rlks with complete domains were performed using clustalx v.1.83   using the default settings . there were some studies of lrr - rlks in many plants , such as a. thaliana , s. lycopersicum , p. trichocarpa , and o. sativa ; however , much more information about the functional classification was reported in a. thaliana . to compare the evolutionary relationships of lrr - rlks between vernicia species and a. thaliana and roughly predict the functions of lrr - rlks in vernicia species , multiple sequence alignments were performed for vmlrr - rlk , vflrr - rlk , and 35 atlrr - rlks with known functions using the amino acid sequences of the rlk domains . the phylogenetic trees were constructed with the neighbor - joining method using mega 5.1 software   with position correction , pairwise deletion , and 1000 bootstrap replicates indicated at each node . the conserved motifs of lrr - rlk protein sequences in two vernicia species were identified using the motif - based sequence analysis tool , multiple expectation maximization for motif elicitation ( meme ) suite version 4.10.0 ( http://meme.nbcr.net/meme90/tools/meme ) , with the following parameters : any number of repetitions of a motif , maximum number of motifs = 25 . the tung wilt disease pathogen f. oxysporum was cultivated in potato dextrose broth ( pdb , 1/4 strength ) on a shaker at 180  rpm ( 28c ) for 4 days to reach a fungal titer of 10  spores / ml . roots of 2-month - old seedlings were dug from the soil , rinsed with water , then soaked in 75% alcohol for 1  min , 0.5% sodium hypochlorite for 3  min , 90% alcohol for 30  s , and then rinsed three times in sterile water . the roots were wounded with a sterile knife , dipped in 100  ml spore liquid , and then replanted in soil . after this infection process , the plants were cultivated in an artificial climate chamber ( 8  h light/16  h dark ) at 26c with 95% relative humidity . roots of plants were collected , and the stage of infection was determined according to the symptoms of the seedlings . the primers used for rt - qpcr were designed using primer premier 5.0 with the following criteria : product size between 100 and 250  bp ; melting temperature around 60c ; 4060% gc content ; and primer length of 1821  bp . subsequently , lrr - rlk gene - specific primers ( table 1 ) were used to amplify the corresponding genes . the qrt - pcrs were carried out using an sybr premix ex taq kit ( takara , tokyo , japan ) according to the manufacturer 's protocol . each pcr mixture ( 20  l ) consisted of 2  l 4-fold diluted 1st - strand cdna , 10  l 2x sybr premix ex taq , 0.4  l 10  m forward and reverse primers , 0.4  l 50x rox reference dye , and 6.8  l depc - treated water . reactions were performed on an abi 7300 real - time quantitative instrument ( applied biosystems , foster city , ca , usa ) . the cycling parameters were as follows : 95c for 30  s , 40 cycles of 95c for 5  s , and 60c for 31  s. a melting curve analysis was performed after the pcr cycling to verify the specificity of the amplification . a total of 286 and 260 candidate genes in the lrr - rlk superfamily were obtained based on annotations of rna - seq data . then , 236 and 230 sequences in v. fordii and v. montana with at least one characteristic domain were positively identified as members of the lrr - rlk superfamily . all of the 466 sequences were submitted to the ncbi by our laboratory , and the accession numbers were listed in table 2 . the lrr - rlk family proteins contained at least one full or partial characteristic domain ( lrr , tm , and/or rlk domains ) . according to the structural characteristics of the lrr - rlks in the two vernicia species , the proteins were classified into seven groups ( table 3 ) : group 1 with an lrr domain ; group 2 with a tm domain ; group 3 with an rlk domain ; group 4 with lrr and tm domains ; group 5 with lrr and rlk domains ; group 6 with tm and rlk domains ; and group 7 with lrr , tm , and rlk domains . as shown in table 3 , groups 1 , 3 , and 5 had the most members and group 4 had the fewest members ( three in v. fordii and five in v. montana ) . the number of members in each group was similar between v. fordii and v. montana , possibly because of the close genetic relationship between these two species . approximately 223 , 234 , 309 , and 379 lrr - rlk genes were identified in the a. thaliana , o. sativa , s. lycopersicum , and p. trichocarpa genomes , respectively . our results showed that there were fewer lrr - rlk members in vernicia species than in o. sativa and p. trichocarpa . the genome sequences also provided information on the different ratios of vernicia homologues to lrr - rlk genes in other species . because of the large differences in length and complexity among the sequences , it was difficult to conduct alignments for all of the lrr - rlks identified in these two vernicia species . therefore , we conducted alignments for the protein groups with the most members . first , we analyzed proteins with the lrr domain , since these were the most abundant . when the lrr domain was selected for the alignment the consistency was approximately 3.90% . therefore , we selected different sequences , trimmed both ends of the sequences , and tried the alignment again . the consistency reached 9.51% , which was still too low to build a phylogenetic tree . the low consistency of lrr domains suggested a high degree of sequence complexity and diversity between vflrr - rlks and vmlrr - rlks . therefore , we selected the rlk domain amino acids sequence containing 53394 amino acids from 201 lrr - rlk genes in vernicia species for alignment . the consistency among these sequences was 23.81% ( supplementary figure  1 in supplementary material available online at http://dx.doi.org/10.1155/2015/823427 ) . to analyze the evolutionary relationships of the lrr - rlk superfamily in these two vernicia species , an unrooted nj phylogenetic tree was constructed based on the multiple sequence alignments of 106 vflrr - rlks and 95 vmlrr - rlks containing the rlk domain ( figure 1 ) . , these proteins were usually classified into different subfamilies according to clades in the phylogenetic tree . therefore , we grouped the vflrr - rlks and vmlrr - rlks into 16 subclades according to the phylogenetic tree ( figure 1 , subclades 116 ) . subclades 14 , 15 , and 16 had only one member , indicating that these subfamilies had few members or their members were too different to group into the same subclade in the tree . to confirm the reliability of the phylogenetic tree , a phylogenetic tree was constructed for each of the two species , using the sequences of 106 vflrr - rlk ( supplementary figure  2 ) and 95 vmlrr - rlk rlk ( supplementary figure  3 ) proteins . the genes showing close relationships in the tree constructed for a single species also showed close relationships in the tree combining both species . some vflrr - rlk or vmlrr - rlk proteins classified into the same clade in the tree for each single species grouped into different clades in the tree combining the two species , possibly because of the more elaborate classification in the larger tree . to predict the function of lrr - rlks in vernicia species , 35 arabidopsis lrr - rlks with known functions ( table 4 ) were compared with vflrr - rlks and vmlrr - rlks ( figure 2 ) . almost every lrr - rlk subfamily in a. thaliana corresponded to an lrr - rlk subclade in vernicia species . the members of subclade 7 in v. fordii and v. montana ( figures 1 and 2 ) grouped together with members of subfamily ii in a. thaliana , suggesting that they may share the same function . these proteins may participate in brassinosteroid signaling , pathogen responses , cell death , and male sporogenesis . similarly , members of subclade 6 in vernicia species may be related to the plant brassinosteroid receptor , vascular differentiation , abscisic acid signaling , embryonic pattern formation , another development , cell death , and innate immunity . subclade 10 members in v. fordii and v. montana may play a role in the pathogen response . interestingly , the members of subclade 9 in vernicia species corresponded to two different subclades in a. thaliana : atlrr - rlkxiii and atlrr - rlkxi . based on the roles of atlrr - rlkxiii and atlrr - rlkxi proteins in arabidopsis , the members of subclade 9 in vernicia species may be involved in meristem differentiation , epidermal surface formation during embryogenesis , floral organ abscission , determination of seed size , cell wall biosynthesis , organ growth , and stomatal patterning and differentiation . to further reveal the diversification and potential functions of lrr - rlks in vernicia , we selected 20 vf / vmlrr - rlks ( table 5 ) with full characteristic domain and investigated their conserved motifs using meme version 4.10.0 . a total of 25 conserved motifs were identified and numbered 125 ( figure 3 ) . among the 20 vf / vmlrr - rlks , there were six different motifs at the n - terminal and six at the c - terminal . the six motifs at the n - terminal were motifs 19 , 1 , 8 , 22 , 17 , and 23 . ten of the 20 lrr - rlks ( 50% ) had motif 19 at the n - terminal , and most of these lrr - rlks were in subclades 1 and 4 ( figure 4 ) . the other five motifs were present in one to three of the 20 lrr - rlks . interestingly , motif 17 was present at the n - terminal of two lrr - rlks , both of which were in subclade 2 . there were too few members of subfamilies 16 and 5 to make accurate predictions about their motif structure . the six motifs at the c - terminal were motifs 6 , 20 , 16 , 4 , 9 , and 7 . motif 6 was present in 11 of the 20 lrr - rlks ( 55% ) , and in almost every subclade . subclade 5 had only one member , which had motif 4 at its c - terminal . motif 16 was present in four of the 20 lrr - rlks , all of which were in subclade 1 . the other c - terminal motifs were detected in only one or two of the 20 lrr - rlks . this motif was present in motifs 22 , 8 , and 1 . among them , motif 1 was the most representative of the basic lrr structural skeleton , with the sequence lxxlxlxxnxlxgxipxxlxxw / lxx . this sequence matched well the plant lrr consensus sequence ( lxxlxlxxnxlxgxipxxlxxlxx ) but differed at the third last amino acid ( w or l ) . motifs 12 , 15 , 3 , and 5 corresponded to the tm domain , and motifs 10 , 4 , 9 , 20 , 2 , 13 , and 6 corresponded to the rlk domain . among all of the motifs , the most conserved structure of lrr - rlks in vernicia species was the rlk domain containing motifs 4 , 9 , 20 , 2 , 13 , and 6 . fusarium wilt disease of tung oil tree is a devastating fungal soil - borne disease that severely affects tree growth . v. fordii , which is the main oil - producing species , is susceptible to this disease , while v. montana ( wood oil tree ) is resistant . to investigate the responses of vm / vflrr - rlks to the fusarium wilt pathogen , we collected roots from plants before infection ( stage 0 ) , at an early stage of f. oxysporum infection ( stage 1 ) , and at a late stage of f. oxysporum infection ( stage 2 ) . we randomly selected 22 vm / vflrr - rlk orthologous genes and monitored their transcript levels by rt - pcr . the 22 orthologous genes were vflrr - rlk2/vmlrr - rlk18 , vflrr - rlk6/vmlrr - rlk17 , vflrr - rlk7/vmlrr - rlk30 , vflrr - rlk9/vmlrr - rlk29 , vflrr - rlk11/vmlrr - rlk111 , vflrr - rlk13/vmlrr - rlk241 , vflrr - rlk159/vmlrr - rlk178 , vflrr - rlk172/vmlrr - rlk164 , vflrr - rlk256/vmlrr - rlk206 , vflrr - rlk260/vmlrr - rlk202 , and vflrr - rlk271/vmlrr - rlk210 . the qrt - pcr results showed that although there were some differences in transcript levels between pairs of orthologous genes , most of them showed similar transcription profiles in response to fusarium wilt disease in both v. fordii and v. montana during the infection period ( figure 5 ) . this result suggests that many vf / vmlrr - rlks have similar functions during pathogen infection . four pairs of orthologous genes ( vflrr - rlk7/vmlrr - rlk30 , vflrr - rlk159/vmlrr - rlk178 , vflrr - rlk256/vmlrr - rlk206 , and vflrr - rlk271/vmlrr - rlk210 ) showed opposite expression patterns between v. montana and v. fordii . in v. montana , the transcript levels of vmlrr - rlk30 , 178 , 206 , and 210 increased at the early stage of infection , whereas those of the corresponding orthologous genes in v. fordii , vflrr - rlk7 , 159 , 256 , and 271 , decreased . this finding suggests that these four vmlrr - rlk genes participate in resistance to f. oxysporum in v. montana . to investigate the tissue specificity of vflrr - rlks and vmlrr - rlrs expression and further analyze genes related to fusarium wilt disease , we analyzed the transcript levels of the 22 genes described above in seven tissues of v. fordii and v. montana by qrt - pcr ( figure 6 ) . among them , vflrr - rlk260 and vflrr - rlk159 showed similar expression patterns in all seven tissues of v. fordii . both showed higher transcript levels in leaves and kernels and lower transcript levels in roots , stems , buds , and ovaries . however , compared with vflrr - rlk260 , vflrr - rlk159 was more strongly expressed in petals , suggesting that it may have a special function in floral development . vflrr - rlk2 was expressed in roots , stems , and leaves and strongly expressed in petals , but not in vascular tissues . vflrr - rlk172 was expressed most strongly in petals , followed by leaves , but expressed at low levels in the other tissues . vflrr - rlk6 was specifically expressed in petals , vflrr - rlk9 in ovaries , and vflrr - rlk11 in roots . together , these results suggest that vflrr - rlks play various roles in the development of tung tree . compared with vflrr - rlks , most vmlrr - rlks showed higher transcript levels in the seven tissues analyzed . six vmlrr - rlks ( vmlrr - rlk18 , vmlrr - rlk29 , vmlrr - rlk202 , vmlrr - rlk30 , vmlrr - rlk178 , and vmlrr - rlk210 ) showed the same expression patterns as their orthologous genes in v. fordii . this may indicate that they share the same function in v. fordii and v. montana . vmlrr - rlk111 was mainly expressed in leaves and had similar transcript levels in other tissues . vmlrr - rlk164 and vmlrr - rlk241 showed peak expression in kernels , but vmlrr - rlk164 was also expressed at high levels in the stems . both vmlrr - rlk17 and vmlrr - rlk206 showed the highest transcript levels in roots and lower levels in other tissues . the different expression patterns in v. montana may reflect functional differentiation during evolution . among the seven pairs of orthologous genes showing similar trends in gene expression in v. montana and v. fordii in response to fusarium infection , three pairs also showed similar expression patterns in the tissues ( vflrr - rlk2/vmlrr - rlk18 , vflrr - rlk9/vmlrr - rlk29 , and vflrr - rlk260/vmlrr - rlk202 ) . of the four pairs of orthologous genes showing opposite responses to fusarium infection in v. montana and v. fordii ( figure 5 ) , three pairs showed similar expression patterns in the tissues , and one pair ( vflrr - rlk256 and vmlrr - rlk206 ) showed different expression patterns in the tissues ( figure 6 ) . there were high transcript levels of vflrr - rlk256 in kernels and vmlrr - rlk206 in the roots . given that the fusarium pathogen invades via the roots of tung tree , these results suggest that vmlrr - rlk206 may play a role in resistance to fusarium wilt disease . this is the first extensive evaluation of the lrr - rlk superfamily in tung oil tree and wood tung tree . phylogenetic analyses , conserved motif analyses , and expression analyses of vflrr - rlks and vmlrr - rlks in different tissues and in response to fusarium infection were conducted . characterization of lrr - rlk genes in a ligneous oil plant will improve our understanding of the evolutionary processes and functions of this gene superfamily . the results of this study provide important information for further research on the diversity and functions of the lrr - rlk gene family in tung tree .
leucine - rich repeat receptor - like kinases ( lrr - rlks ) make up the largest group of rlks in plants and play important roles in many key biological processes such as pathogen response and signal transduction . to date , most studies on lrr - rlks have been conducted on model plants . here , we identified 236 and 230 lrr - rlks in two industrial oil - producing trees : vernicia fordii and vernicia montana , respectively . 
 sequence alignment analyses showed that the homology of the rlk domain ( 23.81% ) was greater than that of the lrr domain ( 9.51% ) among the vf / vmlrr - rlks . 
 the conserved motif of the lrr domain in vf / vmlrr - rlks matched well the known plant lrr consensus sequence but differed at the third last amino acid ( w or l ) . 
 phylogenetic analysis revealed that vf / vmlrr - rlks were grouped into 16 subclades . 
 we characterized the expression profiles of vf / vmlrr - rlks in various tissue types including root , leaf , petal , and kernel . 
 further investigation revealed that vf / vmlrr - rlk orthologous genes mainly showed similar expression patterns in response to tree wilt disease , except 4 pairs of vf / vmlrr - rlks that showed opposite expression trends . 
 these results represent an extensive evaluation of lrr - rlks in two industrial oil trees and will be useful for further functional studies on these proteins .