Research ArticleGenome-Wide Identification and Characterization of theLRR-RLK Gene Family in Two Vernicia Species
Huiping Zhu,1,2 Yangdong Wang,1,2 Hengfu Yin,1,2 Ming Gao,1,2
Qiyan Zhang,1,2 and Yicun Chen1,2
1State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China2Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China
Correspondence should be addressed to Yicun Chen; [email protected]
Received 15 October 2015; Accepted 17 November 2015
Academic Editor: Henry Heng
Copyright © 2015 Huiping Zhu et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Leucine-rich repeat receptor-like kinases (LRR-RLKs) make up the largest group of RLKs in plants and play important roles inmany key biological processes such as pathogen response and signal transduction. To date, most studies on LRR-RLKs have beenconducted on model plants. Here, we identified 236 and 230 LRR-RLKs in two industrial oil-producing trees: Vernicia fordii andVernicia montana, respectively. Sequence alignment analyses showed that the homology of the RLK domain (23.81%) was greaterthan that of the LRR domain (9.51%) among the Vf/VmLRR-RLKs. The conserved motif of the LRR domain in Vf/VmLRR-RLKsmatched well the known plant LRR consensus sequence but differed at the third last amino acid (W or L). Phylogenetic analysisrevealed that Vf/VmLRR-RLKs were grouped into 16 subclades. We characterized the expression profiles of Vf/VmLRR-RLKs invarious tissue types including root, leaf, petal, and kernel. Further investigation revealed that Vf/VmLRR-RLK orthologous genesmainly showed similar expression patterns in response to tree wilt disease, except 4 pairs ofVf/VmLRR-RLKs that showed oppositeexpression trends. These results represent an extensive evaluation of LRR-RLKs in two industrial oil trees and will be useful forfurther functional studies on these proteins.
1. Introduction
Plants and animals respond to changes in their environmentvia cell surface receptors, which allow them to sense bothexternal and internal signals and adapt accordingly. Receptor-like protein kinases (RLKs) are one of the most importantgroups of cell surface receptors. These proteins have specialstructural features that make them particularly suitable forcell-to-cell signaling. Since the first RLK was identified inmaize [1], many studies have functionally characterized RLKsfrom various plants, including rice, poplar, soybean, andpotato, and have shown that the RLKsmake up a superfamilyin 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.RLKs can be classified according to their extracellular N-terminal domain.The RLKs with a leucine-rich-repeat (LRR)N-terminal domain, the LRR-RLKs, are the largest group ofproteins in the RLK superfamily.
LRR-RLK proteins in various organisms contain a con-sensus motif of 20–30 amino acid residues [2] that is tan-demly repeated to build the domain [3]. The distinguishingfeature of an LRR motif is an 11-amino acid consensussequence, LxxLxLxxNxL, where x is any amino acid [4]. Thisdomain can bind to ligands or participate in protein-proteininteractions [4].
The protein kinase (PK) domain of LRR-RLKs usuallyconsists of approximately 250–300 amino acid residues [3]and has a cytoplasmic PK domain [5]. LRR-RLKs can beclassified into three types depending on their cytoplasmicPK domain: (1) protein Ser/Thr kinases, (2) protein tyrosinekinases, and (3) protein histidine kinases [6]. The Ser/Thrkinases have been well studied in plants.The Ser/Thr domaintransduces signals downstream via autophosphorylation andthen phosphorylates specific substrates [7].
Previous studies have shown that the LRR-RLK familyhas 216 members in Arabidopsis thaliana [7], 234 members
Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2015, Article ID 823427, 17 pageshttp://dx.doi.org/10.1155/2015/823427
2 International Journal of Genomics
in Solanum lycopersicum [8], 379 members in Populus tri-chocarpa [9], and 309 members in Oryza sativa [3]. Thisextreme expansion in plant genomes reflects their functionalsignificance [10]. Members of the LRR-RLK family have beenshown to play critical and diverse roles in physiological proc-esses such as secondary wall formation [11], embryogen-esis [12], meristematic growth [13], maintaining vasculartissue polarity [14], germination speed [15], regulation oforgan shape [16], pollen self-incompatibility [17], negativeregulator-programmed cell death [18], signaling pathways[19], abscisic acid (ABA) early signaling [20], brassinosteroidsignaling [21], hormone regulation [22], pathogen defense[23], tolerance to oxidative stress [15], and tolerance to saltand heat stress [10].
To date, most LRR-RLK genes have been isolated frommodel plants and herbs, rather than woody oil plants.Tung oil tree (Vernicia fordii) and wood oil tree (Verniciamontana) are important industrial oil plants belonging to theEuphorbiaceae family.The oil extracted from tung seeds is anexcellent drying oil that is renewable, safe, and environmen-tally friendly. This oil is widely used in industrial productssuch as paints, plasticizers, resins,medicine, synthetic rubber,and printing ink [24], and as a raw material for biodieselproduction [25]. China produces approximately 70–80% ofthe tung oil on the global market. However, tung trees aresusceptible to Fusarium wilt disease. Interestingly, the twodifferent species of Vernicia show different degrees of resis-tance to this disease;V. fordii, which is themain oil-producingspecies, is susceptible to the disease, while wood oil tree (V.montana) is resistant. A previous study showed that manyLRR-RLKs are defense-related [10]; therefore, studies on theLRR-RLKs of these two Vernicia species may help to clarifywhy one species is more resistant than the other.
In this study, we identified the LRR-RLKs in two Verniciaspecies and conducted multiple sequence alignments, phylo-genetic analyses, and conservedmotif analyses of theVfLRR-RLK and VmLRR-RLK gene families. We selected severalLRR-RLK genes for gene expression analyses in varioustissues of V. fordii and V. montana. Finally, we investigatedthe changes in expression of 22 Vm/fLRR-RLK genes duringinfection with Fusarium oxysporum. These results will beuseful for further studies on the functions of LRR-RLKs inwoody oil trees.
2. Materials and Methods
2.1. Plant Materials. Samples of V. fordii and V. montanawere collected from Fuyang Urban Forest Park, Hangzhoucity, Zhejiang Province, China, and then separated into roots,stems, leaves, flower buds, ovaries, and kernels. No specificpermits were required to collect the samples from the park.Three replicates were collected for all samples. The sampleswere immediately frozen in liquid nitrogen and stored at−80∘C until use.
2.2. Total RNA Isolation and cDNA Synthesis. Total RNAwas extracted separately from each sample using an RN38-EASY Spin Plus Plant RNA kit (Aidlab Biotech, Beijing,
China) following the manufacturer’s instructions. The con-centration of purified RNA was determined by agarose gelelectrophoresis and spectrophotometry (NanoDrop 5000,Thermo Scientific, Waltham, MA, USA). Only RNA sampleswith a 260/280 wavelength ratio between 2.0 and 2.2 and a260/230wavelength ratio greater than 1.8were used for cDNAsynthesis. The cDNA was synthesized using Superscript IIIRT (Invitrogen, Carlsbad, CA, USA) following the manu-facturer’s instructions. All cDNA synthesis reactions wereperformed at the same time so that the efficiency of reversetranscription was approximately equal among the samples.The cDNAs were diluted 1 : 10 with nuclease-free water forRT-PCR and amplification.
2.3. Screening for LRR-RLK Genes in V. fordii and V. montana.The members of the LRR-RLK superfamily in the twoVernicia species were first identified from transcriptomedata using look-up function of computer and using “LRR”as the key word; then we sought the selected genes oneby one according to their descriptions of annotations. Allhit genes were considered to be the purpose genes. Then,the corresponding ORF and amino acid sequences wereidentified. For all of the obtained protein sequences, thepresence of characteristic domains (LRR, TM, and RLKdomains) was confirmed using the Conserved Domain Data-base of NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Controversial sequences were used as search que-ries at PBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO-GRAM=blastp&PAGE TYPE=BlastSearch&LINK LOC=blasthom). Sequences not belonging to the LRR-RLK familywere rejected. The Simple Modular Architecture ResearchTool (SMART) (http://smart.embl-heidelberg.de/) [26] wasused as a secondary method to confirm the presence of thedomain(s). All of the obtained sequences were submitted tothe NCBI. Arabidopsis LRR-RLK amino acid sequences withknown functions were downloaded from the NCBI database.
2.4. Sequence Alignment and Construction of PhylogeneticTrees. Multiple sequence alignments of amino acid sequen-ces of RLK domains and full-length amino acid sequences ofVernicia LRR-RLKs with complete domains were performedusing ClustalX v.1.83 [27] using the default settings. DNA-MAN v.5.5.2 was used as a secondary method for aligningsequences and rechecking results.
There were some studies of LRR-RLKs in many plants,such as A. thaliana, S. lycopersicum, P. trichocarpa, and O.sativa; however,muchmore information about the functionalclassification was reported in A. thaliana. To compare theevolutionary relationships of LRR-RLKs between Verniciaspecies and A. thaliana and roughly predict the functionsof LRR-RLKs in Vernicia species, multiple sequence align-ments were performed for VmLRR-RLK, VfLRR-RLK, and35 AtLRR-RLKs with known functions using the amino acidsequences of the RLK domains.
The phylogenetic trees were constructed with the neigh-bor-joiningmethod usingMEGA 5.1 software [28] with posi-tion correction, pairwise deletion, and 1000 bootstrap repli-cates indicated at each node.
International Journal of Genomics 3
Table 1: Sequences of primers specific for VfLRR-RLK and VmLRR-RLK genes amplification used qRT-PCR.
Primer names Sequences (5-3) 𝑇𝑚
Amplicons length (bp)VfLRR-RLK2 GGCTCAATCCCAGAATCAT/CCAACAGAAACGAAACATC 51.9 166VfLRR-RLK6 TTTAGACTTGCTGCCTGAC/TAACACCACCAGTGACCTG 54.1 121VfLRR-RLK7 TCAGGCAGTGAGGTAGAAG/GAAGCACGAGAAAGATTAA 48.9 182VfLRR-RLK9 ATTTCGCCACCATAGAGTC/ATTTCATCCTGCGTAAGTG 51.9 167VfLRR-RLK11 AGGAGAACAGGAAGCCCACT/GAAGGTCCATAAATGTATCA 53.3 198VfLRR-RLK13 GACCTGAAAACTGGAAATG/TATGTAACCAATGGAGCCT 49.9 147VfLRR-RLK159 TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA 49.75 197VfLRR-RLK172 GTCCAATTTCGGTCGGTTGC/AGGAATGGTGTTCGGGTTT 55.2 132VfLRR-RLK256 CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG 58.6 181VfLRR-RLK260 CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG 58.6 181VfLRR-RLK271 CAACAGTCTCAACGGAAGC/AATGGATGATGGAATGGGT 53.0 106VmLRR-RLK18 TTGCCTCATGGAAATCCGACA/GGCCTGTAAGATTGGTTAAT 53.45 156VmLRR-RLK17 GGACCAGCAGTTGTGAGT/CTTTCTGTTGGGTGGAGA 53.75 168VmLRR-RLK30 ATGTCAGGCAGTGAGGTAG/GAAGCACGAGAAAGATTAA 51.95 135VmLRR-RLK29 CGCCACCATAGAGTCATAG/CACTTACGCAGGATGAAAT 53.0 163VmLRR-RLK111 TTCTTCTGGAGATCCCATTT/GTAAACCATCCTTTGCCTC 52.15 151VmLRR-RLK241 TGACCTGAAACCTGGAAAT/GCCACCCATACCATACTCT 53 172VmLRR-RLK178 TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA 49.75 197VmLRR-RLK164 CTATGGAGGGTCCTATTC/TTAAGCCAGTGATTGAGC 51.45 159VmLRR-RLK206 TCGCAAATCGCCTTTATTC/ATGGCTATGCTAGGGTCAA 51.9 118VmLRR-RLK202 CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG 58.6 181VmLRR-RLK210 TCATAGGCCCAGAACACTC/TCCTGGTGCTTATGTGAGT 54.1 231ACT CGATGAAGCACAGTCCAAAAG/GTTGAGAGGAGCCTCAGTG 58.85 170
2.5. Motif Recognition of LRR-RLKs in Vernicia Species. Theconserved motifs of LRR-RLK protein sequences in twoVernicia species were identified using the motif-basedsequence analysis tool, Multiple Expectation Maximizationfor Motif Elicitation (MEME) Suite version 4.10.0 (http://meme.nbcr.net/meme90/tools/meme) [29], with the follow-ing parameters: any number of repetitions of a motif, maxi-mum number of motifs = 25.
2.6. Inoculation of V. fordii and V. montana with FusariumPathogen. The tung wilt disease pathogen F. oxysporum wascultivated in potato dextrose broth (PDB, 1/4 strength) on ashaker at 180 rpm (28∘C) for 4 days to reach a fungal titerof 106 spores/mL. Roots of 2-month-old seedlings were dugfrom the soil, rinsed with water, then soaked in 75% alcoholfor 1min, 0.5% sodium hypochlorite for 3min, 90% alcoholfor 30 s, and then rinsed three times in sterile water.The rootswere wounded with a sterile knife, dipped in 100mL sporeliquid, 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 26∘C with 95% relative humidity. Theplants were observed regularly and the disease incidence wasrecorded [30]. Roots of plants were collected, and the stage ofinfection was determined according to the symptoms of theseedlings.
2.7. Real-Time Quantitative PCR (RT-qPCR). The primersused for RT-qPCR were designed using Primer Premier
5.0 with the following criteria: product size between 100and 250 bp; melting temperature around 60∘C; 40–60% GCcontent; and primer length of 18–21 bp. Primers specific forACT7 (Actin7a) [31] were used to standardize the cDNA.Subsequently, LRR-RLK gene-specific primers (Table 1) wereused to amplify the corresponding genes. The qRT-PCRswere carried out using an SYBR Premix Ex Taq Kit (TaKaRa,Tokyo, Japan) according to the manufacturer’s protocol. EachPCR mixture (20 𝜇L) consisted of 2𝜇L 4-fold diluted 1st-strand cDNA, 10 𝜇L 2x SYBR Premix Ex Taq, 0.4 𝜇L 10 𝜇Mforward and reverse primers, 0.4 𝜇L 50x ROX reference dye,and 6.8 𝜇L DEPC-treated water. Reactions were performedon an ABI 7300 Real-Time quantitative instrument (AppliedBiosystems, Foster City, CA, USA). The cycling parameterswere as follows: 95∘C for 30 s, 40 cycles of 95∘C for 5 s, and60∘C for 31 s. A melting curve analysis was performed afterthe PCR cycling to verify the specificity of the amplification.
3. Results and Discussion
3.1. Identification of VfLRR-RLKs and VmLRR-RLKs in V.fordii and V. montana. A total of 286 and 260 candidategenes in the LRR-RLK superfamily were obtained based onannotations of RNA-seq data. Then, 236 and 230 sequencesin V. fordii and V. montana with at least one characteristicdomain were positively identified as members of the LRR-RLK superfamily. All of the 466 sequences were submitted tothe NCBI by our laboratory, and the accession numbers werelisted in Table 2.
4 International Journal of Genomics
Table 2: GenBank accession numbers of VfLRR-RLK and VmLRR-RLK genes.
Vernicia fordii Vernicia montanaGene ID GenBank accession number Gene ID GenBank accession numberVfLRR-RLK1-VfLRR-RLK14 c805427-KT805440 VmLRR-RLK1-VmLRR-RLK9 KT805663-KT805671VfLRR-RLK18-VfLRR-RLK19 KT805441-KT805442 VmLRR-RLK11-VmLRR-RLK18 KT805672-KT805679VfLRR-RLK22-VfLRR-RLK24 KT805443-KT805445 VmLRR-RLK20-VmLRR-RLK32 KT805680-KT805692VfLRR-RLK26-VfLRR-RLK28 KT805446-KT805448 VmLRR-RLK34 KT805693VfLRR-RLK30-VfLRR-RLK46 KT805449-KT805465 VmLRR-RLK36-VmLRR-RLK39 KT805694-KT805697VfLRR-RLK48 KT805466 VmLRR-RLK41-VmLRR-RLK67 KT805698-KT805724VfLRR-RLK50-VfLRR-RLK53 KT805467-KT805470 VmLRR-RLK69-VmLRR-RLK83 KT805725-KT805739VfLRR-RLK55-VfLRR-RLK57 KT805471-KT805473 VmLRR-RLK86-VmLRR-RLK90 KT805740-KT805744VfLRR-RLK59-VfLRR-RLK60 KT805474-KT805475 VmLRR-RLK92 KT805745VfLRR-RLK63-VfLRR-RLK65 KT805476-KT805478 VmLRR-RLK94-VmLRR-RLK108 KT805746-KT805760VfLRR-RLK68-VfLRR-RLK70 KT805479-KT805481 VmLRR-RLK110-VmLRR-RLK115 KT805761-KT805766VfLRR-RLK72 KT805482 VmLRR-RLK117-VmLRR-RLK118 KT805767-KT805768VfLRR-RLK74-VfLRR-RLK90 KT805483-KT805499 VmLRR-RLK120 KT805769VfLRR-RLK92-VfLRR-RLK93 KT805500-KT805501 VmLRR-RLK122-VmLRR-RLK123 KT805770-KT805771VfLRR-RLK95-VfLRR-RLK97 KT805502-KT805504 VmLRR-RLK125-VmLRR-RLK129 KT805772-KT805776VfLRR-RLK99-VfLRR-RLK104 KT805505-KT805510 VmLRR-RLK131-VmLRR-RLK142 KT805777-KT805788VfLRR-RLK106-VfLRR-RLK124 KT805511-KT805529 VmLRR-RLK144-VmLRR-RLK151 KT805789-KT805796VfLRR-RLK126 KT805530 VmLRR-RLK153-VmLRR-RLK157 KT805797-KT805801VfLRR-RLK128 KT805531 VmLRR-RLK159-VmLRR-RLK187 KT805802-KT805830VfLRR-RLK130-VfLRR-RLK140 KT805532-KT805542 VmLRR-RLK190 KT805831VfLRR-RLK143 KT805543 VmLRR-RLK192-VmLRR-RLK195 KT805832-KT805835VfLRR-RLK145-VfLRR-RLK146 KT805544-KT805545 VmLRR-RLK197-VmLRR-RLK199 KT805836-KT805838VfLRR-RLK148 KT805546 VmLRR-RLK202-VmLRR-RLK206 KT805839-KT805843VfLRR-RLK150-VfLRR-RLK156 KT805547-KT805553 VmLRR-RLK208-VmLRR-RLK222 KT805844-KT805858VfLRR-RLK158-VfLRR-RLK159 KT805554-KT805555 VmLRR-RLK225-VmLRR-RLK245 KT805859-KT805879VfLRR-RLK161-VfLRR-RLK170 KT805556-KT805565 VmLRR-RLK247-VmLRR-RLK257 KT805880-KT805890VfLRR-RLK172-VfLRR-RLK190 KT805566-KT805584 VmLRR-RLK259-VmLRR-RLK260 KT805891-KT805892VfLRR-RLK193-VfLRR-RLK197 KT805585-KT805589VfLRR-RLK199-VfLRR-RLK202 KT805590-KT805593VfLRR-RLK204-VfLRR-RLK208 KT805594-KT805598VfLRR-RLK210 KT805599VfLRR-RLK212-VfLRR-RLK214 KT805600-KT805602VfLRR-RLK216-VfLRR-RLK220 KT805603-KT805607VfLRR-RLK222-VfLRR-RLK223 KT805608-KT805609VfLRR-RLK225 KT805610VfLRR-RLK227-VfLRR-RLK230 KT805611-KT805614VfLRR-RLK232-VfLRR-RLK237 KT805615-KT805620VfLRR-RLK239-VfLRR-RLK246 KT805621-KT805628VfLRR-RLK248-VfLRR-RLK258 KT805629-KT805639VfLRR-RLK260-VfLRR-RLK264 KT805640-KT805644VfLRR-RLK266-VfLRR-RLK276 KT805645-KT805655VfLRR-RLK279-VfLRR-RLK281 KT805656-KT805658VfLRR-RLK283-VfLRR-RLK286 KT805659-KT805662
The LRR-RLK family proteins contained at least one fullor partial characteristic domain (LRR, TM, and/or RLKdomains). According to the structural characteristics of theLRR-RLKs in the two Vernicia species, the proteins wereclassified into seven groups (Table 3): group 1 with an LRRdomain; group 2 with a TM domain; group 3 with an RLK
domain; group 4 with LRR and TM domains; group 5 withLRR and RLK domains; group 6 with TM and RLK domains;and group 7 with LRR, TM, and RLK domains. As shown inTable 3, groups 1, 3, and 5 had the most members and group4 had the fewest members (three in V. fordii and five in V.montana).The number ofmembers in each groupwas similar
International Journal of Genomics 5
Table 3: The number of LRR-RLK genes containing different conserved domains in V. fordii and V. montana.
Species Number of Total Number ofLRRNumber of
TMNumber of
RLKNumber ofLRR-TM
Number ofLRR-RLK
Number ofTM-RLK
Number ofLRR-TM-
RLKV. fordii 236 75 12 73 3 52 11 10V. montana 229 75 6 68 5 55 10 10
between V. fordii and V. montana, possibly because of theclose genetic relationship between these two species.
Approximately 223, 234, 309, and 379 LRR-RLK geneswere identified in the A. thaliana, O. sativa, S. Lycopersicum,and P. trichocarpa genomes, respectively [7]. Our resultsshowed that there were fewer LRR-RLK members in Verniciaspecies than in O. sativa and P. trichocarpa. This may berelated to interspecific differences or functional differenti-ation of LRR-RLKs. The genome sequences also providedinformation on the different ratios of Vernicia homologuesto LRR-RLK genes in other species.
3.2. Alignment and Evolutionary Analysis of VfLRR-RLKs andVmLRR-RLKs. Because of the large differences in length andcomplexity among the sequences, it was difficult to conductalignments for all of the LRR-RLKs identified in these twoVernicia species. Therefore, we conducted alignments for theprotein groups with the most members. First, we analyzedproteins with the LRR domain, since these were the mostabundant. When the LRR domain was selected for the align-ment the consistency was approximately 3.90%. Therefore,we selected different sequences, trimmed both ends of thesequences, and tried the alignment again. The consistencyreached 9.51%, which was still too low to build a phylogenetictree. The low consistency of LRR domains suggested a highdegree of sequence complexity and diversity between VfLRR-RLKs and VmLRR-RLKs. Therefore, we selected the RLKdomain amino acids sequence containing 53–394 aminoacids from 201 LRR-RLK genes in Vernicia species for align-ment. The consistency among these sequences was 23.81%(Supplementary Figure 1 in SupplementaryMaterial availableonline at http://dx.doi.org/10.1155/2015/823427). To analyzethe evolutionary relationships of the LRR-RLK superfamily inthese two Vernicia species, an unrooted NJ phylogenetic treewas constructed based on the multiple sequence alignmentsof 106 VfLRR-RLKs and 95 VmLRR-RLKs containing theRLK domain (Figure 1). There is no standard classificationmethod for LRR-RLKs. In previous studies, these proteinswere usually classified into different subfamilies according toclades in the phylogenetic tree. Therefore, we grouped theVfLRR-RLKs and VmLRR-RLKs into 16 subclades accordingto the phylogenetic tree (Figure 1, subclades 1–16). Subclades14, 15, and 16 had only one member, indicating that thesesubfamilies had few members or their members were toodifferent to group into the same subclade in the tree.
To confirm the reliability of the phylogenetic tree, a phy-logenetic tree was constructed for each of the two species,using the sequences of 106 VfLRR-RLK (SupplementaryFigure 2) and 95 VmLRR-RLK RLK (Supplementary Figure3) proteins. The evolutionary relationships were generally
consistent among the three trees. The genes showing closerelationships in the tree constructed for a single speciesalso showed close relationships in the tree combining bothspecies. Some VfLRR-RLK or VmLRR-RLK proteins classi-fied into the same clade in the tree for each single speciesgrouped into different clades in the tree combining the twospecies, possibly because of the more elaborate classificationin 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. thalianacorresponded 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 ofsubfamily II in A. thaliana, suggesting that they may sharethe same function. These proteins may participate in brass-inosteroid signaling, pathogen responses, cell death, andmale sporogenesis. Similarly, members of subclade 6 in Ver-nicia species may be related to the plant brassinosteroidreceptor, vascular differentiation, abscisic acid signaling,embryonic pattern formation, another development, celldeath, and innate immunity. Subclade 10members inV. fordiiand V. montana may play a role in the pathogen response.Interestingly, the members of subclade 9 in Vernicia speciescorresponded to two different subclades in A. thaliana:AtLRR-RLKXIII and AtLRR-RLKXI. This may reflect func-tional differentiation of LRR-RLKs in A. thaliana. Based onthe roles of AtLRR-RLKXIII and AtLRR-RLKXI proteins inArabidopsis, the members of subclade 9 in Vernicia speciesmay be involved in meristem differentiation, epidermal sur-face formation during embryogenesis, floral organ abscission,determination of seed size, cell wall biosynthesis, organgrowth, and stomatal patterning and differentiation.
3.3. Motif Analysis of Vf/VmLRR-RLKs. To further revealthe diversification and potential functions of LRR-RLKs inVernicia, we selected 20 Vf/VmLRR-RLKs (Table 5) with fullcharacteristic domain and investigated their conservedmotifs using MEME version 4.10.0. A total of 25 conservedmotifs were identified and numbered 1–25 (Figure 3).
Among the 20 Vf/VmLRR-RLKs, there were six differentmotifs at the N-terminal and six at the C-terminal. The sixmotifs at the N-terminal were Motifs 19, 1, 8, 22, 17, and23. Ten of the 20 LRR-RLKs (50%) had Motif 19 at the N-terminal, and most of these LRR-RLKs were in subclades 1and 4 (Figure 4). The other five motifs were present in one tothree of the 20 LRR-RLKs. Interestingly, Motif 17 was presentat the N-terminal of two LRR-RLKs, both of which werein subclade 2. This may indicate that Motif 17 is specific to
6 International Journal of Genomics
VMLR
R-RL
K209
VFLR
R-RL
K261
19
VFLR
R-RL
K258
65
VMLR
R-RL
K215
99
VFLR
R-RL
K172
93
VFLR
R-RL
K271
VMLR
R-RL
K208
VFLR
R-RL
K269
VMLR
R-RL
K210
2729
99
61
VFL
RR-R
LK26
2
VFL
RR-R
LK25
5
VM
LRR-
RLK1
84
VM
LRR-
RLK2
04
9756
93
36
VM
LRR-
RLK1
72
VFL
RR-R
LK23
4V
MLR
R-RL
K153
9799
99
VFL
RR-R
LK24
8V
MLR
R-RL
K137
69V
FLRR
-RLK
220
99V
MLR
R-RL
K222
VFL
RR-R
LK27
5V
MLR
R-RL
K221
6899
9972
VM
LRR-
RLK1
01V
MLR
R-RL
K132
99
VFL
RR-R
LK18
5
99
VFL
RR-R
LK24
1
99
VFL
RR-R
LK14
3V
MLR
R-RL
K48
VFLR
R-RL
K130
VFLR
R-RL
K114
VMLR
R-RL
K156
8999
9972
43
97
VMLR
R-RL
K216
VMLR
R-RL
K220
99
VFLR
R-RL
K274
99
VFLR
R-RL
K254
VMLR
R-RL
K170
99
VFLR
R-RL
K193
99
VMLR
R-RL
K202
VFLR
R-RL
K256
VMLR
R-RL
K207
VFLR
R-RL
K260
3232999999
63
VFLR
R-RL
K140
VFLR
R-RL
K176
99
VFLR
R-RL
K145
49
VMLR
R-RLK
122
VMLR
R-RLK
133
53VF
LRR-R
LK251
VFLR
R-RLK
249
41VM
LRR-R
LK176
99
VFLR
R-RLK
119
VFLRR
-RLK13
4
9999
VFLRR
-RLK10
4
VFLRR-
RLK182
VMLRR
-RLK5
977528
VFLRR-
RLK77
VFLRR-
RLK222
VMLRR-R
LK166
99 VFLRR-R
LK190
99 VMLRR-
RLK194
VMLRR-R
LK203
99 VMLRR-RLK
103
74 VMLRR-RLK
179
VFLRR-RLK19
7
VMLRR-RLK13
4
994411
97
6
00
0
VFLRR-RLK156
VFLRR-RLK24463
VFLRR-RLK243
VFLRR-RLK246
99
66
VFLRR-RLK170VMLRR-RLK47
34
VFLRR-RLK10199
VMLRR-RLK56VMLRR-RLK97
32
VFLRR-RLK155
99
VMLRR-RLK187VFLRR-RLK257VMLRR-RLK175VMLRR-RLK21333
9663
9899
6
0
VFLRR-RLK245VMLRR-RLK98VMLRR-RLK135
36
VFLRR-RLK187
99
VFLRR-RLK158VMLRR-RLK87
99
VFLRR-RLK165VMLRR-RLK140
VFLRR-RLK167
3499
99
99
13
0
VFLRR-RLK148
VMLRR-RLK104
76
VMLRR-RLK78
99
VMLRR-RLK70
31
VFLRR-RLK219
VMLRR-RLK114
99
9
VMLRR-RLK51
VMLRR-RLK141
99
VFLRR-RLK18
VFLRR-RLK59
88
VMLRR-RLK74
99
VFLRR-RLK174
VMLRR-RLK106
99VFLRR-RLK175
99
VFLRR-RLK194
VFLRR-RLK195
53VM
LRR-RLK218
99VFLRR-RLK218
VFLRR-RLK273
VMLRR-RLK192
VMLRR-RLK197
9999
7737
3076
77
2
0
VFLRR-RLK166
VMLRR-RLK161
70VFLRR-RLK189
99VM
LRR-RLK128
VMLRR-RLK139
VFLRR-RLK230
3599
99V
MLRR-RLK229
VM
LRR-RLK237
99
12V
FLRR-RLK250
VM
LRR-RLK16099
VFLRR-RLK206
VM
LRR-RLK155
99
48
VFLRR-RLK150
VM
LRR-RLK899
VFLRR-RLK131
VM
LRR-RLK15V
FLRR-RLK19
9930
VFLRR-RLK138
VM
LRR-RLK86
99
VM
LRR-RLK90V
FLRR-RLK41V
MLRR-RLK60
VFLRR-RLK123
5580
9944
1328
27
1
0
VFL
RR-R
LK24
0
VM
LRR-
RLK1
74
99
VFL
RR-R
LK16
8
73
VFLR
R-RL
K48
VFLR
R-RL
K109
65
VMLR
R-RL
K148
99
VMLR
R-RL
K165
VFLR
R-RL
K164
VMLR
R-RL
K167
99
VFLR
R-RL
K205
VMLR
R-RL
K159
89
VFLR
R-RL
K204
99
VMLR
R-RL
K198
VFLR
R-RL
K208
VMLR
R-RL
K212
5399 76
VMLR
R-RL
K225
VFLR
R-RL
K161
VMLR
R-RL
K205
57
VMLR
R-RL
K144
94
VFLR
R-RLK
186
VMLR
R-RLK
145
99 98
VFLR
R-RLK
84
55
VMLR
R-RLK
81
VMLR
R-RLK
131
56
VFLR
R-RLK
111
51
VFLRR
-RLK31
99
VFLRR-
RLK177
VMLRR
-RLK11
8
96
VFLRR-
RLK178
99
VFLRR-
RLK99
VFLRR-
RLK100
99VFL
RR-RLK1
63VFL
RR-RLK18
8
99 78 6951
4436 47 29 65
16
20
0
VFLRR-RL
K3VML
RR-RLK11
99
VFLRR-RLK
121
53
VFLRR-RLK1
18VFLRR-RLK25
2VMLRR-RLK16
3
99 95
18VFLRR-RLK200VM
LRR-RLK14 66VFLRR-R
LK199 99VMLRR-RLK147
VFLRR-RLK201VFLRR-RLK225
74 99
99
VFLRR-RLK102VMLRR-RLK64
93
VFLRR-RLK108
59
VMLRR-RLK252
99
VMLRR-RLK55
VMLRR-RLK67
99
VFLRR-RLK228
99
VFLRR-RLK154
VMLRR-RLK59
99
VMLRR-RLK108
VFLRR-RLK212
VMLRR-RLK77
5299
9989
59
34
10
5
VFLRR-RLK183
VMLRR-RLK136
99
VMLRR-RLK142
VFLRR-RLK242
VMLRR-RLK99
9999
99
16
VFLRR-RLK266
VMLRR-RLK195
99
VFLRR-RLK270
VMLRR-RLK154
VMLRR-RLK168
9994
99
37
VMLRR-RLK66
42
VFLRR-RLK213
VFLRR-RLK232
Figure 1: Phylogenetic tree based on the RLK sequences of Vf/VmLRR-RLKs. The phylogenetic tree was constructed by MEGA package v5.1using neighbor-joining method. The numbers at each branch point represent the bootstrap scores (1,000 replicates). The VfLRR-RLKs weresigned by circle filled with green, and the VmLRR-RLKs were signed by circle filled with yellow. Amino acid sequences of RLK domain usedwere listed in supplementary Data Set 1.
subclade 2.Therewere too fewmembers of subfamilies 16 and5 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. All members of subclade 4 hadMotif 6 at their C-terminal. Subclade 5 had only onemember,which had Motif 4 at its C-terminal. Motif 16 was present infour of the 20 LRR-RLKs, all of which were in subclade 1.Theother C-terminal motifs were detected in only one or two ofthe 20 LRR-RLKs.
The motifs of different domains were detected accordingto their sequences and sites. The most obvious motif was thatof the LRR domain, characterized by repeated “L” residues.This motif was present in Motifs 22, 8, and 1. Amongthem, Motif 1 was the most representative of the basic LRRstructural skeleton, with the sequence LxxLxLxxNxLxGx-IPxxLxxW/Lxx. This sequence matched well the plant LRRconsensus sequence (LxxLxLxxNxLxGxIPxxLxxLxx) butdiffered at the third last amino acid (W or L). Motifs 12, 15,3, and 5 corresponded to the TM domain, andMotifs 10, 4, 9,
International Journal of Genomics 7
Table4:Subclassificatio
nof
LRR-RL
KgenesinA.
thaliana
,V.fordii,andV.
Montana
.
Subgroup
inA.
thaliana
Genen
ame(accessionnu
mberin
GenBa
nk)
Functio
nsRe
ference
Subgroup
inV.
fordii
Subgroup
inV.
montana
LRRI
LRRP
K(At4g29990)
Lightsignaltransdu
ction
[1]
VfLR
R-RL
K182
(sub
clade
5)Vm
LRR-RL
K5(sub
clade
5)
LRRII
BAK1
/AtSER
K3(At4g33430);
BKK1
/AtSER
K4(At2g13790);
AtSE
RK1(At1g71830);A
tSER
K2(At1g
34210);N
IK1(At5g1600
0);
NIK
2(At3g25560);NIK
3(At1g
60800)
Antivira
ldefense
respon
se;B
Rsig
nalin
g;celldeath;male
sporogenesis;
andpathogen
respon
se
[32–34]
VfLR
R-RL
K187
(sub
clade
7)Vm
LRR-RL
K135
(sub
clade
7);
VmLR
R-RL
K98(sub
clade
7)
LRRV
SRF4
(At3g13065);
Scrambled/SRF
9/SU
B/Strubb
elig
(At1g
11130)
Cellm
orph
ogenesis;
leafsiz
econtrol;organdevelopm
ent;
positionalsignalin
g;androot
epidermispatte
rning
[13,21,35,36]
VfLR
R-RL
K232
(sub
clade
16)
VmLR
R-RL
K122
(sub
clade
5)Vm
LRR-RL
K133
(sub
clade
5)
LRRX
BRI1(At4g39400);BR
L1(At1g
55610);B
RL2/VH1
(At2g01950);BR
L3(At3g13380);
RPK1
/TOAD1(At1g69270);
RPK2
/TOAD2(At3g02130);
EMS1/EXS
(At5g07280);BIR1
(At5g48380)
Abscisica
cidsig
nalin
g;anther
developm
ent;brassin
osteroid
receptor;celld
eath
andinnate
immun
ity;embryonicp
attern
form
ation;
andvascular
different
[20,22,37–40
]
VfLR
R-RL
K155
(sub
clade
6);
VfLR
R-RL
K257
(sub
clade
6);
VfLR
R-RL
K244
(sub
clade
6);
VfLR
R-RL
K3(sub
clade
9);
VfLR
R-RL
K121
(sub
clade
9);
VfLR
R-RL
K118
(sub
clade
11);
VfLR
R-RL
K252
(sub
clade
11)
VmLR
R-RL
K56(sub
clade
6)Vm
LRR-RL
K97(sub
clade
6)Vm
LRR-RL
K187
(sub
clade
6)Vm
LRR-RL
K175
(sub
clade
6)Vm
LRR-RL
K213
(sub
clade
6)Vm
LRR-RL
K11(subclade
9)Vm
LRR-RL
K163
(sub
clade
11)
LRRXI
GSO
1(At4g20140);G
SO2
(At5g44700);CL
V1(At1g75820);
BAM1(At5g65700);B
AM2
(At3g49670);BA
M3(At4g20270);
SOBIR1
(At2g31880);HAES
A(At4g28490);IK
U2(At3g19700);
PXY/TD
Rv(At5g61480)
Antherd
evelo
pment;celldeathand
innateim
mun
ity;epiderm
alsurfa
ceEm
bryogenesis
;formationdu
ring
floralorgan
abscission;
meristem
differentiatio
n;andseed
size
[40–
44]
VfLR
R-RL
K243
(sub
clade
6);
VfLR
R-RL
K246
(sub
clade
6);
VfLR
R-RL
K156
(sub
clade
6);
VfLR
R-RL
K206
(sub
clade
9);
VfLR
R-RL
K19(sub
clade
9)
VmLR
R-RL
K229
(sub
clade
9)Vm
LRR-RL
K237
(sub
clade
9)Vm
LRR-RL
K155
(sub
clade
9)Vm
LRR-RL
K15(sub
clade
9)
LRRXII
FLS2
(At5g46330);EF
R(At5g204
80)
Pathogen
respon
se[45]
VfLR
R-RL
K164
(sub
clade
10)
VfLR
R-RL
K48(sub
clade
10)
VfLR
R-RL
K109
(sub
clade
10)
VmLR
R-RL
K167
(sub
clade
10)
VmLR
R-RL
K148
(sub
clade
10)
LRRXIII
FEI1(At1g
31420);FEI2(At2g35620);
EREC
TA(At2g26330);ER
L1(At5g62230);ER
L2(At5g07180)
Cellw
allbiosynthesis;organ
grow
th;and
stomatalpatte
rning
anddifferentiatio
n[46–
48]
VfLR
R-RL
K230
(sub
clade
9)Vm
LRR-RL
K139
(sub
clade
9)Vm
LRR-RL
K128
(sub
clade
9)
8 International Journal of Genomics
VMLR
R-RL
K81
VMLR
R-RL
K131
58
VFLR
R-RL
K111
45
VFLR
R-RL
K31
99
VFLR
R-RL
K178
VFLR
R-RL
K177
VMLR
R-RL
K118
95100
18
VFLR
R-RL
K99
VFLR
R-RL
K100
99
VFLR
R-RL
K163
VFL
RR-R
LK18
8
10083
39
VFL
RR-R
LK84
VFL
RR-R
LK18
6
VM
LRR-
RLK1
45
100
VM
LRR-
RLK1
44
VFL
RR-R
LK16
1
VM
LRR-
RLK2
05
539698
63
28
VM
LRR-
RLK2
25
21
VFL
RR-R
LK20
5V
MLR
R-RL
K159
92
402KLR- RRLFV100
891KLR-RRLMV V
FLRR
-RLK
208
VM
LRR-
RLK2
1249100
08
13
At5g
2048
0V
FLRR
-RLK
164
VM
LRR-
RLK1
6710
0
1712
VM
LRR-
RLK1
65
23
VM
LRR-
RLK1
94V
MLR
R-RL
K203
100
VM
LRR-
RLK1
03
27
At5g
4633
0V
MLR
R-RL
K148
VFLR
R-RL
K48
VFLR
R-RL
K109
81100
99
510
VMLR
R-RL
K51
VMLR
R-RL
K141
100
VFLR
R-RL
K18
VFLR
R-RL
K59
88
VMLR
R-RL
K74
100
VFLR
R-RL
K174
VMLR
R-RL
K106
100 V
FLRR
-RLK
175
100
VFLR
R-RL
K194
VFLR
R-RL
K195
65
VMLR
R-RL
K218
100
VFLR
R-RL
K218
VFLR
R-RL
K273
VMLR
R-RL
K192
VMLR
R-RL
K197
99100
774835
70812
VFLR
R-RL
K170
VMLR
R-RLK
47
VFLR
R-RLK
101
100
At1G6
9270
VMLR
R-RLK
56
VMLR
R-RLK
97
VFLR
R-RLK
155
100 VMLR
R-RLK
187
At3g02
130
VFLRR
-RLK25
7
VMLRR
-RLK17
5
VMLRR
-RLK21
3
428073617
69698
0 At2g3
1880
VFLRR-
RLK243
VFLRR-
RLK246
100 VFLRR-
RLK156
At5g0728
0
At2g0195
0
At4g3940
0
VFLRR-RL
K244
At1g55610
At3g13380
10010098100
57333421
0
VFLRR-RLK2
45
VMLRR-RLK
207
VFLRR-RLK26026
VFLRR-RLK25624
VMLRR-RLK202100
VFLRR-RLK193
VFLRR-RLK254
VMLRR-RLK1709910
098
VFLRR-RLK274VMLRR-RLK216VMLRR-RLK220100100
99
VFLRR-RLK114VMLRR-RLK156
90
VFLRR-RLK130
100
VMLRR-RLK48
100
VFLRR-RLK143
74
VFLRR-RLK241VFLRR-RLK185VMLRR-RLK101VMLRR-RLK13299
10099
48
VFLRR-RLK248VMLRR-RLK137
63
VFLRR-RLK220
100
VMLRR-RLK222VFLRR-RLK275
VMLRR-RLK221
58100
100
VFLRR-RLK234VMLRR-RLK153
97
VMLRR-RLK172
100
VMLRR-RLK184
VMLRR-RLK204
99
VFLRR-RLK255
58
VFLRR-RLK262
92
VFLRR-RLK269
VMLRR-RLK210
30
VMLRR-RLK208
25
VFLRR-RLK271
100
VFLRR-RLK172
VMLRR-RLK215
VFLRR-RLK261
VFLRR-RLK258
VMLRR-RLK209
296199
95
63
45
100
70
99
67
1
0
VMLRR-RLK139
VFLRR-RLK230
VMLRR-RLK128
100At1g31420
At2g35620
73100
VFLRR-RLK189
VFLRR-RLK166
VMLRR-RLK161
69100
100
At2g26330
At5g62230At5g07180
99
9933
VMLRR-RLK229
VMLRR-RLK237
100At4g20140
At5g44700
99100
17
VFLRR-RLK3
VMLRR-RLK11
100VFLRR-RLK121
78
At5g48380
VFLRR-RLK118V
FLRR-RLK252
VM
LRR-RLK163
100
7549
5
0V
FLRR-RLK240
VM
LRR-RLK174100
VFLRR-RLK168
64V
FLRR-RLK250
VM
LRR-RLK160
10021
At3g197005
VFLRR-RLK206
VM
LRR-RLK155100
At4g2849053
131K
LR-R
RLFVVM
LRR-RLK1569
At4g20270
93
At1g75820At5g65700At3g49670
10062
66
VFLRR-RLK19
43
At5g61480
40
VFLRR-RLK150
VM
LRR-RLK8
100
VFLRR-RLK138
VM
LRR-RLK86
100VMLRR-RLK90
VMLRR-RLK60
VFLRR-RLK41VFLRR-RLK123
52 87100
70
61
54
26
1
0
0
VFLR
R-RL
K140
VFLR
R-RL
K176
100
VFLR
R-RL
K145
54
VMLR
R-RL
K98
VMLR
R-RL
K135
VFLR
R-RL
K187
100
At3g
2556
0
94
At1g
6080
0
97
At1g
7183
0
At1g
3421
0
89
At4g
3343
0
97
At2g
1379
0
100
VFLR
R-RL
K158
VMLR
R-RL
K87
100
VFLR
R-RL
K165
VMLR
R-RLK
140
VFLR
R-RLK
167
100
100
56 100
25
0
VMLR
R-RLK
122
VMLR
R-RLK
133
56
At1g11
130
23
VFLR
R-RLK
222
VMLR
R-RLK
166
100
VFLRR
-RLK19
0
100
VMLRR
-RLK17
9
VFLRR-
RLK197
VMLRR
-RLK13
4
100
56 30
VFLRR-
RLK77
14
VFLRR-
RLK182
VMLRR
-RLK5
85
At4g2999
0
80
VFLRR-R
LK104
92
VFLRR-RL
K119VFLR
R-RLK134
100
VMLRR-R
LK176
VFLRR-RLK
251VFLRR-RLK
249
47 100 100
4817
2
0
VFLRR-RLK2
19VMLRR-RLK11
4 100
VMLRR-RLK70
VMLRR-RLK78VFL
RR-RLK148VMLR
R-RLK10474 100
32 11
0
VFLRR-RLK200VMLRR-RLK14 56
VFLRR-RLK199 100
VMLRR-RLK147VFLRR-RLK201VFLRR-RLK225
73100
100
VFLRR-RLK102VMLRR-RLK64
91
VMLRR-RLK252
69
VFLRR-RLK108
100
VMLRR-RLK55
VMLRR-RLK67
100
VFLRR-RLK228
100
VFLRR-RLK154
VMLRR-RLK59
100
VMLRR-RLK108
VFLRR-RLK212
VMLRR-RLK77 57100
100
88
36
16
17
VFLRR-RLK266
VMLRR-RLK195
100
VFLRR-RLK270
VMLRR-RLK154
VMLRR-RLK168
100
96
100
20
VFLRR-RLK183
VMLRR-RLK136
100
VMLRR-RLK142
VFLRR-RLK242
VMLRR-RLK99
100
100
99
34
VMLRR-RLK66
65
VFLRR-RLK213
96
At5g16000
VFLRR-RLK232At3g13065
69
96
0.2
Figure 2: Phylogenetic tree based on the RLK sequences of LRR-RLK gene family both in V. fordii, V. montana, and A. thaliana. Thephylogenetic tree was constructed by MEGA package v5.1 using neighbor-joining method. The numbers at each branch point representthe bootstrap scores (1,000 replicates). The LRR-RLKs of V. Fordii were signed by circle filled with green, the LRR-RLKs of V. montana weresigned by circle filled with yellow, and the LRR-RLKs ofArabidopsis thalianawere signed by circle filled with blue.The accession number andthe amino acid sequences of the A. thaliana used were listed in supplementary Data Set 1.
International Journal of Genomics 9
Motif 1
Motif 13
Motif 19
Motif 14
Motif 15 Motif 16
Motif 17
Motif 10
Motif 11 Motif 12
Motif 18
Motif 20
Motif 7 Motif 8
Motif 2
Motif 3
Motif 5 Motif 6
Motif 9
Motif 4
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
4
2
0
(bits
)
41
39
37
35
33
31
29
27
25
23
21
19
17
15
13
1197531
37
35
33
31
29
27
25
23
21
19
17
15
13
1197531
4
2
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10 International Journal of Genomics
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Figure 3: Display of conserved motifs of Vf/VmLRR-RLK gene family. The conserved motifs were searched in 20 Vf/VmLRR-RLKswhich contained full characteristic domains (the amino acid sequences were listed in supplementary Data Set 2) by Multiple ExpectationMaximization for Motif Elicitation (MEME) Suite version 4.10.0. Overall height in each stack indicates the sequence conservation at thatposition; height of each residue letter indicates relative frequency of the corresponding residue.
Table 5: Basic information of some VfLRR-RLK and VmLRR-RLK family genes.
Gene name Amino acids length L content (%) PI Molecular mass (KD) LRR-Domain TM-Domain RLK-DomainBasic information of some VfLRR-RLK family genes
VfLRR-RLK159 567 11.88 9.06 62669.6 25–204 269–453 534–564VfLRR-RLK220 782 11.75 8.29 86377.1 187–333 384–569 656–781VfLRR-RLK233 836 10.16 7.01 93442.0 389–446 5–312 566–780VfLRR-RLK248 1002 11.93 6.71 110540.3 187–333 384–569 655–922VfLRR-RLK255 719 13.04 8.41 79622.9 6–220 297–481 564–708VfLRR-RLK256 991 12.10 6.91 111107.1 155–311 445–624 701–967VfLRR-RLK258 986 10.48 6.15 108664.5 248–319 380–561 644–913VfLRR-RLK261 984 10.62 6.05 108287.2 248–319 380–561 644–913VfLRR-RLK271 1010 11.04 5.93 112529.3 159–302 378–553 634–901VfLRR-RLK275 1036 11.08 5.03 113890.5 292–351 428–612 700–963
Basic information of some VmLRR-RLK family genesVmLRR-RLK172 1028 11.97 8.31 112996.4 116–342 413–600 683–950VmLRR-RLK178 567 11.87 9.19 62741.8 269–453 25–205 518–558VmLRR-RLK179 659 11.08 7.02 74326.8 389–446 5–312 566–651VmLRR-RLK202 935 12.04 6.95 105218.5 155–311 390–569 646–912VmLRR-RLK208 1018 10.94 5.42 113540.2 280–302 378–561 642–909VmLRR-RLK209 986 10.69 6.19 108573.5 259–319 380–561 644–913VmLRR-RLK210 1010 10.83 5.50 112606.3 280–302 378–553 634–901VmLRR-RLK215 742 9.60 7.49 82247.5 2–60 99–280 363–669VmLRR-RLK216 1061 13.01 8.29 117955.4 147–276 514–695 772–1050VmLRR-RLK220 847 12.16 8.51 94738.0 19–221 293–474 551–829
20, 2, 13, and 6 corresponded to the RLK domain. Among allof the motifs, the most conserved structure of LRR-RLKs inVernicia species was the RLK domain containing Motifs 4, 9,20, 2, 13, and 6.
3.4. Expression of VfLRR-RLKs and VmLRR-RLKs in Responseto Fusarium Infection. Fusarium wilt disease of tung oil treeis a devastating fungal soil-borne disease that severely affects
tree growth.V. fordii, which is themain oil-producing species,is susceptible to this disease, whileV.montana (wood oil tree)is resistant. To investigate the responses of Vm/VfLRR-RLKsto the Fusariumwilt pathogen, we collected roots from plantsbefore infection (stage 0), at an early stage of F. oxysporuminfection (stage 1), and at a late stage of F. oxysporuminfection (stage 2). We randomly selected 22 Vm/VfLRR-RLK orthologous genes and monitored their transcript
International Journal of Genomics 11
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Figure 4: Conserve motifs of different subclades of LRR-RLKs in Vernicia species. The conserve motifs of each LRR-RLK gene weresearched by Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0. Different colors and different lengthsboxes represent different motifs.
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. Allgenes were amplified reliably.
The qRT-PCR results showed that although there weresome differences in transcript levels between pairs of orthol-ogous genes, most of them showed similar transcription pro-files in response to Fusarium wilt disease in both V. fordiiand V. montana during the infection period (Figure 5). This
result suggests that many Vf/VmLRR-RLKs have similarfunctions during pathogen infection. Four pairs of orthol-ogous genes (VfLRR-RLK7/VmLRR-RLK30, VfLRR-RLK159/VmLRR-RLK178, VfLRR-RLK256/VmLRR-RLK206, andVfLRR-RLK271/VmLRR-RLK210) showed opposite expres-sion patterns between V. montana and V. fordii. In V. mon-tana, the transcript levels of VmLRR-RLK30, 178, 206, and210 increased at the early stage of infection, whereas thoseof the corresponding orthologous genes in V. fordii, VfLRR-RLK7, 159, 256, and 271, decreased.This finding suggests thatthese four VmLRR-RLK genes participate in resistance to F.oxysporum in V. montana.
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Figure 5: Expression analysis of 22 Vm/Vf LRR-RLK genes in roots of Vernicia during infection with Fusarium. Vertical axis representsgene transcript levels. Primary axis represents transcript levels of Vf LRR-RLKs; secondary axis represents transcript levels of VmLRR-RLKs.Standard errors are shown (𝑛 = 3 biological samples). Each sample was analyzed by real-time PCR in triplicate. 0, before infection; 1, earlystage of infection; 2, late stage of infection.
3.5. Transcription Patterns of VfLRR-RLKs and VmLRR-RLRsin Various Tissues. To investigate the tissue specificity ofVfLRR-RLKs and VmLRR-RLRs expression and further ana-lyze genes related to Fusarium wilt disease, we analyzed thetranscript levels of the 22 genes described above in seventissues of V. fordii and V. montana by qRT-PCR (Figure 6).Among them, VfLRR-RLK260 and VfLRR-RLK159 showedsimilar expression patterns in all seven tissues of V. fordii.Both showed higher transcript levels in leaves and kernelsand lower transcript levels in roots, stems, buds, and ovaries.However, compared with VfLRR-RLK260, VfLRR-RLK159was more strongly expressed in petals, suggesting that itmay have a special function in floral development. VfLRR-RLK2 was expressed in roots, stems, and leaves and stronglyexpressed in petals, but not in vascular tissues. VfLRR-RLK172 was expressed most strongly in petals, followed byleaves, but expressed at low levels in the other tissues.VfLRR-RLK13 showed the highest transcript level in ovaries, followedby leaves. VfLRR-RLK271 showed similar expression patternsin all tissues. The other five genes showed tissue-specificexpression patterns. VfLRR-RLK6 was specifically expressedin petals,VfLRR-RLK9 in ovaries, andVfLRR-RLK11 in roots.Both VfLRR-RLK7 and VfLRR-RLK256 were specificallyexpressed in kernels. Together, these results suggest that
VfLRR-RLKs play various roles in the development of tungtree.
Compared with VfLRR-RLKs, most VmLRR-RLKsshowed 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 theirorthologous genes in V. fordii. This may indicate that theyshare the same function inV. fordii andV.montana.Theotherfive VmLRR-RLKs showed different expression patterns in V.montana.VmLRR-RLK111wasmainly expressed in leaves andhad similar transcript levels in other tissues. VmLRR-RLK164and VmLRR-RLK241 showed peak expression in kernels,but VmLRR-RLK164 was also expressed at high levels in thestems. Both VmLRR-RLK17 and VmLRR-RLK206 showedthe highest transcript levels in roots and lower levels in othertissues. The different expression patterns in V. montana mayreflect functional differentiation during evolution.
Among the seven pairs of orthologous genes showingsimilar trends in gene expression in V. montana and V.fordii in response to Fusarium infection, three pairs alsoshowed similar expression patterns in the tissues (VfLRR-RLK2/VmLRR-RLK18, VfLRR-RLK9/VmLRR-RLK29, andVfLRR-RLK260/VmLRR-RLK202). The other four pairs
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nFigure 6: Transcript levels of 22Vm/Vf LRR-RLK genes in various tissues. Columnheight shows gene transcript levels. Primary axis representstranscript levels of Vf LRR-RLKs; secondary axis represents transcript levels of VmLRR-RLKs. Standard errors are shown (𝑛 = 3 biologicalsamples). Each sample was analyzed by real-time PCR in triplicate.
showed different expression patterns in the seven tissuesanalyzed. Of the four pairs of orthologous genes showingopposite responses to Fusarium infection in V. montana andV. fordii (Figure 5), three pairs showed similar expressionpatterns in the tissues, and one pair (VfLRR-RLK256 andVmLRR-RLK206) showed different expression patterns inthe tissues (Figure 6). There were high transcript levels ofVfLRR-RLK256 in kernels and VmLRR-RLK206 in the roots.Given that the Fusarium pathogen invades via the roots oftung tree, these results suggest that VmLRR-RLK206 mayplay a role in resistance to Fusarium wilt disease.
4. Conclusion
This is the first extensive evaluation of the LRR-RLK super-family in tung oil tree and wood tung tree. Phylogeneticanalyses, conserved motif analyses, and expression analysesof VfLRR-RLKs and VmLRR-RLKs in different tissues and inresponse to Fusarium infection were conducted. Characteri-zation of LRR-RLK genes in a ligneous oil plant will improveour understanding of the evolutionary processes and func-tions of this gene superfamily.The results of this study provideimportant information for further research on the diversityand functions of the LRR-RLK gene family in tung tree.
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper.
Authors’ Contribution
Huiping Zhu and YangdongWang contributed equally to thepaper.
Acknowledgment
The work was supported by the National Natural ScienceFoundation of China (31200485).
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