# Genome-Wide Identification and Characterization of theLRR-RLK Gene Family in TwoVernicia Species

Authors: Huiping Zhu; Yangdong Wang; Hengfu Yin; Ming Gao; Qiyan Zhang; Yicun Chen
Journal: International Journal of Genomics
 (2015)

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## Abstract

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 230LRR-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 greater than that of the LRR domain (9.51%) among theVf/VmLRR-RLKs. The conserved motif of the LRR domain inVf/VmLRR-RLKs matched well the known plant LRR consensus sequence but differed at the third last amino acid (W or L). Phylogenetic analysis revealed thatVf/VmLRR-RLKs were grouped into 16 subclades. We characterized the expression profiles ofVf/VmLRR-RLKs in various tissue types including root, leaf, petal, and kernel. Further investigation revealed thatVf/VmLRR-RLK orthologous genes mainly showed similar expression patterns in response to tree wilt disease, except 4 pairs ofVf/VmLRR-RLKs that showed opposite expression trends. These results represent an extensive evaluation ofLRR-RLKs in two industrial oil trees and will be useful for further functional studies on these proteins.


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## Body

## 1. Introduction

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 [1], 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. 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 of proteins in the RLK superfamily.LRR-RLK proteins in various organisms contain a consensus motif of 20–30 amino acid residues [2] that is tandemly repeated to build the domain [3]. The distinguishing feature of an LRR motif is an 11-amino acid consensus sequence, LxxLxLxxNxL, where x is any amino acid [4]. This domain can bind to ligands or participate in protein-protein interactions [4].The protein kinase (PK) domain of LRR-RLKs usually consists of approximately 250–300 amino acid residues [3] and has a cytoplasmic PK domain [5]. 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 [6]. The Ser/Thr kinases have been well studied in plants. The Ser/Thr domain transduces signals downstream via autophosphorylation and then phosphorylates specific substrates [7].Previous studies have shown that the LRR-RLK family has 216 members inArabidopsis thaliana [7], 234 members inSolanum lycopersicum [8], 379 members inPopulus trichocarpa [9], and 309 members inOryza sativa [3]. This extreme expansion in plant genomes reflects their functional significance [10]. Members of the LRR-RLK family have been shown to play critical and diverse roles in physiological processes such as secondary wall formation [11], embryogenesis [12], meristematic growth [13], maintaining vascular tissue polarity [14], germination speed [15], regulation of organ shape [16], pollen self-incompatibility [17], negative regulator-programmed cell death [18], signaling pathways [19], abscisic acid (ABA) early signaling [20], brassinosteroid signaling [21], hormone regulation [22], pathogen defense [23], tolerance to oxidative stress [15], and tolerance to salt and heat stress [10].To date, mostLRR-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 [24], and as a raw material for biodiesel production [25]. China produces approximately 70–80% of the tung oil on the global market. However, tung trees are susceptible toFusarium wilt disease. Interestingly, the two different species ofVernicia 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 [10]; therefore, studies on the LRR-RLKs of these twoVernicia species may help to clarify why one species is more resistant than the other.In this study, we identified theLRR-RLKs in twoVernicia species and conducted multiple sequence alignments, phylogenetic analyses, and conserved motif analyses of theVfLRR-RLK andVmLRR-RLK gene families. We selected severalLRR-RLK genes for gene expression analyses in various tissues ofV. fordii andV. montana. Finally, we investigated the changes in expression of 22Vm/fLRR-RLK genes during infection withFusarium oxysporum. These results will be useful for further studies on the functions of LRR-RLKs in woody oil trees.


## 2. Materials and Methods

### 2.1. Plant Materials

Samples ofV. fordii andV. 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. No specific permits were required to collect the samples from the park. Three replicates were collected for all samples. The samples were immediately frozen in liquid nitrogen and stored at −80°C until use.


### 2.2. Total RNA Isolation and cDNA Synthesis

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.


### 2.3. Screening forLRR-RLK Genes inV. fordii andV. montana

The members of the LRR-RLK superfamily in the twoVernicia 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. All hit genes were considered to be the purpose genes. Then, the corresponding ORF and amino acid sequences were identified. 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). Controversial sequences were used as search queries at PBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthom). Sequences not belonging to the LRR-RLK family were rejected. The Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) [26] was used as a secondary method to confirm the presence of the domain(s). All of the obtained sequences were submitted to the NCBI. Arabidopsis LRR-RLK amino acid sequences with known functions were downloaded from the NCBI database.


### 2.4. Sequence Alignment and Construction of Phylogenetic Trees

Multiple sequence alignments of amino acid sequences of RLK domains and full-length amino acid sequences ofVernicia LRR-RLKs with complete domains were performed using ClustalX v.1.83 [27] using the default settings. DNAMAN v.5.5.2 was used as a secondary method for aligning sequences and rechecking results.There were some studies of LRR-RLKs in many plants, such asA. thaliana,S. lycopersicum,P. trichocarpa, andO. sativa; however, much more information about the functional classification was reported inA. thaliana. To compare the evolutionary relationships of LRR-RLKs betweenVernicia species andA. thaliana and roughly predict the functions of LRR-RLKs inVernicia species, multiple sequence alignments were performed forVmLRR-RLK,VfLRR-RLK, and 35AtLRR-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 [28] with position correction, pairwise deletion, and 1000 bootstrap replicates indicated at each node.


### 2.5. Motif Recognition of LRR-RLKs inVernicia Species

The conserved motifs of LRR-RLK protein sequences in twoVernicia 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) [29], with the following parameters: any number of repetitions of a motif, maximum number of motifs = 25.


### 2.6. Inoculation ofV. fordii andV. montana withFusarium Pathogen

The tung wilt disease pathogenF. oxysporum was cultivated in potato dextrose broth (PDB, 1/4 strength) on a shaker at 180 rpm (28°C) for 4 days to reach a fungal titer of 106 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 26°C with 95% relative humidity. The plants were observed regularly and the disease incidence was recorded [30]. Roots of plants were collected, and the stage of infection was determined according to the symptoms of the seedlings.


### 2.7. Real-Time Quantitative PCR (RT-qPCR)

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 60°C; 40–60% GC content; 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) 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 μL) consisted of 2 μL 4-fold diluted 1st-strand cDNA, 10 μL 2x SYBR Premix Ex Taq, 0.4 μL 10 μM forward and reverse primers, 0.4 μL 50x ROX reference dye, and 6.8 μL 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: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 31 s. A melting curve analysis was performed after the PCR cycling to verify the specificity of the amplification.Table 1
Sequences of primers specific forVfLRR-RLK and VmLRR-RLK genes amplification used qRT-PCR.
Primer names
Sequences (5′-3′)
T
m
Amplicons length (bp)
VfLRR-RLK2
GGCTCAATCCCAGAATCAT/CCAACAGAAACGAAACATC
51.9
166
VfLRR-RLK6
TTTAGACTTGCTGCCTGAC/TAACACCACCAGTGACCTG
54.1
121
VfLRR-RLK7
TCAGGCAGTGAGGTAGAAG/GAAGCACGAGAAAGATTAA
48.9
182
VfLRR-RLK9
ATTTCGCCACCATAGAGTC/ATTTCATCCTGCGTAAGTG
51.9
167
VfLRR-RLK11
AGGAGAACAGGAAGCCCACT/GAAGGTCCATAAATGTATCA
53.3
198
VfLRR-RLK13
GACCTGAAAACTGGAAATG/TATGTAACCAATGGAGCCT
49.9
147
VfLRR-RLK159
TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA
49.75
197
VfLRR-RLK172
GTCCAATTTCGGTCGGTTGC/AGGAATGGTGTTCGGGTTT
55.2
132
VfLRR-RLK256
CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG
58.6
181
VfLRR-RLK260
CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG
58.6
181
VfLRR-RLK271
CAACAGTCTCAACGGAAGC/AATGGATGATGGAATGGGT
53.0
106
VmLRR-RLK18
TTGCCTCATGGAAATCCGACA/GGCCTGTAAGATTGGTTAAT
53.45
156
VmLRR-RLK17
GGACCAGCAGTTGTGAGT/CTTTCTGTTGGGTGGAGA
53.75
168
VmLRR-RLK30
ATGTCAGGCAGTGAGGTAG/GAAGCACGAGAAAGATTAA
51.95
135
VmLRR-RLK29
CGCCACCATAGAGTCATAG/CACTTACGCAGGATGAAAT
53.0
163
VmLRR-RLK111
TTCTTCTGGAGATCCCATTT/GTAAACCATCCTTTGCCTC
52.15
151
VmLRR-RLK241
TGACCTGAAACCTGGAAAT/GCCACCCATACCATACTCT
53
172
VmLRR-RLK178
TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA
49.75
197
VmLRR-RLK164
CTATGGAGGGTCCTATTC/TTAAGCCAGTGATTGAGC
51.45
159
VmLRR-RLK206
TCGCAAATCGCCTTTATTC/ATGGCTATGCTAGGGTCAA
51.9
118
VmLRR-RLK202
CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG
58.6
181
VmLRR-RLK210
TCATAGGCCCAGAACACTC/TCCTGGTGCTTATGTGAGT
54.1
231
ACT
CGATGAAGCACAGTCCAAAAG/GTTGAGAGGAGCCTCAGTG
58.85
170


## 2.1. Plant Materials

Samples ofV. fordii andV. 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. No specific permits were required to collect the samples from the park. Three replicates were collected for all samples. The samples were immediately frozen in liquid nitrogen and stored at −80°C until use.


## 2.2. Total RNA Isolation and cDNA Synthesis

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.


## 2.3. Screening forLRR-RLK Genes inV. fordii andV. montana

The members of the LRR-RLK superfamily in the twoVernicia 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. All hit genes were considered to be the purpose genes. Then, the corresponding ORF and amino acid sequences were identified. 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). Controversial sequences were used as search queries at PBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthom). Sequences not belonging to the LRR-RLK family were rejected. The Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) [26] was used as a secondary method to confirm the presence of the domain(s). All of the obtained sequences were submitted to the NCBI. Arabidopsis LRR-RLK amino acid sequences with known functions were downloaded from the NCBI database.


## 2.4. Sequence Alignment and Construction of Phylogenetic Trees

Multiple sequence alignments of amino acid sequences of RLK domains and full-length amino acid sequences ofVernicia LRR-RLKs with complete domains were performed using ClustalX v.1.83 [27] using the default settings. DNAMAN v.5.5.2 was used as a secondary method for aligning sequences and rechecking results.There were some studies of LRR-RLKs in many plants, such asA. thaliana,S. lycopersicum,P. trichocarpa, andO. sativa; however, much more information about the functional classification was reported inA. thaliana. To compare the evolutionary relationships of LRR-RLKs betweenVernicia species andA. thaliana and roughly predict the functions of LRR-RLKs inVernicia species, multiple sequence alignments were performed forVmLRR-RLK,VfLRR-RLK, and 35AtLRR-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 [28] with position correction, pairwise deletion, and 1000 bootstrap replicates indicated at each node.


## 2.5. Motif Recognition of LRR-RLKs inVernicia Species

The conserved motifs of LRR-RLK protein sequences in twoVernicia 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) [29], with the following parameters: any number of repetitions of a motif, maximum number of motifs = 25.


## 2.6. Inoculation ofV. fordii andV. montana withFusarium Pathogen

The tung wilt disease pathogenF. oxysporum was cultivated in potato dextrose broth (PDB, 1/4 strength) on a shaker at 180 rpm (28°C) for 4 days to reach a fungal titer of 106 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 26°C with 95% relative humidity. The plants were observed regularly and the disease incidence was recorded [30]. Roots of plants were collected, and the stage of infection was determined according to the symptoms of the seedlings.


## 2.7. Real-Time Quantitative PCR (RT-qPCR)

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 60°C; 40–60% GC content; 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) 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 μL) consisted of 2 μL 4-fold diluted 1st-strand cDNA, 10 μL 2x SYBR Premix Ex Taq, 0.4 μL 10 μM forward and reverse primers, 0.4 μL 50x ROX reference dye, and 6.8 μL 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: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 31 s. A melting curve analysis was performed after the PCR cycling to verify the specificity of the amplification.Table 1
Sequences of primers specific forVfLRR-RLK and VmLRR-RLK genes amplification used qRT-PCR.
Primer names
Sequences (5′-3′)
T
m
Amplicons length (bp)
VfLRR-RLK2
GGCTCAATCCCAGAATCAT/CCAACAGAAACGAAACATC
51.9
166
VfLRR-RLK6
TTTAGACTTGCTGCCTGAC/TAACACCACCAGTGACCTG
54.1
121
VfLRR-RLK7
TCAGGCAGTGAGGTAGAAG/GAAGCACGAGAAAGATTAA
48.9
182
VfLRR-RLK9
ATTTCGCCACCATAGAGTC/ATTTCATCCTGCGTAAGTG
51.9
167
VfLRR-RLK11
AGGAGAACAGGAAGCCCACT/GAAGGTCCATAAATGTATCA
53.3
198
VfLRR-RLK13
GACCTGAAAACTGGAAATG/TATGTAACCAATGGAGCCT
49.9
147
VfLRR-RLK159
TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA
49.75
197
VfLRR-RLK172
GTCCAATTTCGGTCGGTTGC/AGGAATGGTGTTCGGGTTT
55.2
132
VfLRR-RLK256
CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG
58.6
181
VfLRR-RLK260
CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG
58.6
181
VfLRR-RLK271
CAACAGTCTCAACGGAAGC/AATGGATGATGGAATGGGT
53.0
106
VmLRR-RLK18
TTGCCTCATGGAAATCCGACA/GGCCTGTAAGATTGGTTAAT
53.45
156
VmLRR-RLK17
GGACCAGCAGTTGTGAGT/CTTTCTGTTGGGTGGAGA
53.75
168
VmLRR-RLK30
ATGTCAGGCAGTGAGGTAG/GAAGCACGAGAAAGATTAA
51.95
135
VmLRR-RLK29
CGCCACCATAGAGTCATAG/CACTTACGCAGGATGAAAT
53.0
163
VmLRR-RLK111
TTCTTCTGGAGATCCCATTT/GTAAACCATCCTTTGCCTC
52.15
151
VmLRR-RLK241
TGACCTGAAACCTGGAAAT/GCCACCCATACCATACTCT
53
172
VmLRR-RLK178
TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA
49.75
197
VmLRR-RLK164
CTATGGAGGGTCCTATTC/TTAAGCCAGTGATTGAGC
51.45
159
VmLRR-RLK206
TCGCAAATCGCCTTTATTC/ATGGCTATGCTAGGGTCAA
51.9
118
VmLRR-RLK202
CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG
58.6
181
VmLRR-RLK210
TCATAGGCCCAGAACACTC/TCCTGGTGCTTATGTGAGT
54.1
231
ACT
CGATGAAGCACAGTCCAAAAG/GTTGAGAGGAGCCTCAGTG
58.85
170


## 3. Results and Discussion

### 3.1. Identification ofVfLRR-RLKs andVmLRR-RLKs inV. fordii andV. montana

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 inV. fordii andV. 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.Table 2
GenBank accession numbers ofVfLRR-RLK and VmLRR-RLK genes.
Vernicia fordii
Vernicia montana
Gene ID
GenBank accession number
Gene ID
GenBank accession number
VfLRR-RLK1-VfLRR-RLK14
c805427-KT805440
VmLRR-RLK1-VmLRR-RLK9
KT805663-KT805671
VfLRR-RLK18-VfLRR-RLK19
KT805441-KT805442
VmLRR-RLK11-VmLRR-RLK18
KT805672-KT805679
VfLRR-RLK22-VfLRR-RLK24
KT805443-KT805445
VmLRR-RLK20-VmLRR-RLK32
KT805680-KT805692
VfLRR-RLK26-VfLRR-RLK28
KT805446-KT805448
VmLRR-RLK34
KT805693
VfLRR-RLK30-VfLRR-RLK46
KT805449-KT805465
VmLRR-RLK36-VmLRR-RLK39
KT805694-KT805697
VfLRR-RLK48
KT805466
VmLRR-RLK41-VmLRR-RLK67
KT805698-KT805724
VfLRR-RLK50-VfLRR-RLK53
KT805467-KT805470
VmLRR-RLK69-VmLRR-RLK83
KT805725-KT805739
VfLRR-RLK55-VfLRR-RLK57
KT805471-KT805473
VmLRR-RLK86-VmLRR-RLK90
KT805740-KT805744
VfLRR-RLK59-VfLRR-RLK60
KT805474-KT805475
VmLRR-RLK92
KT805745
VfLRR-RLK63-VfLRR-RLK65
KT805476-KT805478
VmLRR-RLK94-VmLRR-RLK108
KT805746-KT805760
VfLRR-RLK68-VfLRR-RLK70
KT805479-KT805481
VmLRR-RLK110-VmLRR-RLK115
KT805761-KT805766
VfLRR-RLK72
KT805482
VmLRR-RLK117-VmLRR-RLK118
KT805767-KT805768
VfLRR-RLK74-VfLRR-RLK90
KT805483-KT805499
VmLRR-RLK120
KT805769
VfLRR-RLK92-VfLRR-RLK93
KT805500-KT805501
VmLRR-RLK122-VmLRR-RLK123
KT805770-KT805771
VfLRR-RLK95-VfLRR-RLK97
KT805502-KT805504
VmLRR-RLK125-VmLRR-RLK129
KT805772-KT805776
VfLRR-RLK99-VfLRR-RLK104
KT805505-KT805510
VmLRR-RLK131-VmLRR-RLK142
KT805777-KT805788
VfLRR-RLK106-VfLRR-RLK124
KT805511-KT805529
VmLRR-RLK144-VmLRR-RLK151
KT805789-KT805796
VfLRR-RLK126
KT805530
VmLRR-RLK153-VmLRR-RLK157
KT805797-KT805801
VfLRR-RLK128
KT805531
VmLRR-RLK159-VmLRR-RLK187
KT805802-KT805830
VfLRR-RLK130-VfLRR-RLK140
KT805532-KT805542
VmLRR-RLK190
KT805831
VfLRR-RLK143
KT805543
VmLRR-RLK192-VmLRR-RLK195
KT805832-KT805835
VfLRR-RLK145-VfLRR-RLK146
KT805544-KT805545
VmLRR-RLK197-VmLRR-RLK199
KT805836-KT805838
VfLRR-RLK148
KT805546
VmLRR-RLK202-VmLRR-RLK206
KT805839-KT805843
VfLRR-RLK150-VfLRR-RLK156
KT805547-KT805553
VmLRR-RLK208-VmLRR-RLK222
KT805844-KT805858
VfLRR-RLK158-VfLRR-RLK159
KT805554-KT805555
VmLRR-RLK225-VmLRR-RLK245
KT805859-KT805879
VfLRR-RLK161-VfLRR-RLK170
KT805556-KT805565
VmLRR-RLK247-VmLRR-RLK257
KT805880-KT805890
VfLRR-RLK172-VfLRR-RLK190
KT805566-KT805584
VmLRR-RLK259-VmLRR-RLK260
KT805891-KT805892
VfLRR-RLK193-VfLRR-RLK197
KT805585-KT805589
VfLRR-RLK199-VfLRR-RLK202
KT805590-KT805593
VfLRR-RLK204-VfLRR-RLK208
KT805594-KT805598
VfLRR-RLK210
KT805599
VfLRR-RLK212-VfLRR-RLK214
KT805600-KT805602
VfLRR-RLK216-VfLRR-RLK220
KT805603-KT805607
VfLRR-RLK222-VfLRR-RLK223
KT805608-KT805609
VfLRR-RLK225
KT805610
VfLRR-RLK227-VfLRR-RLK230
KT805611-KT805614
VfLRR-RLK232-VfLRR-RLK237
KT805615-KT805620
VfLRR-RLK239-VfLRR-RLK246
KT805621-KT805628
VfLRR-RLK248-VfLRR-RLK258
KT805629-KT805639
VfLRR-RLK260-VfLRR-RLK264
KT805640-KT805644
VfLRR-RLK266-VfLRR-RLK276
KT805645-KT805655
VfLRR-RLK279-VfLRR-RLK281
KT805656-KT805658
VfLRR-RLK283-VfLRR-RLK286
KT805659-KT805662The 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 twoVernicia 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 inV. fordii and five inV. montana). The number of members in each group was similar betweenV. fordii andV. montana, possibly because of the close genetic relationship between these two species.Table 3
The number of LRR-RLK genes containing different conserved domains inV. fordiiand V. montana.
Species
Number of Total
Number of LRR
Number of TM
Number of RLK
Number of LRR-TM
Number of LRR-RLK
Number of TM-RLK
Number of LRR-TM-RLK
V. fordii
236
75
12
73
3
52
11
10
V. montana
229
75
6
68
5
55
10
10Approximately 223, 234, 309, and 379LRR-RLK genes were identified in theA. thaliana,O. sativa,S. Lycopersicum, andP. trichocarpa genomes, respectively [7]. Our results showed that there were fewerLRR-RLK members inVernicia species than inO. sativa andP. trichocarpa. This may be related to interspecific differences or functional differentiation ofLRR-RLKs. The genome sequences also provided information on the different ratios ofVernicia homologues toLRR-RLK genes in other species.


### 3.2. Alignment and Evolutionary Analysis of VfLRR-RLKs and VmLRR-RLKs

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 twoVernicia 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 53–394 amino acids from 201 LRR-RLK genes inVernicia 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 twoVernicia 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). There is no standard classification method for LRR-RLKs. In previous studies, 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 1–16). 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.Figure 1
Phylogenetic tree based on the RLK sequences ofVf/VmLRR-RLKs. The phylogenetic tree was constructed by MEGA package v5.1 using neighbor-joining method. The numbers at each branch point represent the bootstrap scores (1,000 replicates). The VfLRR-RLKs were signed by circle filled with green, and the VmLRR-RLKs were signed by circle filled with yellow. Amino acid sequences of RLK domain used were listed in supplementary Data Set 1.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 evolutionary relationships were generally consistent among the three trees. 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 inVernicia 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 inA. thaliana corresponded to an LRR-RLK subclade inVernicia species. The members of subclade 7 inV. fordii andV. montana (Figures 1 and 2) grouped together with members of subfamily II inA. 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 inVernicia 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 inV. fordii andV. montana may play a role in the pathogen response. Interestingly, the members of subclade 9 inVernicia species corresponded to two different subclades inA. thaliana: AtLRR-RLKXIII and AtLRR-RLKXI. This may reflect functional differentiation of LRR-RLKs inA. thaliana. Based on the roles of AtLRR-RLKXIII and AtLRR-RLKXI proteins in Arabidopsis, the members of subclade 9 inVernicia 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.Table 4
Subclassification of LRR-RLK genes inA. thaliana, V. fordii, and V. Montana.
Subgroup inA. thaliana
Gene name (accession number in GenBank)
Functions
Reference
Subgroup inV. fordii
Subgroup inV. montana
LRR I
LRRPK  (At4g29990)
Light signal transduction
[1]
VfLRR-RLK182 (subclade 5)
VmLRR-RLK5 (subclade 5)
LRR II
BAK1/AtSERK3 (At4g33430);  BKK1/AtSERK4 (At2g13790);  AtSERK1  (At1g71830);  AtSERK2  (At1g34210);  NIK1 (At5g16000);  NIK2  (At3g25560);  NIK3  (At1g60800)
Antiviral defense response;  BR signaling;  cell death;  male sporogenesis;  and pathogen response
[32–34]
VfLRR-RLK187 (subclade 7)
VmLRR-RLK135 (subclade 7);  VmLRR-RLK98  (subclade 7)
LRR V
SRF4  (At3g13065);  Scrambled/SRF9/SUB/Strubbelig  (At1g11130)
Cell morphogenesis;  leaf size control; organ development; positional signaling; and root epidermis patterning
[13, 21, 35, 36]
VfLRR-RLK232 (subclade 16)
VmLRR-RLK122 (subclade 5)VmLRR-RLK133 (subclade 5)
LRR X
BRI1  (At4g39400);  BRL1  (At1g55610);  BRL2/VH1  (At2g01950);  BRL3  (At3g13380);   RPK1/TOAD1  (At1g69270);  RPK2/TOAD2  (At3g02130);  EMS1/EXS  (At5g07280);  BIR1  (At5g48380)
Abscisic acid signaling;  anther development;  brassinosteroid receptor;  cell death and innateimmunity;  embryonic pattern formation;  and vascular different
[20, 22, 37–40]
VfLRR-RLK155 (subclade 6);  VfLRR-RLK257 (subclade 6);  VfLRR-RLK244 (subclade 6);  VfLRR-RLK3 (subclade 9);  VfLRR-RLK121 (subclade 9);  VfLRR-RLK118 (subclade 11);  VfLRR-RLK252 (subclade 11)
VmLRR-RLK56 (subclade 6)VmLRR-RLK97 (subclade 6)VmLRR-RLK187 (subclade 6)VmLRR-RLK175 (subclade 6)VmLRR-RLK213 (subclade 6)VmLRR-RLK11 (subclade 9)VmLRR-RLK163 (subclade 11)
LRR XI
GSO1  (At4g20140);  GSO2  (At5g44700);  CLV1  (At1g75820);  BAM1  (At5g65700);  BAM2  (At3g49670);  BAM3  (At4g20270);  SOBIR1  (At2g31880);  HAESA  (At4g28490);  IKU2  (At3g19700);  PXY/TDRv  (At5g61480)
Anther development;  cell death and innate immunity;  epidermal surfaceEmbryogenesis;  formation during floral organ abscission;  meristem differentiation;  and seed size
[40–44]
VfLRR-RLK243 (subclade 6); VfLRR-RLK246 (subclade 6); VfLRR-RLK156 (subclade 6); VfLRR-RLK206 (subclade 9); VfLRR-RLK19  (subclade 9)
VmLRR-RLK229 (subclade 9)VmLRR-RLK237 (subclade 9)VmLRR-RLK155 (subclade 9)VmLRR-RLK15 (subclade 9)
LRR XII
FLS2  (At5g46330);  EFR  (At5g20480)
Pathogen response
[45]
VfLRR-RLK164 (subclade 10)VfLRR-RLK48 (subclade 10)VfLRR-RLK109 (subclade 10)
VmLRR-RLK167 (subclade 10)VmLRR-RLK148 (subclade 10)
LRR XIII
FEI1  (At1g31420);  FEI2  (At2g35620);  ERECTA  (At2g26330);  ERL1  (At5g62230);  ERL2  (At5g07180)
Cell wall biosynthesis;  organ growth;  and stomatal patterning and differentiation
[46–48]
VfLRR-RLK230 (subclade 9)
VmLRR-RLK139 (subclade 9)VmLRR-RLK128 (subclade 9)Figure 2
Phylogenetic tree based on the RLK sequences of LRR-RLK gene family both inV. fordii,V. montana, andA. thaliana. The phylogenetic tree was constructed by MEGA package v5.1 using neighbor-joining method. The numbers at each branch point represent the bootstrap scores (1,000 replicates). The LRR-RLKs ofV. Fordii were signed by circle filled with green, the LRR-RLKs ofV. montana were signed by circle filled with yellow, and the LRR-RLKs ofArabidopsis thaliana were signed by circle filled with blue. The accession number and the amino acid sequences of theA. thaliana used were listed in supplementary Data Set 1.


### 3.3. Motif Analysis of Vf/VmLRR-RLKs

To further reveal the diversification and potential functions of LRR-RLKs inVernicia, 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 1–25 (Figure 3).Table 5
Basic information of someVfLRR-RLK and VmLRR-RLK family genes.
Gene name
Amino acids length
L content (%)
PI
Molecular mass (KD)
LRR-Domain
TM-Domain
RLK-Domain
Basic information of someVfLRR-RLK family genes
VfLRR-RLK159
567
11.88
9.06
62669.6
25–204
269–453
534–564
VfLRR-RLK220
782
11.75
8.29
86377.1
187–333
384–569
656–781
VfLRR-RLK233
836
10.16
7.01
93442.0
389–446
5–312
566–780
VfLRR-RLK248
1002
11.93
6.71
110540.3
187–333
384–569
655–922
VfLRR-RLK255
719
13.04
8.41
79622.9
6–220
297–481
564–708
VfLRR-RLK256
991
12.10
6.91
111107.1
155–311
445–624
701–967
VfLRR-RLK258
986
10.48
6.15
108664.5
248–319
380–561
644–913
VfLRR-RLK261
984
10.62
6.05
108287.2
248–319
380–561
644–913
VfLRR-RLK271
1010
11.04
5.93
112529.3
159–302
378–553
634–901
VfLRR-RLK275
1036
11.08
5.03
113890.5
292–351
428–612
700–963
Basic information of someVmLRR-RLK family genes
VmLRR-RLK172
1028
11.97
8.31
112996.4
116–342
413–600
683–950
VmLRR-RLK178
567
11.87
9.19
62741.8
269–453
25–205
518–558
VmLRR-RLK179
659
11.08
7.02
74326.8
389–446
5–312
566–651
VmLRR-RLK202
935
12.04
6.95
105218.5
155–311
390–569
646–912
VmLRR-RLK208
1018
10.94
5.42
113540.2
280–302
378–561
642–909
VmLRR-RLK209
986
10.69
6.19
108573.5
259–319
380–561
644–913
VmLRR-RLK210
1010
10.83
5.50
112606.3
280–302
378–553
634–901
VmLRR-RLK215
742
9.60
7.49
82247.5
2–60
99–280
363–669
VmLRR-RLK216
1061
13.01
8.29
117955.4
147–276
514–695
772–1050
VmLRR-RLK220
847
12.16
8.51
94738.0
19–221
293–474
551–829Figure 3
Display of conserved motifs ofVf/VmLRR-RLK gene family. The conserved motifs were searched in 20 Vf/VmLRR-RLKs which contained full characteristic domains (the amino acid sequences were listed in supplementary Data Set 2) by Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0. Overall height in each stack indicates the sequence conservation at that position; height of each residue letter indicates relative frequency of the corresponding residue.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 (Figure4). 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. This may indicate that Motif 17 is specific to subclade 2. There were too few members of subfamilies 16 and 5 to make accurate predictions about their motif structure.Figure 4
Conserve motifs of different subclades of LRR-RLKs inVernicia species. The conserve motifs of each LRR-RLK gene were searched by Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0. Different colors and different lengths boxes represent different motifs.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 had Motif 6 at their C-terminal. 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.The motifs of different domains were detected according to their sequences and sites. The most obvious motif was that of the LRR domain, characterized by repeated “L” residues. 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 inVernicia species was the RLK domain containing Motifs 4, 9, 20, 2, 13, and 6.


### 3.4. Expression ofVfLRR-RLKs andVmLRR-RLKs in Response toFusarium Infection

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, whileV. montana (wood oil tree) is resistant. To investigate the responses ofVm/VfLRR-RLKs to theFusarium wilt pathogen, we collected roots from plants before infection (stage 0), at an early stage ofF. oxysporum infection (stage 1), and at a late stage ofF. oxysporum infection (stage 2). We randomly selected 22Vm/VfLRR-RLK orthologous genes and monitored their transcript levels by RT-PCR. The 22 orthologous genes wereVfLRR-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, andVfLRR-RLK271/VmLRR-RLK210. All genes were amplified reliably.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 toFusarium wilt disease in bothV. fordii andV. montana during the infection period (Figure 5). This result suggests that manyVf/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, andVfLRR-RLK271/VmLRR-RLK210) showed opposite expression patterns betweenV. montana andV. fordii. InV. montana, the transcript levels ofVmLRR-RLK30,178,206, and210 increased at the early stage of infection, whereas those of the corresponding orthologous genes inV. fordii,VfLRR-RLK7,159,256, and271, decreased. This finding suggests that these fourVmLRR-RLK genes participate in resistance toF. oxysporum inV. montana.Figure 5
Expression analysis of 22Vm/Vf LRR-RLK genes in roots ofVernicia during infection withFusarium. Vertical axis represents gene transcript levels. Primary axis represents transcript levels ofVf LRR-RLKs; secondary axis represents transcript levels ofVmLRR-RLKs. Standard errors are shown (n=3 biological samples). Each sample was analyzed by real-time PCR in triplicate. 0, before infection; 1, early stage of infection; 2, late stage of infection.


### 3.5. Transcription Patterns ofVfLRR-RLKs andVmLRR-RLRs in Various Tissues

To investigate the tissue specificity ofVfLRR-RLKs andVmLRR-RLRs expression and further analyze genes related toFusarium wilt disease, we analyzed the transcript levels of the 22 genes described above in seven tissues ofV. fordii andV. montana by qRT-PCR (Figure 6). Among them,VfLRR-RLK260 andVfLRR-RLK159 showed similar expression patterns in all seven tissues ofV. fordii. Both showed higher transcript levels in leaves and kernels and lower transcript levels in roots, stems, buds, and ovaries. However, compared withVfLRR-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-RLK13 showed the highest transcript level in ovaries, followed by leaves.VfLRR-RLK271 showed similar expression patterns in all tissues. The other five genes showed tissue-specific expression patterns.VfLRR-RLK6 was specifically expressed in petals,VfLRR-RLK9 in ovaries, andVfLRR-RLK11 in roots. BothVfLRR-RLK7 andVfLRR-RLK256 were specifically expressed in kernels. Together, these results suggest thatVfLRR-RLKs play various roles in the development of tung tree.Figure 6
Transcript levels of 22Vm/Vf LRR-RLK genes in various tissues. Column height shows gene transcript levels. Primary axis represents transcript levels ofVf LRR-RLKs; secondary axis represents transcript levels ofVmLRR-RLKs. Standard errors are shown (n=3 biological samples). Each sample was analyzed by real-time PCR in triplicate.Compared withVfLRR-RLKs, mostVmLRR-RLKs showed higher transcript levels in the seven tissues analyzed. SixVmLRR-RLKs (VmLRR-RLK18,VmLRR-RLK29,VmLRR-RLK202,VmLRR-RLK30,VmLRR-RLK178, andVmLRR-RLK210) showed the same expression patterns as their orthologous genes inV. fordii. This may indicate that they share the same function inV. fordii andV. montana. The other fiveVmLRR-RLKs showed different expression patterns inV. montana.VmLRR-RLK111 was mainly expressed in leaves and had similar transcript levels in other tissues.VmLRR-RLK164 andVmLRR-RLK241 showed peak expression in kernels, butVmLRR-RLK164 was also expressed at high levels in the stems. BothVmLRR-RLK17 andVmLRR-RLK206 showed the highest transcript levels in roots and lower levels in other tissues. The different expression patterns inV. montana may reflect functional differentiation during evolution.Among the seven pairs of orthologous genes showing similar trends in gene expression inV. montana andV. fordii in response toFusarium infection, three pairs also showed similar expression patterns in the tissues (VfLRR-RLK2/VmLRR-RLK18,VfLRR-RLK9/VmLRR-RLK29, andVfLRR-RLK260/VmLRR-RLK202). The other four pairs showed different expression patterns in the seven tissues analyzed. Of the four pairs of orthologous genes showing opposite responses toFusarium infection inV. montana andV. fordii (Figure 5), three pairs showed similar expression patterns in the tissues, and one pair (VfLRR-RLK256 andVmLRR-RLK206) showed different expression patterns in the tissues (Figure 6). There were high transcript levels ofVfLRR-RLK256 in kernels andVmLRR-RLK206 in the roots. Given that theFusarium pathogen invades via the roots of tung tree, these results suggest thatVmLRR-RLK206 may play a role in resistance toFusarium wilt disease.


## 3.1. Identification ofVfLRR-RLKs andVmLRR-RLKs inV. fordii andV. montana

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 inV. fordii andV. 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.Table 2
GenBank accession numbers ofVfLRR-RLK and VmLRR-RLK genes.
Vernicia fordii
Vernicia montana
Gene ID
GenBank accession number
Gene ID
GenBank accession number
VfLRR-RLK1-VfLRR-RLK14
c805427-KT805440
VmLRR-RLK1-VmLRR-RLK9
KT805663-KT805671
VfLRR-RLK18-VfLRR-RLK19
KT805441-KT805442
VmLRR-RLK11-VmLRR-RLK18
KT805672-KT805679
VfLRR-RLK22-VfLRR-RLK24
KT805443-KT805445
VmLRR-RLK20-VmLRR-RLK32
KT805680-KT805692
VfLRR-RLK26-VfLRR-RLK28
KT805446-KT805448
VmLRR-RLK34
KT805693
VfLRR-RLK30-VfLRR-RLK46
KT805449-KT805465
VmLRR-RLK36-VmLRR-RLK39
KT805694-KT805697
VfLRR-RLK48
KT805466
VmLRR-RLK41-VmLRR-RLK67
KT805698-KT805724
VfLRR-RLK50-VfLRR-RLK53
KT805467-KT805470
VmLRR-RLK69-VmLRR-RLK83
KT805725-KT805739
VfLRR-RLK55-VfLRR-RLK57
KT805471-KT805473
VmLRR-RLK86-VmLRR-RLK90
KT805740-KT805744
VfLRR-RLK59-VfLRR-RLK60
KT805474-KT805475
VmLRR-RLK92
KT805745
VfLRR-RLK63-VfLRR-RLK65
KT805476-KT805478
VmLRR-RLK94-VmLRR-RLK108
KT805746-KT805760
VfLRR-RLK68-VfLRR-RLK70
KT805479-KT805481
VmLRR-RLK110-VmLRR-RLK115
KT805761-KT805766
VfLRR-RLK72
KT805482
VmLRR-RLK117-VmLRR-RLK118
KT805767-KT805768
VfLRR-RLK74-VfLRR-RLK90
KT805483-KT805499
VmLRR-RLK120
KT805769
VfLRR-RLK92-VfLRR-RLK93
KT805500-KT805501
VmLRR-RLK122-VmLRR-RLK123
KT805770-KT805771
VfLRR-RLK95-VfLRR-RLK97
KT805502-KT805504
VmLRR-RLK125-VmLRR-RLK129
KT805772-KT805776
VfLRR-RLK99-VfLRR-RLK104
KT805505-KT805510
VmLRR-RLK131-VmLRR-RLK142
KT805777-KT805788
VfLRR-RLK106-VfLRR-RLK124
KT805511-KT805529
VmLRR-RLK144-VmLRR-RLK151
KT805789-KT805796
VfLRR-RLK126
KT805530
VmLRR-RLK153-VmLRR-RLK157
KT805797-KT805801
VfLRR-RLK128
KT805531
VmLRR-RLK159-VmLRR-RLK187
KT805802-KT805830
VfLRR-RLK130-VfLRR-RLK140
KT805532-KT805542
VmLRR-RLK190
KT805831
VfLRR-RLK143
KT805543
VmLRR-RLK192-VmLRR-RLK195
KT805832-KT805835
VfLRR-RLK145-VfLRR-RLK146
KT805544-KT805545
VmLRR-RLK197-VmLRR-RLK199
KT805836-KT805838
VfLRR-RLK148
KT805546
VmLRR-RLK202-VmLRR-RLK206
KT805839-KT805843
VfLRR-RLK150-VfLRR-RLK156
KT805547-KT805553
VmLRR-RLK208-VmLRR-RLK222
KT805844-KT805858
VfLRR-RLK158-VfLRR-RLK159
KT805554-KT805555
VmLRR-RLK225-VmLRR-RLK245
KT805859-KT805879
VfLRR-RLK161-VfLRR-RLK170
KT805556-KT805565
VmLRR-RLK247-VmLRR-RLK257
KT805880-KT805890
VfLRR-RLK172-VfLRR-RLK190
KT805566-KT805584
VmLRR-RLK259-VmLRR-RLK260
KT805891-KT805892
VfLRR-RLK193-VfLRR-RLK197
KT805585-KT805589
VfLRR-RLK199-VfLRR-RLK202
KT805590-KT805593
VfLRR-RLK204-VfLRR-RLK208
KT805594-KT805598
VfLRR-RLK210
KT805599
VfLRR-RLK212-VfLRR-RLK214
KT805600-KT805602
VfLRR-RLK216-VfLRR-RLK220
KT805603-KT805607
VfLRR-RLK222-VfLRR-RLK223
KT805608-KT805609
VfLRR-RLK225
KT805610
VfLRR-RLK227-VfLRR-RLK230
KT805611-KT805614
VfLRR-RLK232-VfLRR-RLK237
KT805615-KT805620
VfLRR-RLK239-VfLRR-RLK246
KT805621-KT805628
VfLRR-RLK248-VfLRR-RLK258
KT805629-KT805639
VfLRR-RLK260-VfLRR-RLK264
KT805640-KT805644
VfLRR-RLK266-VfLRR-RLK276
KT805645-KT805655
VfLRR-RLK279-VfLRR-RLK281
KT805656-KT805658
VfLRR-RLK283-VfLRR-RLK286
KT805659-KT805662The 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 twoVernicia 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 inV. fordii and five inV. montana). The number of members in each group was similar betweenV. fordii andV. montana, possibly because of the close genetic relationship between these two species.Table 3
The number of LRR-RLK genes containing different conserved domains inV. fordiiand V. montana.
Species
Number of Total
Number of LRR
Number of TM
Number of RLK
Number of LRR-TM
Number of LRR-RLK
Number of TM-RLK
Number of LRR-TM-RLK
V. fordii
236
75
12
73
3
52
11
10
V. montana
229
75
6
68
5
55
10
10Approximately 223, 234, 309, and 379LRR-RLK genes were identified in theA. thaliana,O. sativa,S. Lycopersicum, andP. trichocarpa genomes, respectively [7]. Our results showed that there were fewerLRR-RLK members inVernicia species than inO. sativa andP. trichocarpa. This may be related to interspecific differences or functional differentiation ofLRR-RLKs. The genome sequences also provided information on the different ratios ofVernicia homologues toLRR-RLK genes in other species.


## 3.2. Alignment and Evolutionary Analysis of VfLRR-RLKs and VmLRR-RLKs

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 twoVernicia 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 53–394 amino acids from 201 LRR-RLK genes inVernicia 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 twoVernicia 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). There is no standard classification method for LRR-RLKs. In previous studies, 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 1–16). 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.Figure 1
Phylogenetic tree based on the RLK sequences ofVf/VmLRR-RLKs. The phylogenetic tree was constructed by MEGA package v5.1 using neighbor-joining method. The numbers at each branch point represent the bootstrap scores (1,000 replicates). The VfLRR-RLKs were signed by circle filled with green, and the VmLRR-RLKs were signed by circle filled with yellow. Amino acid sequences of RLK domain used were listed in supplementary Data Set 1.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 evolutionary relationships were generally consistent among the three trees. 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 inVernicia 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 inA. thaliana corresponded to an LRR-RLK subclade inVernicia species. The members of subclade 7 inV. fordii andV. montana (Figures 1 and 2) grouped together with members of subfamily II inA. 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 inVernicia 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 inV. fordii andV. montana may play a role in the pathogen response. Interestingly, the members of subclade 9 inVernicia species corresponded to two different subclades inA. thaliana: AtLRR-RLKXIII and AtLRR-RLKXI. This may reflect functional differentiation of LRR-RLKs inA. thaliana. Based on the roles of AtLRR-RLKXIII and AtLRR-RLKXI proteins in Arabidopsis, the members of subclade 9 inVernicia 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.Table 4
Subclassification of LRR-RLK genes inA. thaliana, V. fordii, and V. Montana.
Subgroup inA. thaliana
Gene name (accession number in GenBank)
Functions
Reference
Subgroup inV. fordii
Subgroup inV. montana
LRR I
LRRPK  (At4g29990)
Light signal transduction
[1]
VfLRR-RLK182 (subclade 5)
VmLRR-RLK5 (subclade 5)
LRR II
BAK1/AtSERK3 (At4g33430);  BKK1/AtSERK4 (At2g13790);  AtSERK1  (At1g71830);  AtSERK2  (At1g34210);  NIK1 (At5g16000);  NIK2  (At3g25560);  NIK3  (At1g60800)
Antiviral defense response;  BR signaling;  cell death;  male sporogenesis;  and pathogen response
[32–34]
VfLRR-RLK187 (subclade 7)
VmLRR-RLK135 (subclade 7);  VmLRR-RLK98  (subclade 7)
LRR V
SRF4  (At3g13065);  Scrambled/SRF9/SUB/Strubbelig  (At1g11130)
Cell morphogenesis;  leaf size control; organ development; positional signaling; and root epidermis patterning
[13, 21, 35, 36]
VfLRR-RLK232 (subclade 16)
VmLRR-RLK122 (subclade 5)VmLRR-RLK133 (subclade 5)
LRR X
BRI1  (At4g39400);  BRL1  (At1g55610);  BRL2/VH1  (At2g01950);  BRL3  (At3g13380);   RPK1/TOAD1  (At1g69270);  RPK2/TOAD2  (At3g02130);  EMS1/EXS  (At5g07280);  BIR1  (At5g48380)
Abscisic acid signaling;  anther development;  brassinosteroid receptor;  cell death and innateimmunity;  embryonic pattern formation;  and vascular different
[20, 22, 37–40]
VfLRR-RLK155 (subclade 6);  VfLRR-RLK257 (subclade 6);  VfLRR-RLK244 (subclade 6);  VfLRR-RLK3 (subclade 9);  VfLRR-RLK121 (subclade 9);  VfLRR-RLK118 (subclade 11);  VfLRR-RLK252 (subclade 11)
VmLRR-RLK56 (subclade 6)VmLRR-RLK97 (subclade 6)VmLRR-RLK187 (subclade 6)VmLRR-RLK175 (subclade 6)VmLRR-RLK213 (subclade 6)VmLRR-RLK11 (subclade 9)VmLRR-RLK163 (subclade 11)
LRR XI
GSO1  (At4g20140);  GSO2  (At5g44700);  CLV1  (At1g75820);  BAM1  (At5g65700);  BAM2  (At3g49670);  BAM3  (At4g20270);  SOBIR1  (At2g31880);  HAESA  (At4g28490);  IKU2  (At3g19700);  PXY/TDRv  (At5g61480)
Anther development;  cell death and innate immunity;  epidermal surfaceEmbryogenesis;  formation during floral organ abscission;  meristem differentiation;  and seed size
[40–44]
VfLRR-RLK243 (subclade 6); VfLRR-RLK246 (subclade 6); VfLRR-RLK156 (subclade 6); VfLRR-RLK206 (subclade 9); VfLRR-RLK19  (subclade 9)
VmLRR-RLK229 (subclade 9)VmLRR-RLK237 (subclade 9)VmLRR-RLK155 (subclade 9)VmLRR-RLK15 (subclade 9)
LRR XII
FLS2  (At5g46330);  EFR  (At5g20480)
Pathogen response
[45]
VfLRR-RLK164 (subclade 10)VfLRR-RLK48 (subclade 10)VfLRR-RLK109 (subclade 10)
VmLRR-RLK167 (subclade 10)VmLRR-RLK148 (subclade 10)
LRR XIII
FEI1  (At1g31420);  FEI2  (At2g35620);  ERECTA  (At2g26330);  ERL1  (At5g62230);  ERL2  (At5g07180)
Cell wall biosynthesis;  organ growth;  and stomatal patterning and differentiation
[46–48]
VfLRR-RLK230 (subclade 9)
VmLRR-RLK139 (subclade 9)VmLRR-RLK128 (subclade 9)Figure 2
Phylogenetic tree based on the RLK sequences of LRR-RLK gene family both inV. fordii,V. montana, andA. thaliana. The phylogenetic tree was constructed by MEGA package v5.1 using neighbor-joining method. The numbers at each branch point represent the bootstrap scores (1,000 replicates). The LRR-RLKs ofV. Fordii were signed by circle filled with green, the LRR-RLKs ofV. montana were signed by circle filled with yellow, and the LRR-RLKs ofArabidopsis thaliana were signed by circle filled with blue. The accession number and the amino acid sequences of theA. thaliana used were listed in supplementary Data Set 1.


## 3.3. Motif Analysis of Vf/VmLRR-RLKs

To further reveal the diversification and potential functions of LRR-RLKs inVernicia, 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 1–25 (Figure 3).Table 5
Basic information of someVfLRR-RLK and VmLRR-RLK family genes.
Gene name
Amino acids length
L content (%)
PI
Molecular mass (KD)
LRR-Domain
TM-Domain
RLK-Domain
Basic information of someVfLRR-RLK family genes
VfLRR-RLK159
567
11.88
9.06
62669.6
25–204
269–453
534–564
VfLRR-RLK220
782
11.75
8.29
86377.1
187–333
384–569
656–781
VfLRR-RLK233
836
10.16
7.01
93442.0
389–446
5–312
566–780
VfLRR-RLK248
1002
11.93
6.71
110540.3
187–333
384–569
655–922
VfLRR-RLK255
719
13.04
8.41
79622.9
6–220
297–481
564–708
VfLRR-RLK256
991
12.10
6.91
111107.1
155–311
445–624
701–967
VfLRR-RLK258
986
10.48
6.15
108664.5
248–319
380–561
644–913
VfLRR-RLK261
984
10.62
6.05
108287.2
248–319
380–561
644–913
VfLRR-RLK271
1010
11.04
5.93
112529.3
159–302
378–553
634–901
VfLRR-RLK275
1036
11.08
5.03
113890.5
292–351
428–612
700–963
Basic information of someVmLRR-RLK family genes
VmLRR-RLK172
1028
11.97
8.31
112996.4
116–342
413–600
683–950
VmLRR-RLK178
567
11.87
9.19
62741.8
269–453
25–205
518–558
VmLRR-RLK179
659
11.08
7.02
74326.8
389–446
5–312
566–651
VmLRR-RLK202
935
12.04
6.95
105218.5
155–311
390–569
646–912
VmLRR-RLK208
1018
10.94
5.42
113540.2
280–302
378–561
642–909
VmLRR-RLK209
986
10.69
6.19
108573.5
259–319
380–561
644–913
VmLRR-RLK210
1010
10.83
5.50
112606.3
280–302
378–553
634–901
VmLRR-RLK215
742
9.60
7.49
82247.5
2–60
99–280
363–669
VmLRR-RLK216
1061
13.01
8.29
117955.4
147–276
514–695
772–1050
VmLRR-RLK220
847
12.16
8.51
94738.0
19–221
293–474
551–829Figure 3
Display of conserved motifs ofVf/VmLRR-RLK gene family. The conserved motifs were searched in 20 Vf/VmLRR-RLKs which contained full characteristic domains (the amino acid sequences were listed in supplementary Data Set 2) by Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0. Overall height in each stack indicates the sequence conservation at that position; height of each residue letter indicates relative frequency of the corresponding residue.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 (Figure4). 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. This may indicate that Motif 17 is specific to subclade 2. There were too few members of subfamilies 16 and 5 to make accurate predictions about their motif structure.Figure 4
Conserve motifs of different subclades of LRR-RLKs inVernicia species. The conserve motifs of each LRR-RLK gene were searched by Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0. Different colors and different lengths boxes represent different motifs.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 had Motif 6 at their C-terminal. 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.The motifs of different domains were detected according to their sequences and sites. The most obvious motif was that of the LRR domain, characterized by repeated “L” residues. 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 inVernicia species was the RLK domain containing Motifs 4, 9, 20, 2, 13, and 6.


## 3.4. Expression ofVfLRR-RLKs andVmLRR-RLKs in Response toFusarium Infection

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, whileV. montana (wood oil tree) is resistant. To investigate the responses ofVm/VfLRR-RLKs to theFusarium wilt pathogen, we collected roots from plants before infection (stage 0), at an early stage ofF. oxysporum infection (stage 1), and at a late stage ofF. oxysporum infection (stage 2). We randomly selected 22Vm/VfLRR-RLK orthologous genes and monitored their transcript levels by RT-PCR. The 22 orthologous genes wereVfLRR-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, andVfLRR-RLK271/VmLRR-RLK210. All genes were amplified reliably.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 toFusarium wilt disease in bothV. fordii andV. montana during the infection period (Figure 5). This result suggests that manyVf/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, andVfLRR-RLK271/VmLRR-RLK210) showed opposite expression patterns betweenV. montana andV. fordii. InV. montana, the transcript levels ofVmLRR-RLK30,178,206, and210 increased at the early stage of infection, whereas those of the corresponding orthologous genes inV. fordii,VfLRR-RLK7,159,256, and271, decreased. This finding suggests that these fourVmLRR-RLK genes participate in resistance toF. oxysporum inV. montana.Figure 5
Expression analysis of 22Vm/Vf LRR-RLK genes in roots ofVernicia during infection withFusarium. Vertical axis represents gene transcript levels. Primary axis represents transcript levels ofVf LRR-RLKs; secondary axis represents transcript levels ofVmLRR-RLKs. Standard errors are shown (n=3 biological samples). Each sample was analyzed by real-time PCR in triplicate. 0, before infection; 1, early stage of infection; 2, late stage of infection.


## 3.5. Transcription Patterns ofVfLRR-RLKs andVmLRR-RLRs in Various Tissues

To investigate the tissue specificity ofVfLRR-RLKs andVmLRR-RLRs expression and further analyze genes related toFusarium wilt disease, we analyzed the transcript levels of the 22 genes described above in seven tissues ofV. fordii andV. montana by qRT-PCR (Figure 6). Among them,VfLRR-RLK260 andVfLRR-RLK159 showed similar expression patterns in all seven tissues ofV. fordii. Both showed higher transcript levels in leaves and kernels and lower transcript levels in roots, stems, buds, and ovaries. However, compared withVfLRR-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-RLK13 showed the highest transcript level in ovaries, followed by leaves.VfLRR-RLK271 showed similar expression patterns in all tissues. The other five genes showed tissue-specific expression patterns.VfLRR-RLK6 was specifically expressed in petals,VfLRR-RLK9 in ovaries, andVfLRR-RLK11 in roots. BothVfLRR-RLK7 andVfLRR-RLK256 were specifically expressed in kernels. Together, these results suggest thatVfLRR-RLKs play various roles in the development of tung tree.Figure 6
Transcript levels of 22Vm/Vf LRR-RLK genes in various tissues. Column height shows gene transcript levels. Primary axis represents transcript levels ofVf LRR-RLKs; secondary axis represents transcript levels ofVmLRR-RLKs. Standard errors are shown (n=3 biological samples). Each sample was analyzed by real-time PCR in triplicate.Compared withVfLRR-RLKs, mostVmLRR-RLKs showed higher transcript levels in the seven tissues analyzed. SixVmLRR-RLKs (VmLRR-RLK18,VmLRR-RLK29,VmLRR-RLK202,VmLRR-RLK30,VmLRR-RLK178, andVmLRR-RLK210) showed the same expression patterns as their orthologous genes inV. fordii. This may indicate that they share the same function inV. fordii andV. montana. The other fiveVmLRR-RLKs showed different expression patterns inV. montana.VmLRR-RLK111 was mainly expressed in leaves and had similar transcript levels in other tissues.VmLRR-RLK164 andVmLRR-RLK241 showed peak expression in kernels, butVmLRR-RLK164 was also expressed at high levels in the stems. BothVmLRR-RLK17 andVmLRR-RLK206 showed the highest transcript levels in roots and lower levels in other tissues. The different expression patterns inV. montana may reflect functional differentiation during evolution.Among the seven pairs of orthologous genes showing similar trends in gene expression inV. montana andV. fordii in response toFusarium infection, three pairs also showed similar expression patterns in the tissues (VfLRR-RLK2/VmLRR-RLK18,VfLRR-RLK9/VmLRR-RLK29, andVfLRR-RLK260/VmLRR-RLK202). The other four pairs showed different expression patterns in the seven tissues analyzed. Of the four pairs of orthologous genes showing opposite responses toFusarium infection inV. montana andV. fordii (Figure 5), three pairs showed similar expression patterns in the tissues, and one pair (VfLRR-RLK256 andVmLRR-RLK206) showed different expression patterns in the tissues (Figure 6). There were high transcript levels ofVfLRR-RLK256 in kernels andVmLRR-RLK206 in the roots. Given that theFusarium pathogen invades via the roots of tung tree, these results suggest thatVmLRR-RLK206 may play a role in resistance toFusarium wilt disease.


## 4. Conclusion

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 ofVfLRR-RLKs andVmLRR-RLKs in different tissues and in response toFusarium infection were conducted. Characterization ofLRR-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 theLRR-RLK gene family in tung tree.



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*Source: 823427-2015-12-13.xml*