International Journal of Genomics

International Journal of Genomics / 2015 / Article

Research Article | Open Access

Volume 2015 |Article ID 823427 |

Huiping Zhu, Yangdong Wang, Hengfu Yin, Ming Gao, Qiyan Zhang, Yicun Chen, "Genome-Wide Identification and Characterization of the LRR-RLK Gene Family in Two Vernicia Species", International Journal of Genomics, vol. 2015, Article ID 823427, 17 pages, 2015.

Genome-Wide Identification and Characterization of the LRR-RLK Gene Family in Two Vernicia Species

Academic Editor: Henry Heng
Received15 Oct 2015
Accepted17 Nov 2015
Published13 Dec 2015


Leucine-rich repeat receptor-like kinases (LRR-RLKs) make up the largest group of RLKs in plants and play important roles in many key biological processes such as pathogen response and signal transduction. To date, most studies on LRR-RLKs have been conducted on model plants. Here, we identified 236 and 230 LRR-RLKs in two industrial oil-producing trees: Vernicia fordii and Vernicia montana, respectively. Sequence alignment analyses showed that the homology of the RLK domain (23.81%) was greater than that of the LRR domain (9.51%) among the Vf/VmLRR-RLKs. The conserved motif of the LRR domain in Vf/VmLRR-RLKs matched well the known plant LRR consensus sequence but differed at the third last amino acid (W or L). Phylogenetic analysis revealed that Vf/VmLRR-RLKs were grouped into 16 subclades. We characterized the expression profiles of Vf/VmLRR-RLKs in various tissue types including root, leaf, petal, and kernel. Further investigation revealed that Vf/VmLRR-RLK orthologous genes mainly showed similar expression patterns in response to tree wilt disease, except 4 pairs of Vf/VmLRR-RLKs that showed opposite expression trends. These results represent an extensive evaluation of LRR-RLKs in two industrial oil trees and will be useful for further functional studies on these proteins.

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 in Arabidopsis thaliana [7], 234 members in Solanum lycopersicum [8], 379 members in Populus trichocarpa [9], and 309 members in Oryza 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, most LRR-RLK genes have been isolated from model plants and herbs, rather than woody oil plants. Tung oil tree (Vernicia fordii) and wood oil tree (Vernicia montana) are important industrial oil plants belonging to the Euphorbiaceae family. The oil extracted from tung seeds is an excellent drying oil that is renewable, safe, and environmentally friendly. This oil is widely used in industrial products such as paints, plasticizers, resins, medicine, synthetic rubber, and printing ink [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 to Fusarium wilt disease. Interestingly, the two different species of Vernicia show different degrees of resistance to this disease; V. fordii, which is the main oil-producing species, is susceptible to the disease, while wood oil tree (V. montana) is resistant. A previous study showed that many LRR-RLKs are defense-related [10]; therefore, studies on the LRR-RLKs of these two Vernicia species may help to clarify why one species is more resistant than the other.

In this study, we identified the LRR-RLKs in two Vernicia species and conducted multiple sequence alignments, phylogenetic analyses, and conserved motif analyses of the VfLRR-RLK and VmLRR-RLK gene families. We selected several LRR-RLK genes for gene expression analyses in various tissues of V. fordii and V. montana. Finally, we investigated the changes in expression of 22 Vm/fLRR-RLK genes during infection with Fusarium oxysporum. These results will be useful for further studies on the functions of LRR-RLKs in woody oil trees.

2. Materials and Methods

2.1. Plant Materials

Samples of V. fordii and V. montana were collected from Fuyang Urban Forest Park, Hangzhou city, Zhejiang Province, China, and then separated into roots, stems, leaves, flower buds, ovaries, and kernels. 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 for LRR-RLK Genes in V. fordii and V. montana

The members of the LRR-RLK superfamily in the two Vernicia species were first identified from transcriptome data using look-up function of computer and using “LRR” as the key word; then we sought the selected genes one by one according to their descriptions of annotations. 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 ( Controversial sequences were used as search queries at PBLAST ( Sequences not belonging to the LRR-RLK family were rejected. The Simple Modular Architecture Research Tool (SMART) ( [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 of Vernicia 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 as A. thaliana, S. lycopersicum, P. trichocarpa, and O. sativa; however, much more information about the functional classification was reported in A. thaliana. To compare the evolutionary relationships of LRR-RLKs between Vernicia species and A. thaliana and roughly predict the functions of LRR-RLKs in Vernicia species, multiple sequence alignments were performed for VmLRR-RLK, VfLRR-RLK, and 35 AtLRR-RLKs with known functions using the amino acid sequences of the RLK domains.

The phylogenetic trees were constructed with the neighbor-joining method using MEGA 5.1 software [28] with position correction, pairwise deletion, and 1000 bootstrap replicates indicated at each node.

2.5. Motif Recognition of LRR-RLKs in Vernicia Species

The conserved motifs of LRR-RLK protein sequences in two Vernicia species were identified using the motif-based sequence analysis tool, Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0 ( [29], with the following parameters: any number of repetitions of a motif, maximum number of motifs = 25.

2.6. Inoculation of V. fordii and V. montana with Fusarium Pathogen

The tung wilt disease pathogen F. 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 for ACT7 (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.

Primer namesSequences (5′-3′)Amplicons length (bp)


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 candidate genes in the LRR-RLK superfamily were obtained based on annotations of RNA-seq data. Then, 236 and 230 sequences in V. fordii and V. montana with at least one characteristic domain were positively identified as members of the LRR-RLK superfamily. All of the 466 sequences were submitted to the NCBI by our laboratory, and the accession numbers were listed in Table 2.

Vernicia fordiiVernicia montana
Gene IDGenBank accession numberGene IDGenBank accession number


The LRR-RLK family proteins contained at least one full or partial characteristic domain (LRR, TM, and/or RLK domains). According to the structural characteristics of the LRR-RLKs in the two Vernicia species, the proteins were classified into seven groups (Table 3): group 1 with an LRR domain; group 2 with a TM domain; group 3 with an RLK domain; group 4 with LRR and TM domains; group 5 with LRR and RLK domains; group 6 with TM and RLK domains; and group 7 with LRR, TM, and RLK domains. As shown in Table 3, groups 1, 3, and 5 had the most members and group 4 had the fewest members (three in V. fordii and five in V. montana). The number of members in each group was similar between V. fordii and V. montana, possibly because of the close genetic relationship between these two species.

SpeciesNumber of TotalNumber of LRRNumber of TMNumber of RLKNumber of LRR-TMNumber of LRR-RLKNumber of TM-RLKNumber of LRR-TM-RLK

V. fordii2367512733521110
V. montana229756685551010

Approximately 223, 234, 309, and 379 LRR-RLK genes were identified in the A. thaliana, O. sativa, S. Lycopersicum, and P. trichocarpa genomes, respectively [7]. Our results showed that there were fewer LRR-RLK members in Vernicia species than in O. sativa and P. trichocarpa. This may be related to interspecific differences or functional differentiation of LRR-RLKs. The genome sequences also provided information on the different ratios of Vernicia homologues to LRR-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 two Vernicia species. Therefore, we conducted alignments for the protein groups with the most members. First, we analyzed proteins with the LRR domain, since these were the most abundant. When the LRR domain was selected for the alignment the consistency was approximately 3.90%. Therefore, we selected different sequences, trimmed both ends of the sequences, and tried the alignment again. The consistency reached 9.51%, which was still too low to build a phylogenetic tree. The low consistency of LRR domains suggested a high degree of sequence complexity and diversity between VfLRR-RLKs and VmLRR-RLKs. Therefore, we selected the RLK domain amino acids sequence containing 53–394 amino acids from 201 LRR-RLK genes in Vernicia species for alignment. The consistency among these sequences was 23.81% (Supplementary Figure  1 in Supplementary Material available online at To analyze the evolutionary relationships of the LRR-RLK superfamily in these two Vernicia species, an unrooted NJ phylogenetic tree was constructed based on the multiple sequence alignments of 106 VfLRR-RLKs and 95 VmLRR-RLKs containing the RLK domain (Figure 1). 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.

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 in Vernicia species, 35 Arabidopsis LRR-RLKs with known functions (Table 4) were compared with VfLRR-RLKs and VmLRR-RLKs (Figure 2). Almost every LRR-RLK subfamily in A. thaliana corresponded to an LRR-RLK subclade in Vernicia species. The members of subclade 7 in V. fordii and V. montana (Figures 1 and 2) grouped together with members of subfamily II in A. thaliana, suggesting that they may share the same function. These proteins may participate in brassinosteroid signaling, pathogen responses, cell death, and male sporogenesis. Similarly, members of subclade 6 in Vernicia species may be related to the plant brassinosteroid receptor, vascular differentiation, abscisic acid signaling, embryonic pattern formation, another development, cell death, and innate immunity. Subclade 10 members in V. fordii and V. montana may play a role in the pathogen response. Interestingly, the members of subclade 9 in Vernicia species corresponded to two different subclades in A. thaliana: AtLRR-RLKXIII and AtLRR-RLKXI. This may reflect functional differentiation of LRR-RLKs in A. thaliana. Based on the roles of AtLRR-RLKXIII and AtLRR-RLKXI proteins in Arabidopsis, the members of subclade 9 in Vernicia species may be involved in meristem differentiation, epidermal surface formation during embryogenesis, floral organ abscission, determination of seed size, cell wall biosynthesis, organ growth, and stomatal patterning and differentiation.

Subgroup in A. thalianaGene name (accession number in GenBank)FunctionsReferenceSubgroup in V. fordiiSubgroup in V. montana

LRR ILRRPK (At4g29990)Light signal transduction[1]VfLRR-RLK182 (subclade 5)VmLRR-RLK5 (subclade 5)

LRR IIBAK1/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[3234]VfLRR-RLK187 (subclade 7)VmLRR-RLK135 (subclade 7); VmLRR-RLK98 (subclade 7)

LRR VSRF4 (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 XBRI1 (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 innate
immunity; embryonic pattern formation; and vascular different
[20, 22, 3740]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 XIGSO1 (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 surface
Embryogenesis; formation during floral organ abscission; meristem differentiation; and seed size
[4044]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 XIIFLS2 (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 XIIIFEI1 (At1g31420); FEI2 (At2g35620); ERECTA (At2g26330); ERL1 (At5g62230); ERL2 (At5g07180)Cell wall biosynthesis; organ growth; and stomatal patterning and differentiation[4648]VfLRR-RLK230 (subclade 9)VmLRR-RLK139 (subclade 9)
VmLRR-RLK128 (subclade 9)

3.3. Motif Analysis of Vf/VmLRR-RLKs

To further reveal the diversification and potential functions of LRR-RLKs in Vernicia, we selected 20 Vf/VmLRR-RLKs (Table 5) with full characteristic domain and investigated their conserved motifs using MEME version 4.10.0. A total of 25 conserved motifs were identified and numbered 1–25 (Figure 3).

Gene nameAmino acids lengthL content (%)PIMolecular mass (KD)LRR-DomainTM-DomainRLK-Domain

Basic information of some VfLRR-RLK family genes

Basic information of some VmLRR-RLK family genes

Among the 20 Vf/VmLRR-RLKs, there were six different motifs at the N-terminal and six at the C-terminal. The six motifs at the N-terminal were Motifs 19, 1, 8, 22, 17, and 23. Ten of the 20 LRR-RLKs (50%) had Motif 19 at the N-terminal, and most of these LRR-RLKs were in subclades 1 and 4 (Figure 4). The other five motifs were present in one to three of the 20 LRR-RLKs. Interestingly, Motif 17 was present at the N-terminal of two LRR-RLKs, both of which were in subclade 2. 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.

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 in Vernicia species was the RLK domain containing Motifs 4, 9, 20, 2, 13, and 6.

3.4. Expression of VfLRR-RLKs and VmLRR-RLKs in Response to Fusarium 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, while V. montana (wood oil tree) is resistant. To investigate the responses of Vm/VfLRR-RLKs to the Fusarium wilt pathogen, we collected roots from plants before infection (stage 0), at an early stage of F. oxysporum infection (stage 1), and at a late stage of F. oxysporum infection (stage 2). We randomly selected 22 Vm/VfLRR-RLK orthologous genes and monitored their transcript levels by RT-PCR. The 22 orthologous genes were VfLRR-RLK2/VmLRR-RLK18, VfLRR-RLK6/VmLRR-RLK17, VfLRR-RLK7/VmLRR-RLK30, VfLRR-RLK9/VmLRR-RLK29, VfLRR-RLK11/VmLRR-RLK111, VfLRR-RLK13/VmLRR-RLK241, VfLRR-RLK159/VmLRR-RLK178, VfLRR-RLK172/VmLRR-RLK164, VfLRR-RLK256/VmLRR-RLK206, VfLRR-RLK260/VmLRR-RLK202, and VfLRR-RLK271/VmLRR-RLK210. 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 to Fusarium wilt disease in both V. fordii and V. montana during the infection period (Figure 5). This result suggests that many Vf/VmLRR-RLKs have similar functions during pathogen infection. Four pairs of orthologous genes (VfLRR-RLK7/VmLRR-RLK30, VfLRR-RLK159/VmLRR-RLK178, VfLRR-RLK256/VmLRR-RLK206, and VfLRR-RLK271/VmLRR-RLK210) showed opposite expression patterns between V. montana and V. fordii. In V. montana, the transcript levels of VmLRR-RLK30, 178, 206, and 210 increased at the early stage of infection, whereas those of the corresponding orthologous genes in V. fordii, VfLRR-RLK7, 159, 256, and 271, decreased. This finding suggests that these four VmLRR-RLK genes participate in resistance to F. oxysporum in V. montana.

3.5. Transcription Patterns of VfLRR-RLKs and VmLRR-RLRs in Various Tissues

To investigate the tissue specificity of VfLRR-RLKs and VmLRR-RLRs expression and further analyze genes related to Fusarium wilt disease, we analyzed the transcript levels of the 22 genes described above in seven tissues of V. fordii and V. montana by qRT-PCR (Figure 6). Among them, VfLRR-RLK260 and VfLRR-RLK159 showed similar expression patterns in all seven tissues of V. fordii. Both showed higher transcript levels in leaves and kernels and lower transcript levels in roots, stems, buds, and ovaries. However, compared with VfLRR-RLK260, VfLRR-RLK159 was more strongly expressed in petals, suggesting that it may have a special function in floral development. VfLRR-RLK2 was expressed in roots, stems, and leaves and strongly expressed in petals, but not in vascular tissues. VfLRR-RLK172 was expressed most strongly in petals, followed by leaves, but expressed at low levels in the other tissues. VfLRR-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, and VfLRR-RLK11 in roots. Both VfLRR-RLK7 and VfLRR-RLK256 were specifically expressed in kernels. Together, these results suggest that VfLRR-RLKs play various roles in the development of tung tree.

Compared with VfLRR-RLKs, most VmLRR-RLKs showed higher transcript levels in the seven tissues analyzed. Six VmLRR-RLKs (VmLRR-RLK18, VmLRR-RLK29, VmLRR-RLK202, VmLRR-RLK30, VmLRR-RLK178, and VmLRR-RLK210) showed the same expression patterns as their orthologous genes in V. fordii. This may indicate that they share the same function in V. fordii and V. montana. The other five VmLRR-RLKs showed different expression patterns in V. montana. VmLRR-RLK111 was mainly expressed in leaves and had similar transcript levels in other tissues. VmLRR-RLK164 and VmLRR-RLK241 showed peak expression in kernels, but VmLRR-RLK164 was also expressed at high levels in the stems. Both VmLRR-RLK17 and VmLRR-RLK206 showed the highest transcript levels in roots and lower levels in other tissues. The different expression patterns in V. montana may reflect functional differentiation during evolution.

Among the seven pairs of orthologous genes showing similar trends in gene expression in V. montana and V. fordii in response to Fusarium infection, three pairs also showed similar expression patterns in the tissues (VfLRR-RLK2/VmLRR-RLK18, VfLRR-RLK9/VmLRR-RLK29, and VfLRR-RLK260/VmLRR-RLK202). The other four pairs showed different expression patterns in the seven tissues analyzed. Of the four pairs of orthologous genes showing opposite responses to Fusarium infection in V. montana and V. fordii (Figure 5), three pairs showed similar expression patterns in the tissues, and one pair (VfLRR-RLK256 and VmLRR-RLK206) showed different expression patterns in the tissues (Figure 6). There were high transcript levels of VfLRR-RLK256 in kernels and VmLRR-RLK206 in the roots. Given that the Fusarium pathogen invades via the roots of tung tree, these results suggest that VmLRR-RLK206 may play a role in resistance to Fusarium wilt disease.

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 of VfLRR-RLKs and VmLRR-RLKs in different tissues and in response to Fusarium infection were conducted. Characterization of LRR-RLK genes in a ligneous oil plant will improve our understanding of the evolutionary processes and functions of this gene superfamily. The results of this study provide important information for further research on the diversity and functions of the LRR-RLK gene family in tung tree.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Huiping Zhu and Yangdong Wang contributed equally to the paper.


The work was supported by the National Natural Science Foundation of China (31200485).

Supplementary Materials

Supplementary Fig.1 Multiple alignment of RLK domain for LRR-RLK family from V. fordii and V. montana. The multiple alignment was performed using Clustal X v.1.83 with the default settings, and the figure was output by DNAMAN v.5.5.2. Residues highlighted in different color represent different amino acids identity, respectively. Amino acid sequences of RLK domain used were list in supplementary Data Set 1.

Supplementary Fig.2 Phylogenetic tree based on the RLK sequences of VfLRR-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). Amino acid sequences of RLK domain used were list in supplementary Data Set 1.

Supplementary Fig.3 Phylogenetic tree based on the RLK sequences of 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). Amino acid sequences of RLK domain used were list in supplementary Data Set 1.

Supplementary Data Set 1 Amino acid sequences of RLK domain from V. fordii , V. Montana and A. Thaliana. All of the amino acid sequences used to conduct multiple sequence alignments and construction of phylogenetic trees were listed in this data set.

Supplementary Data Set 2 Amino acid sequences for motif analysis. All of the amino acid sequences used to perform motif analysis were listed in this data set.

  1. Supplemantary Material


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