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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2015) 39: 775-789© TÜBİTAKdoi:10.3906/biy-1502-38

Analysis of structure and differential expression of Pseudomonas syringae 5-like(RPS5-like) genes in pathogen-infected Vitis flexuosa

Md. Zaherul ISLAM, Soon Young AHN, Hae Keun YUN*Department of Horticulture and Life Science, Yeungnam University, Gyeongsan, Republic of Korea

* Correspondence: haekeun@ynu.ac.kr

1. IntroductionGrapevine (Vitis spp.) is grown worldwide and exposed to many diseases caused by fungi, bacteria, and viruses (Wang et al., 2011). The diseases caused by fungi, which include anthracnose by Elsinoe ampelina (Kim et al., 2008; Kono et al., 2009), grey mold by Botrytis cinerea (Gullino, 1992; De Waard et al., 1993; Barka et al., 2000), and ripe rot by Colletotrichum gloeosporioides (Lee, 1962) and C. acutatum (Melksham et al., 2002; Hong et al., 2008), are destructive and affect fruits, vegetables, ornamental plants, and even field crops. These diseases cause significant economic losses during the production of grape berries. Grape crown gall, caused by the bacterium Rhizobium vitis, is a common and devastating disease that leads to reduced growth and yield by obstructing vascular tissue and restricting the movement of water and nutrients into the vine above the tumor (Eastwell et al., 2006).

Disease resistance (R) genes are thought to confer resistance to specific diseases caused by fungi, bacteria, viruses, oomycetes, or nematodes (Hammond-Kosack and Jones, 2000; Taler et al., 2004). The products of R-genes

act as receptors that specifically bind ligands encoded by the corresponding pathogen avirulence factors in a gene during the gene recognition process (Baker et al., 1997; Hammond-Kosack and Jones, 1997). Interaction between an R-gene product and a pathogen avirulence factor triggers downstream signaling pathways, subsequently activating plant defense mechanisms (Hammond-Kosack and Jones, 1996). Several resistant states such as oxidative burst, cell-wall strengthening, induction of defense gene expression, and rapid cell death at the site of infection (hypersensitive response) occur in downstream cellular events (Morel and Dangl, 1997). These local hypersensitive responses can trigger long-lasting systemic responses (systemic acquired resistance (SAR)) that prime the plant for resistance against a broad spectrum of pathogens (Dong, 2001; Metraux, 2001).

Recognized major classes of R-genes are as follows: nucleotide binding site–leucine-rich repeats (NBS-LRRs); extracellular LRRs anchored to the transmembrane domain; receptor like kinase (RLK); a membrane protein domain (TrD) fused to a putative coiled-coil (CC) domain;

Abstract: Nucleotide binding site-leucine-rich repeat (NBS-LRR) genes are the largest gene resistance family in plants. Resistance to Pseudomonas syringae 5 (RPS5) is a member of the NBS-LRR family. RPS5 and its homologs are important disease resistance genes in plants including Arabidopsis. This study identified nine loci of RPS5-like genes in Vitis flexuosa that showed differential expression from transcriptome analysis by next generation sequencing (NGS) of V. flexuosa infected with Elsinoe ampelina based on structural analyses: VfRPS5-like1059 (Vitis flexuosa resistance to Pseudomonas syringae 5-like 1059), VfRPS5-like1833, VfRPS5-like4135, VfRPS5-like4833, VfRPS5-like6172, VfRPS5-like13564, VfRPS5-like20585, VfRPS5-like55532, and VfRPS5-like62178. These genes, 2236–4762 bp in length, exhibited the structure of a coiled coil-nucleotide binding site-leucine-rich repeat (CC-NBS-LRR) resistance gene. The predicted amino acid sequences of all nine genes contained typical NBS domains. Chromosomal localization revealed that the nine genes were located in six chromosomes of the V. flexuosa pseudo-genome. Of the nine loci, only VfRPS5-like55532 showed upregulated expression against all tested pathogens at all tested time points, except 24 h postinoculation (hpi) for E. ampelina. All tested genes also showed differential expression against major grapevine fungal and bacterial pathogens. The results presented herein will also serve as a useful reference dataset for functional analysis and utilization in molecular breeding of resistant grapevines.

Key words: R-gene, Vitis, resistance to Pseudomonas syringae 5, Elsinoe ampelina, Rhizobium vitis, Botrytis cinerea, Colletotrichum gloesporioides, Colletotrichum acutatum, CC-NBS-LRR

Received: 13.02.2015 Accepted/Published Online: 01.06.2015 Printed: 30.09.2015

Research Article

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extracellular LRRs, along with transmembrane, fused to a PEST (Pro-Glu-Ser-Thr) domain; and enzymatic R-genes containing intracellular Helminthosporium carbonum toxin reductase enzyme (Joshi and Nayak, 2011; Gururani et al., 2012). NBS-LRR genes, which comprise the largest class of R-genes, are thought to be involved in the recognition of specific pathogen effectors and activation of defense responses (Kortekamp et al., 2008). More than 50 novel NBS-LRR class resistance genes have been isolated and characterized in different plant species (Joshi and Nayak, 2011). For example, the Arabidopsis genome sequence contains 149 NBS-LRR genes (Meyers et al., 2003), the rice genome contains more than 500 NBS coding sequences (Bai et al., 2002; Meyers et al., 2002; Zhou et al., 2004), and the apple genome contains 1015 NBS-LRR genes (Arya et al., 2014). Furthermore, NBS-LRR class R-genes have been categorized into three major classes based on the N-terminus motif: TIR-NBS-LRR (TNL), which contains a domain resembling the drosophila toll and mammalian interleukin-1 receptors (TIR); CC-NBS-LRR (CNL), which contains a coiled-coil (CC) or a leucine-zipper (LZ) motif; and the nonmotif group (Meyers et al., 1999; Pan et al., 2000; Liu et al., 2007; Joshi and Nayak, 2011). The CC structures consist of two or more α-helices with a superhelical twist (Lupas, 1996), and CC-type NBS R-genes are widespread in both dicots and monocots (Pan et al., 2000; Meyers et al., 2003; Monosi et al., 2004). The CC structures are important for protein–protein interactions (Nooren et al., 1999; Burkhard et al., 2001). Specifically, these domains play an important role in R-Avr specific recognition to activate downstream defense signaling in the nonrace specific disease resistance 1 (NDR1) mediated signaling pathway (Shirasu et al., 2003). The NBS domain plays a significant role in plant defense signaling. In plant R-proteins, the NBS region is a conserved domain that is responsible for binding and hydrolysis of ATP and GTP (Tameling et al., 2002). The NBS domains contain several conserved motifs, with kinase 1a or P-loop, kinase 2, kinase 3a, and GLPL being the most common motifs (Traut, 1994; Meyers et al., 1999; Pan et al., 2000). The kinase 1a motif with the consensus sequence of GXXXXGK (T/S) acts as a phosphate binding motif. The kinase 2 motif contains four consecutive hydrophobic amino acids followed by a conserved aspartate (D) residue that coordinates the metal ion on Mg-ATP. The kinase 3a motif is involved in purine or ribose binding (Traut, 1994). Typically, an LRR is composed of around 20–30 amino acids with a characteristic repetitive sequence pattern unusually rich in the hydrophobic amino acid leucine, which is found in all life forms (Bella et al., 2008). The leucine-rich repeat (LRR) plays a role in protein phosphorylation and protein–protein interactions (Kobe and Deisenhofer, 1994; Staskawicz et al., 1995). Arabidopsis resistance to

Pseudomonas syringae 5 (RPS5) gene belongs to the CC-NBS-LRR gene family. RPS5 mediates resistance against the bacterial pathogen Pseudomonas syringae, and its protein contains a putative NBS domain composed of kinase-1a (or P-loop), kinase-2a and kinase-3a subdomains (Saraste et al., 1990; Traut, 1994; Grant et al., 1995), 13 imperfect LRRs in the C-terminal region, and a potential leucine zipper at the N-terminal region (Warren et al., 1998).

The most extensively cultivated grape species, V. vinifera L., is highly susceptible to many diseases caused by fungi, bacteria, and viruses (Wang et al., 2011), whereas wild cultivars such as V. riparia, V. rupestris, and V. rotundifolia are highly resistant to several important grapevine diseases (Eibach et al., 1989; Kortekamp et al., 2008; Reisch et al., 2012). A Korean wild species, Vitis flexuosa, was reported to have a number of RPS5-like genes with different expression levels that imparted resistance to grape anthracnose caused by Elsinoe ampelina (Ahn et al., 2014). Therefore, in this study, we identified and characterized nine RPS5-like genes in Vitis flexuosa by sequence analysis. Specifically, we conducted structural analysis, phylogenetic analysis, homology analysis, and conserved motif analysis and evaluated the chromosomal distribution and gene duplication. We also characterized the gene expression pattern of nine VfRPS5-like genes against several fungi (B. cinerea, C. gloeosporioides, C. acutatum, and E. ampelina) and a bacterium (R. vitis).

2. Materials and methods2.1. Plant materials and pathogensGrapevines (V. flexuosa VISKO001, native to Korea) were maintained in a greenhouse at Yeungnam University, Gyeongsan. Leaves were subjected to pathogen infection with one of the following virulent strains for gene expression analysis: E. ampelina (EA-1), B. cinerea (B1035), or C. gloeosporioides (Co563-1), which were isolated from infected grapes by Dr WK Kim of the National Academy of Agricultural Science, RDA, Korea, and R. vitis strain Cheonan 493, which was kindly provided by Prof JS Cha, Chungbuk National University, Korea.2.2. Inoculation of pathogensSeveral colonies of E. ampelina were transferred to Fries liquid medium and incubated in a shaking incubator (140 rpm) at 28 °C for 21 days. Pellets of cultures were then harvested by centrifugation, ground with a homogenizer in sterile distilled water, poured onto V-8 juice agar medium [20% (v/v) V-8 juice, 2% (w/v) agar] and incubated at 28 °C under a near ultraviolet lamp for 2 days to produce spores (Yun et al., 2007). Spores of E. ampelina were collected by scraping off the plates with sterile distilled water. The concentration of the spore suspension was adjusted to 106 spores/mL and then sprayed onto leaves. After growing mycelia on potato dextrose agar [PDA; potato starch

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(4 g), dextrose (20 g), and agar (15 g/L)], B. cinerea was subjected to fluorescent light (12 h light followed by 12 h dark) for around 10 days. The spores were then collected by scraping off the plates with 0.24% potato dextrose broth (PDB) solution, after which the spore suspensions were adjusted to concentrations of 106 spores/mL and sprayed onto leaves. C. gloeosporioides and C. acutatum were cultivated on PDA and V-8 juice media, respectively, under continuous irradiation of fluorescent light for 3 days. The spores were collected by scraping the plates with sterile distilled water and then used to treat samples, as described above. For bacterial inoculation, bacteria was grown on PDA media in the dark for 3 days, after which a single colony was transferred to YEP medium (yeast extract (10 g), bacto peptone (5 g), NaCl 5 g/L, and pH 7.0) and grown at 28 °C in a shaking incubator for 16 to 18 h. The samples were then spun down by centrifugation and resuspended with sterile water (OD600 = 1). Next, the leaves were injured slightly with a pencil tip and inoculated by dropping 20 μL of R. vitis cell suspensions onto the wounded portion of the leaves. Leaves inoculated with pathogens were subsequently incubated in a moist box at 22–28 °C for 48 h. The leaves were harvested at different time points [0, 1, 6, 12, 24, and 48 hours postinoculation (hpi)], immediately frozen in liquid nitrogen, and stored at −80 °C until subsequent analysis.

2.3. RNA isolation and real-time PCR analysisLeaves of samples were ground in liquid nitrogen using a mortar and pestle; then total RNA was extracted using the modified pine tree method (Chang et al., 1993). RNA quality was measured based on the absorbance at 230, 260, and 280 nm using a Nano Drop spectrophotometer (ACTGene ASP-3700; ACTGene, Inc., Piscataway, NJ, USA). First-strand cDNA was used as a template for PCR and was synthesized from the total RNA (500 ng) using a GoScriptTM reverse transcription system (Promega, Madison, WI, USA). Real-time PCR was performed on a C1000TM thermal cycler (BioRad, Hercules, CA, USA) using SYBR Premix Ex Taq (TaKaRa Bio Inc., Osaka, Japan) as the fluorescent dye. Amplification was conducted by subjecting the samples to 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Transcript levels were calculated using the standard-curve method and normalized against the grapevine actin gene (AB372563) as an internal control with nontreated leaves (at time zero) as a reference. In addition, melting curves of the amplified products were recorded. For each gene, the reference sample was defined as the 1x expression level, and the results were expressed as the fold increase in mRNA level over the reference sample. To reduce error, experiments were replicated three times. The gene-specific primers shown in Table 1 were used for real-time PCR in this experiment.

Table 1. Primers specific for VfRPS5-like genes used for real-time PCR.

Gene Primers Sequences

VfRPS5-like1059FP CTCTCCTTCCCGTAGAGCCAGTRP ACAAATCTGAACGACGTGGGGA

VfRPS5-like1833FP TCAGCTCACATCTCCACAAAGCTRP TCCCCTGGCAGAGAAGTAGAAA

VfRPS5-like4135FP TGCAAGCTTCAAGAAAATCGGGTRP CTCAGCCACATCCACCTTCCTC

VfRPS5-like4832FP TCGCAGGAAGAGCAAGTGTTGTRP GGCTGCCCAACATTCAGGAATT

VfRPS5-like6172FP TCCCCTCCATAGCCCTTTGTCARP GCAAAGGAGCAGAGGGGAATAG

VfRPS5-like13564FP ACACCGGCCCTCAAACAAATCARP CGTCCCTTTCAAACGTAGTAAAGG

VfRPS5-like20585FP CCCTTCCAACCTTTGACCACCARP GAGCTTGACGATTAATTGAGGC

VfRPS5-like55532FP TGAATTTGCTGCACTGAAGACGARP GAAGCGCTAGTATGGGAAGATG

VfRPS5-like62178FP AGAGATCAAAACCAGCAGCAGGARP TGGAGCAGAGAGATGGTGTTGA

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2.4. Sequence analysis of genesTranslation software (http://web.expasy.org/translate/) was used to translate the nucleotide sequences of genes to amino acid sequences. The primary structures of the genes were analyzed using the ExPasy protParam (http://web.expasy.org/protparam/). Secondary structure analysis was performed by SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html), and multiple alignment of protein sequences was performed using ClustalW (http://www.genome.jp/tools/clustalw/). The VfRPS5-like genes were blasted against each other using the NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to check for gene duplication events. Typical domains were analyzed using the web tool SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1) (Letunic et al., 2009). Positional information describing the nine VfRPS5-like genes was obtained from the grape database (http://112.220.192.2/grape/index.php/campbell/blast), with option 20130831_sequence_removeUn.fa. 2.5. Phylogenetic and conserved motif analysisHomologous gene sequences were collected from the NCBI database using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). BLASTp program was used and the database ‘‘nr’’ was selected. A phylogenetic neighbor-joining tree of gene sequences was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) version 6.0 software (Saitou and Nei, 1987; Tamura et al., 2013). Tree branches were evaluated using the bootstrap method (Felsenstein, 1985). To observe the structural divergence of VfRPS5-like genes, the conserved motifs in the encoded proteins were analyzed using the Multiple Expectation Maximization for Motif Elicitation (MEME) online program (http://meme.sdsc.edu/meme/intro.html). The optimized parameters of MEME were employed as follows: maximum number of motifs, 10; minimum motif width, 10; and maximum motif width, 50.

3. Results and discussion3.1. Identification and sequence analysis of RPS5-like genes in grapevineTranscriptome analysis by NGS of V. flexuosa VISKO001 infected with E. ampelina (Ahn et al., 2014) generated nucleotide sequences from 21 RPS5-like genes. Domain analyses of all protein sequences of these genes were then conducted using the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1) to confirm the RPS5-like gene structure in V. flexuosa. Finally, we identified nine RPS5-like genes in Vitis flexuosa that were confirmed to contain the CC, NBS, and LRR domains (Figure 1). In this study, these genes were characterized and referred to as VfRPS5-like1059 (Vitis

flexuosa resistance to Pseudomonas syringae 5-like 1059), VfRPS5-like1833, VfRPS5-like4135, VfRPS5-like4833, VfRPS5-like6172, VfRPS5-like13564, VfRPS5-like20585, VfRPS5-like55532, and VfRPS5-like62178 and deposited in the National Agricultural Biotechnology Information Center (NABIC), Rural Development Administration, Korea, under accession numbers NS-0699-000001, NS-0700-000001, NS-0701-000001, NS-0702-000001, NS-0703-000001, NS-0704-000001, NS-0705-000001, NS-0706-000001, and NS-0707-000002, respectively. Gene duplication events of nine VfRPS5-like genes were then investigated by comparing them to each other using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Among the predicted amino acid sequences of nine genes, the highest similarity (68%) was observed between VfRPS5-like55532 and VfRPS5-like62178 (Table 2). According to Kong et al. (2013), gene duplication has occurred when the similarity of aligned regions covers more than 80%, and the homology of the aligned region is greater than 80%; therefore, no duplicated genes were found among the nine genes tested in V. flexuosa. The primary structure and characteristics of the nine loci of the RPS5-like gene in V. flexuosa were analyzed using several bioinformatic tools (Table 3). The size of the nine VfRPS5-like genes varied from 2236 to 4762 bp, encoding 593 to 1561 amino acids (66.49 to 177.58 kDa), while the predicted isoelectric points ranged from 5.32 to 6.46. Two out of nine proteins predicted from nucleotide sequences of the VfRPS5-like gene showed an instability index of less than 40 (Table 3), indicating that they were stable based on the instability index (II) (Guruprasad et al., 1990). The secondary structures of the predicted proteins from the nine loci of the VfRPS5-like gene were analyzed by the self-optimized prediction method with alignment (SOPMA) (Table 4). The results showed that random coil, alpha helix, beta turn, and extended strands ranged from 20.35%–26.31%, 47.70%–56.40%, 6.54%–9.31%, and 15.90%–19.09%, respectively. The prediction suggested that alpha helices and random coils are abundant structural elements of nine VfRPS5-like proteins, in which random coils are important elements that influence protein flexibility and stabilization of protein folding (Sharmin et al., 2011). Figure 1 shows the results of domain analysis of proteins predicted from nine VfRPS5-like gene sequences conducted using the Simple Modular Architecture Research Tool (SMART). Domain organization showed that all predicted proteins contained the nucleotide binding site (NBS) domain in the conserved region, which ranged from 269 to 285 amino acid residues (Table 5), corresponding to about 30–32 kDa. The NBS domain of the resistance (R) gene contains several conserved motifs (Traut, 1994; Pan et al., 2000; Kortekamp et al., 2008). A typical NBS domain of the R-gene consists of the consensus GXXGXGKTT for the

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S

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VfRPS5-like13564 NBS CC

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VfRPS5-like20585 NBS CC

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VfRPS5-like55532 NBS CC

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VfRPS5-like62178 NBS CC

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Figure 1. Structure of the VfRPS5-like protein predicted by SMART (including the COILS program and TMHMM2). CC indicates coiled-coil, NBS indicates nucleotide binding site, and LRR indicates leucine-rich repeat domains. M and asterisk indicate start codon and stop codon, respectively.

Table 2. Identities (%) of deduced amino acids based on alignment of nine VfRPS5-like genes of Vitis flexuosa.

1 2 3 4 5 6 7 8 9

1 32 31 27 28 31 50 63 62

2 32 30 30 29 66 31 33 30

3 31 30 30 27 28 28 28 27

4 27 30 30 41 28 26 26 27

5 28 29 27 41 28 30 28 27

6 31 66 28 28 28 27 29 28

7 50 31 28 26 30 27 57 60

8 63 33 28 26 28 29 57 68

9 62 30 27 27 27 28 59 68

1 = VfRPS5-like1059, 2 = VfRPS5-like1833, 3 = VfRPS5-like4135, 4 = VfRPS5-like4832, 5 = VfRPS5-like6172, 6 = VfRPS5-like13564, 7 = VfRPS5-like20585, 8 = VfRPS5-like55532, and 9 = VfRPS5-like62178.

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Table 3. Primary structure analysis of nine VfRPS5-like genes of Vitis flexuosa.

Gene Nucleotide length (bp)

Proteinlength (aa) MWa (KDa) pIb Instability

index (II)

VfRPS5-like1059 4689 1562 177.70 6.44 44.45

VfRPS5-like1833 2112 703 81.06 5.99 50.27

VfRPS5-like4135 2814 901 102.19 5.84 42.49

VfRPS5-like4832 3660 1063 120.07 5.32 50.08

VfRPS5-like6172 1785 594 66.60 5.41 32.89

VfRPS5-like13564 3971 1032 119.84 5.89 53.90

VfRPS5-like20585 3328 965 111.05 6.46 37.24

VfRPS5-like55532 3291 968 111.67 5.80 45.53

VfRPS5-like62178 3702 982 111.96 6.30 41.58aMW, molecular weight; bpI, isoelectric point.

Table 4. Secondary structure analysis of nine predicted proteins from the VfRPS5-like gene.

Gene Random coil Alpha helix Beta turn Extended strand

VfRPS5-like1059 26.31% 47.70% 8.19% 17.80%

VfRPS5-like1833 21.34% 56.19% 6.54% 15.93%

VfRPS5-like4135 24.86% 47.72% 8.99% 18.42%

VfRPS5-like4832 23.24% 51.55% 9.31% 15.90%

VfRPS5-like6172 23.91% 50.84% 8.08% 17.17%

VfRPS5-like13564 23.16% 51.16% 6.59% 19.09%

VfRPS5-like20585 20.83% 55.54% 7.36% 16.27%

VfRPS5-like55532 20.35% 56.40% 7.23% 16.01%

VfRPS5-like62178 23.52% 52.04% 7.84% 16.60%

Table 5. In silico analysis of VfRPS5-like genes of Vitis flexuosa.

Gene Strand Chromosomeno.

N-terminal CC NBS domain

Start End Start End

VfRPS5-like1059 + 12 891 919 979 1257

VfRPS5-like1833 + 19 29 96 150 429

VfRPS5-like4135 + 09 31 66 172 456

VfRPS5-like4832 – 11 24 53 158 439

VfRPS5-like6172 + 14 26 61 153 421

VfRPS5-like13564 + 19 21 85 143 424

VfRPS5-like20585 + 06 29 63 146 429

VfRPS5-like55532 – 12 18 52 144 421

VfRPS5-like62178 + 12 35 52 159 435

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kinase 1a or phosphate binding loop (P-loop) motif, while the kinase 2 motif contains four consecutive hydrophobic amino acids followed by two invariant, or nearly invariant, aspartates, which coordinate the metal ion binding (Traut, 1994; Pan et al., 2000). Multiple alignment of NBS domains of the predicted proteins of nine VfRPS5-like genes, and the characterized resistance proteins, Arabidopsis RPS5

and RFL1, showed that all conserved motifs were present, including the most important and common motifs: kinase 1a, kinase 2, and GLPL (Figure 2) (Traut, 1994; Meyers et al., 1999; Pan et al., 2000). Therefore, all nine genes tested in this study contain typical NBS domains that function as ATP and GTP binding sites (Tameling et al., 2002). The SMART program also detected the formation of

Figure 2. Multiple alignment of nucleotide binding site (NBS) domains of nine loci of the VfRPS5-like gene and two Arabidopsis NBS encoding genes. The common motifs are denoted under the motif. Vf1, Vf2, Vf3, Vf4, Vf5, Vf6, Vf7, Vf8, Vf9, AT1, and AT2 indicate VfRPS5-like1059, VfRPS5-like11833, VfRPS5-like4135, VfRPS5-like4832, VfRPS5-like6172, VfRPS5-like13564, VfRPS5-like20585, VfRPS5-like55532, VfRPS5-like62178, Arabidopsis RPS5, and Arabidopsis RFL1, respectively.

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coiled-coil structures at the N-terminal and LRR domain in the C-terminal that were involved in protein-protein interactions (Kobe and Deisenhofer, 1994; Staskawicz et al., 1995; Nooren et al., 1999; Burkhard et al., 2001) in all nine genes (Figure 1). Among the analyzed genes, six showed common structural features of NBS-LRR genes, and 3 genes showed somewhat unusual configurations. VfRPS5-like1059 contained extra 2 LRRs close to the CC domain in N-terminal. VfRPS5-like13564 and VfRPS5-like55532 commonly had one extra CC domain after the LRR domains. This unusual structural configuration of genes was also found in the NCBI database. The homologous sequence (accession: CBI29658.3) (Jaillon et al., 2007) of VfRPS5-like1059 contained one extra NBS domain and three extra LRR domains preceding the CC domain. The homologous sequences of VfRPS5-like13564 (accession: CAN61769.1) and VfRPS5-like55532 (accession: CBI29675.3) contained an extra CC domain

in their C-terminal parts (Jaillon et al., 2007; Velasco et al., 2007). However, structural information pertaining to the nine genes generated by domain analysis and multiple alignments showed that they all belonged to the CC-NBS-LRR class R-genes (Figures 1 and 2).3.2. Phylogenetic analysis, motif composition, and homology study of RPS5-like genes in V. flexuosaHomologous protein sequences of VfRPS5-like genes were collected from the NCBI database using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic trees were then created from full-length amino acid sequences and NBS domain sequences using 28 sequences, including 9 VfRPS5-like genes and a human Apaf1 gene as an out-group sequence, by neighbor-joining using MEGA6 to investigate the relationships among VfRPS5-like and other Vitis species proteins (Figures 3a and 3b). In the phylogenetic tree of full-length sequences, investigated genes were divided into three groups, with

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VfRPS5-like1059 Vv(CBI29658.3) Vv(CBI29675.3) VfRPS5-like55532 Vv(CAN71735.1) Vv(CBI29677.3) VfRPS5-like62178 Vv(CBI29678.3) VfRPS5-like20585 Vv(CAN60524.1) VfRPS5-like1833 Vv(CBI20133.3) Vv (XP_002283414.1) Vv(CAN61769.1) Vv(XP_002283439.2) VfRPS5-like13564 Vv (XP_002281742.2) Vv(CBI36278.3) VfRPS5-like4135 Vv(XP_002267120.1) VfRPS5-like4832 Vv (XP_002263288.1) Vv(XP_002263579.2) VfRPS5-like6172 Vv (XP_003633688.1) Vv(XP_002263761.2) Vv(CBI22343.3) Apaf 1 (H. sapience)

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Figure 3. Phylogenetic analysis of nine predicted proteins (a) and NBS domains (b) from the VfRPS5-like gene with Vitis homologous proteins. The phylogenetic trees were generated using Mega 6. Node numbers indicate bootstrap values from 1000 replications. A human Apaf1 sequence was used as the out-group (Li et al., 1997).

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VfRPS5-like1059, VfRPS5-like20585, VfRPS5-like155532, and VfRPS5-like62178 clustered into group I; VfRPS5-like1833 and VfRPS5-like13564 in group II; and VfRPS5-like4135, VfRPS5-like4832, and VfRPS5-like6172 in group III. The distribution of different motifs of VfRPS5-like genes supported the phylogenetic groups. In most cases, members in the same group showed common motif compositions. Genes such as VfRPS5-like1059, VfRPS5-like20585, VfRPS5-like155532, and VfRPS5-like62178 included in phylogenetic group I commonly contained all described motifs. While motif 7 is partially present in VfRPS5-like1833 and absent from VfRPS5-like13564, motifs 8, 9, and 10 were absent from both genes, which fell into phylogenetic group II. Motifs 1, 2, 3, 6, and 7 were found in VfRPS5-like4832 and VfRPS5-like6172, which were included in phylogenetic group III; motifs 8 and 9

were not present in any of these genes (Figures 3a and 4; Table 6). In the phylogenetic tree constructed from NBS domains, all tested genes were clustered into 3 major groups, which is similar to the tree from proteins (Figure 3b).

A homology study of VfRPS5-like genes was also performed using BLAST searches of the NCBI database. The deduced amino acid sequences from the nine loci of the RPS5-like gene in V. flexuosa showed a high level of similarity with protein sequences of other plant species. All loci of the VfRPS5-like gene were highly homologous with the protein sequences that originated from V. vinifera, with identities of greater than 68% and E values of 0.0, indicating their relatively conserved evolutionary relationship at the protein level (Table 7). A search of positional information of the VfRPS5-like genes was carried out using the grape

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Figure 4. Schematic representation of motif compositions in the VfRPS5-like protein sequences. Different motifs, numbered 1–10 ( , motif 1; , motif 2; , motif 3; , motif 4; , motif 5; , motif 6; , motif 7; , motif 8; , motif 9; , motif 10), are displayed in different boxes. The names of all members are displayed on the left-hand side, while the bottom scale indicates the length of motifs.

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database (http://112.220.192.2/grape/index.php/campbell/blast). In silico chromosomal localization of VfRPS5-like genes showed that nine genes were distributed among six chromosomes of the V. flexuosa pseudo-genome (Table 5), with VfRPS5-like1059, VfRPS5-like55532, and VfRPS5-like62178 positioned on chromosome 12; VfRPS5-like1833 and VfRPS5-like13564 on chromosome 19; and VfRPS5-like4135, VfRPS5-like4832, VfRPS5-like6172, and VfRPS5-like20585 on chromosomes 9, 11, 14, and 6, respectively. Overall, the results of this study revealed that nine loci of the tested VfRPS5-like genes were distributed on six of the 19 chromosomes of the V. flexuosa pseudo-genome.3.3. Expression analysis of VfRPS5-like genesThe expression patterns of the VfRPS5-like genes were analyzed by quantitative real-time PCR using primers specific for each gene and confirmed by showing a single

peak in melting curve analysis (data not shown). To evaluate the activity of VfRPS5-like genes against disease attacks, leaves of V. flexuosa were infected with pathogenic fungi (E. ampelina, B. cinerea, C. acutatum, and C. gloeosporioides) and a pathogenic bacterium (R. vitis) and then incubated and collected at different time points. Expression levels of tested genes were evaluated as relative expression compared to uninoculated control samples. The expression levels of VfRPS5-like1059 against five pathogens are shown in Figure 5a. VfRPS5-like1059 was downregulated in response to all pathogen inoculation at all tested time points except 1 hpi, while it was only upregulated in response to B. cinerea at 1 hpi. VfRPS5-like1833 was downregulated in response to all pathogens at all time points except B. cinerea, under which it was upregulated only at 1 and 12 hpi (Figure 5b). VfRPS5-

Table 6. Best possible matched sequences of motifs (1–10) presented in Figure 4.

Motif no. Best possible matched sequence Length (aa)

1 VWSCLEDEQIRRIGVWGMGGTGKTTIMTHI 30

2 NNYLTAIPNFFFQHMHSLRVLDLHGTNIPSLPPSISNLICLRGLYLNHCP 50

3 DARDRGHAILDALINVCLLERSDKGKCVKMHDVIRDMALKISSQNHDSKF 50

4 KPCFLYCALFPEDYEIYIDYLIECWIAEGFI 31

5 NNHQDVAHMFDHVYWVTVSKEWSTKKLQDAIMQRLK 36

6 IEPIAEQVVKECGGLPLAIDTVARTM 26

7 PCEGLQDFPDHEEWEQATRISLMNNQICTLPETLHC 36

8 FTFQFAVGYHDSTYSHILESFDYPSYNC 28

9 WPSLQRIKISTCHMLKRLPFNNANAAKLRFIEGQQSWWEALVWEDDAIKQ 50

10 SIWQGPIHAGSLAQLTTLTLYKCPQLKKIFSNGMIQQLPHLQHLRVEECN 50

Table 7. Homology of nine loci of the RPS5-like gene from Vitis flexuosa.

Gene Top matched clones Top homologous species Identity (%) Query cover (%) E value

VfRPS5-like1059 CBI29658.3 Vitis vinifera 85 100 0.00

VfRPS5-like1833 XP_002283414.1 Vitis vinifera 68 99 0.00

VfRPS5-like4135 XP_002281742.2 Vitis vinifera 93 99 0.00

VfRPS5-like4832 XP_002263288.1 Vitis vinifera 98 100 0.00

VfRPS5-like6172 XP_003633688.1 Vitis vinifera 91 100 0.00

VfRPS5-like13564 XP_002283439.2 Vitis vinifera 87 97 0.00

VfRPS5-like20585 CAN60524.1 Vitis vinifera 75 95 0.00

VfRPS5-like55532 CBI29675.3 Vitis vinifera 99 100 0.00

VfRPS5-like62178 CBI29678.3 Vitis vinifera 97 85 0.00

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like4135 was upregulated and downregulated at all tested time points in response to infection with E. ampelina and C. acutatum, respectively (Figure 5c). VfRPS5-like4832 was upregulated at all time-points in response to E. ampelina and B. cinerea, whereas it was downregulated at all time-points in response to C. gloeosporioides (Figure 5d). VfRPS5-like6172 showed downregulated expression from 6 to 48 hpi in response to all pathogens, except R. vitis, which induced upregulation at all tested time

points except 1 hpi (Figure 5e). VfRPS5-like13564 was upregulated by B. cinerea inoculation and downregulated in response to R. vitis inoculation at all tested time points (Figure 5f). VfRPS5-like20585 was upregulated under E. ampelina, B. cinerea, and C. gloeosporioides inoculation and downregulated under R. vitis inoculation at all time points (Figure 5g). VfRPS5-like55532 was upregulated at all tested time points in response to all pathogens except 24 hpi under E. ampelina. The highest expression of this gene

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Figure 5. Expression pattern of nine RPS5-like genes of Vitis flexuosa against infection by Elsinoe ampelina ( ), Botrytis cinerea ( ), Colletotrichum acutatum ( ), Colletotrichum gloeosporioides ( ), and Rhizobium vitis ( ). a) VfRPS5-like1059, b) VfRPS5-like1833, c) VfRPS5-like4135, d) VfRPS5-like4832, e) VfRPS5-like6172, f) VfRPS5-like13564, g) VfRPS5-like20585, h) VfRPS5-like55532, and i) VfRPS5-like62178. The error bars represent the standard deviation of the means of three independent replicates.

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was around 1.6-fold greater than that of the control against E. ampelina at 6 hpi (Figure 5h). VfRPS5-like62178 were upregulated at all time points except 48 hpi in response to C. gloeosporioides (Figure 5i).

Defense responses against most pathogens are generally mediated by initiating signal pathways of disease resistance (R) genes in plants. Most R-genes encode proteins containing an N-terminal signaling domain, a nucleotide binding site, and a series of C-terminal leucine-rich repeats. These NBS-LRR genes confer resistance to infection by specific pathogen strains. The NBS domain plays a significant role in plant defense signaling. One class of NBS-LRR genes, CC-NBS-LRR, plays an important role in disease resistance. The Arabidopsis RPS5 gene, which belongs to the CC-NBS-LRR gene family, mediates recognition of the Pseudomonas syringae effector protein, AvrPphB. This gene is activated by AvrPphB-mediated cleavage of the protein kinase PBS1 (Qi et al., 2012). In uninfected cells, RPS5 is generally bound to PBS1 through the CC domain and ADP through the NBS domain so that it is primed to respond to a pathogen attack. PBS1 is cleaved by AvrPphB of Pseudomonas syringae, resulting in a conformational change that leads to the exchange of ATP for ADP. This ATP-bound form of RPS5 engages in downstream signaling molecules that activate the defense response (Ade et al., 2007; Qi et al., 2012). More than 50 NBS-LRR R-genes, including the almost 40 CC-NBS-LRR, conferred resistance against several pathogens in plants such as Arabidopsis, tomato, potato, barley, rice, maize, apple, grape, lettuce, pepper, sunflower, melon, and soybean (Kortekamp et al., 2008; Joshi and Nayak, 2011; Kar et al., 2013; Arya et al., 2014).

To date, there have been few reports describing R-gene expression in plants. Tan et al. (2007) found that expression of most NBS-LRR encoding and related genes was not significantly changed under defense responses or by treatment with signaling molecules, salicylic acid (SA), or jasmonic acid (JA) in a microarray analysis of Arabidopsis. RNA profiling experiments also failed to detect differential expression of R-genes (Maleck et al., 2000; Tao et al., 2003). However, Yoshimura et al. (1998) showed that expression of resistance gene, Xa1, was induced by bacterial inoculation in rice. He et al. (2013) also demonstrated that expression level of ME137 increased in rice 5 days after inoculation with Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. Gong et al. (2013) reported that expression of the wheat CC-NBS-LRR family gene, TdRGA-7Ba, was induced after challenge by Erysiphe graminis f. sp. Tritici, the pathogen of powdery mildew disease. Arya et al. (2014) reported that expression of 18 out of 26 Malus × domestica NBS genes (MdNBS gene) was induced, while expression of other MdNBS genes was not observed under attacks of several pathogens and insects. Kortekamp et

al. (2008) also reported that the CC-NBS-LRR family gene, VRP1, was induced during pathogen attack in the resistant grapevine cultivar ‘Gloire de Montpellier’. It has been reported that CzR1, a CC-NBS-LRR R-gene found in turmeric plants, conferred resistance against Pythium aphanidermatum via upregulated expression under pathogen challenge (Kar et al., 2013). Similarly, transcripts of powdery mildew resistance genes, Mla6 and Mla13, were induced after inoculation of Blumeria graminis f.sp. hordei (Halterman et al., 2003). The expression of R-genes is essential to initiate various signal transductions required for the resistant and defensive responses against pathogen attacks in plants. Although many R-genes were not known to be significantly induced by pathogen attacks in many plants, expressions of some R-genes were induced under pathogen infection in plants. Investigation of expression of R-genes can provide meaningful information for elucidating their function in resistance responses against pathogen infection in plants. In a previous study (Ahn et al., 2014), many candidate RPS5 genes showing different expression levels were preliminarily selected from the transcriptome of V. flexuosa infected with E. ampelina by NGS. Therefore, we selected nine genes to analyze their differential expressions against various pathogens in V. flexuosa grapevines.

In this study, the molecular structure was analyzed to detect all common domains in all nine VfRPS5-like genes based on their predicted amino acid sequences in V. flexuosa. Typical NBS domains with CC and LRR domains were detected by the SMART program in all nine genes tested in this study. In the expression study, all VfRPS5-like genes were expressed in response to infection by all pathogens. Different VfRPS5-like genes exhibited different expression patterns in response to pathogen infections at various times. Although expression levels in the genes tested in this study were not higher, they showed differential expression patterns. The response of all tested VfRPS5-like genes after pathogen infection indicated that they might play a role in defense pathways against pathogen infection in grapevine.

Biotic stress resistance in plants is an important issue, and R-genes are a major weapon used by plants to protect themselves against disease. The existence of R-genes in plants is highly desirable for sustainable crop production. NBS-LRR encoding genes are the most important and largest class of R-genes that enhance resistance responses against various diseases and insect pests. In this study, we identified nine nonduplicated RPS5-like genes from transcriptome analysis by NGS of V. flexuosa inoculated with E. ampelina. These were aligned on six different chromosomes of the V. flexuosa pseudo-genome. Structural analysis and a comparison study confirmed that these genes all belonged to the CC-NBS-LRR class of R-genes. Moreover,

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these genes were found to share high homology with other NBS-encoding genes. We also investigated the expression profile of these VfRPS5-like genes in response to fungal and bacterial pathogen infection through real-time PCR using gene-specific primers. All tested genes showed differential expression in response to the inoculated pathogens, indicating their multicapability in plant resistance responses

against different pathogens in grapevine. The information presented herein will be useful for further characterization of Vitis defense response at the molecular level.

AcknowledgmentThis work was supported by a 2014 Yeungnam University research grant.

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