Transcriptional profiling of ESTs responsive to Rhizobium vitis from ‘Tamnara’ grapevines (Vitis sp.)

Journal of Plant Physiology 167 (2010) 1084–1092

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Journal of Plant Physiology journal homepage: www.elsevier.de/jplph

Transcriptional profiling of ESTs responsive to Rhizobium vitis from ‘Tamnara’ grapevines (Vitis sp.) Youn Jung Choi a , Hae Keun Yun b,∗ , Kyo Sun Park a , Jeong Ho Noh a , Youn Young Heo a , Seung Hui Kim a , Dae Won Kim c , Hee Jae Lee d,e a

Fruits Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 440-706, Republic of Korea Department of Horticultural Science, Yeungnam University, Gyeongsan 712-749, Republic of Korea c Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea d Department of Horticultural Science, Seoul National University, Seoul 151-921, Republic of Korea e Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 22 September 2009 Received in revised form 24 February 2010 Accepted 24 February 2010 Keywords: Crown gall disease Defense response genes EST-derived SSR markers EST mapping

a b s t r a c t Genes related with defense responses were screened from the cDNA library constructed with Rhizobium vitis-inoculated or salicylic acid (SA)-treated ‘Tamnara’ grapevine (Vitis sp.) leaves. Among 13,728 expressed sequence tags (ESTs) from ‘Tamnara’ grapevine upon R. vitis inoculation and SA treatment, 6776 unigenes containing 1915 contigs and 4860 singletons were obtained. In gene ontology analysis, there were about 3200 clones related with biological process, 3555 with molecular function, and 3354 with cellular component genes. Proteins of secretory organ (35%), plasma membrane (30%), endoplasmic reticulum (20%), and vacuole (11%) were predicted. Photosynthesis-related genes and defense-related genes were most abundant. Among ESTs, 199 resistance-related ones were mapped to the genome of Vitis vinifera L. with three markers, GLP1–12, MHD98, and MHD145, which are known to be linked to resistance against powdery mildew. Approximately, 120 simple sequence repeats (SSRs) detected in cDNAs could be used as EST-derived SSR markers in disease resistant grape breeding. © 2010 Elsevier GmbH. All rights reserved.

1. Introduction Crown gall disease, caused by Rhizobium vitis, reduces the yield and vigor of grapevine, especially on Vitis vinifera L. and interspecific hybrids where climatic conditions favor freeze injury (Burr et al., 1998; Schroth et al., 1988). Incidence of crown gall disease was high on V. vinifera L. cultivars, but low on V. labrasca L. and hybrid cultivars (Burr et al., 1998; Park et al., 2000). ‘Tamnara’ grapevines, bred from the cross between ‘Campbell Early’ and ‘Himrod Seedless’, are moderately resistant to R. vitis as determined by controlled inoculations (Park et al., 2004). The inheritance of complex characteristics can be elucidated by establishing their association with linked molecular markers, and identified candidate genes can be available for improving of traditional grapevine cultivars after further functional characterization. Although whole genome sequence of grapevines was completed

Abbreviations: AOS, active oxygen species; CAT, catalase; CHS, chalcone synthase; ESTs, expressed sequence tags; GO, gene ontology; GST, glutathione Stransferase; LOX, lipoxygenase; PESTAS, Pipeline of the EST Analysis Service; PR, pathogenesis-related; PS, photosystem; LRR, leucine rich repeat; RuBP, ribulose1,5-bisphosphate; SA, salicylic acid; TGICL, TIGR Gene Indices Clustering Tools. ∗ Corresponding author. Tel.: +82 53 810 2942; fax: +82 53 810 4659. E-mail address: [email protected] (H.K. Yun). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.02.005

(Jaillon et al., 2007), the information of infection and control of R. vitis remains to be elucidated. Through the profiling expressed sequence tags (ESTs), at the transcriptional level with the marker selection, the efficiency of disease resistant grape breeding is expected to be improved (Doligez et al., 2002; Fischer et al., 2004). Levels of polymorphism and transferability tested by ten grape EST database-derived simple sequence repeats (SSRs) and their availabilities for breeding grapevines resistant to diseases were reported (Scott et al., 2000). In this study, genes related with defense responses were screened and discovered from cDNA library constructed with R. vitis-inoculated or salicylic acid (SA)-treated ‘Tamnara’ grapevine leaves using Pipeline of the EST Analysis Service (PESTAS) system of Genome Research Center (GRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB) (Nam et al., 2009), and transcriptional profiling of ESTs responsive to R. vitis from the grapevine leaves was constructed. 2. Materials and methods 2.1. Plant and pathogen materials ‘Tamnara’ grapevines grown in a greenhouse at 25–30 ◦ C under natural light were sprayed with 0.5 mM salicylic acid (SA) on leaves,

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Table 1 Transcriptome features of ‘Tamnara’ grapevines. Total reads

Total knowna

Total no. of assembled b

Sequenced

High quality sequence

Singleton

Contig

Total

BLASTX

InterPro Scan

Total

13,728

13,191

4860

1915 (883 bp)c

6776 (818 bp)d

6397 (1576)e

4835 (14)f

6411

a b c d e f

BLASTX cutoff E-value, 1e−5 ; InterPro Scan cutoff E-value, 0.01. Cutoff (Phred score 20 and sequence length 100 bp). Average size of contigs. Average size of total ESTs. Annotated BLASTX only. Annotated InterPro Scan only.

and inoculated with Rhizobium vitis Cheonan 493 (Yun et al., 2003). For the inoculation, the R. vitis strain was grown in LB medium and the bacterial cultures were diluted to OD600 approximately 1 (109 cfu/mL). The cultures were injected into the holes drilled on the internode of the grapevines and 0.5 mM SA was treated on both sides of the grapevine leaves. Leaves were harvested at 1, 3, 6, 12, 24, 48, and 72 h after SA treatment and R. vitis inoculation, and then immediately frozen in liquid N2 and stored at −80 ◦ C until used for RNA extraction. All harvested samples from each treatment were pooled and used for RNA extraction, screening differential expression of cDNA, and reverse transcription polymerase chain reaction analysis.

2.4. RNA slot blot analysis Total RNAs of 5 ␮g were used and the RNA mixtures were denatured at 65 ◦ C for 10 min and then blotted membranes using the Bio-Dot SF (Bio-Rad, Hercules, CA, USA). RNA samples were transferred and immobilized to Hybond-N+ nylon membrane with a UV-crosslinker. The digoxigenin (DIG) labelled cDNA probes were generated from total RNA with DIG 11-dUTP (Roche, Penzberg, Germany) and AMV reverse transcriptase. DIG Easy Hyb, DIG Wash and Block Buffer set, and CDP-Star, Ready-to-Use (Roche, Penzberg, Germany) were used for hybridization, washing, and detection. Detected blots were exposed to X-ray films and analysed with a BioRad GS-800 densitometer and Quanity one software (Bio-Rad, Munich, Germany).

2.2. RNA extraction and cDNA library construction Total RNA was extracted separately from R. vitis-inoculated, SAtreated, and control leaves using the modified pine tree method of removing polysaccharides and phenolic compounds (Chang et al., 1993). Total RNAs of all time courses, 0.5, 1, 3, 6, 12, 24, 48, and 72 h, were collected in each tube for each series of the SAtreated and the R. vitis-inoculated grapevines. cDNA libraries were constructed using ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA, USA) from total mRNA isolated from the R. vitis-inoculated leaves.

2.3. Expressed sequence tag (EST) sequencing and data analysis ESTs were sequenced with 3730 Big Dye Terminator v3.1 Cycle Sequencing Kit and Sequencer 3730XL DNA Analyzer (Applied Biosystem, Foster City, CA, USA). PESTAS was used for analyzing the ESTs from the R. vitis-inoculated and the SA-treated leaves. PESTAS is available at http://pestas.kribb.re.kr/. Supplementary information is available at http://pestas.kribb.re.kr/pestas.jsp (Nam et al., 2009). EST sequences were pre-processed using Phred with 20 of cutoff value to extract high quality regions from raw sequence data. Cross Match and SeqClean were used for vector and contaminant trimming, respectively. After repeat masking with the RepeatMasker, EST sequences were clustered and assembled into contigs and singletons to reduce the inherent redundancy and to build unigenes sets, using the TIGR Gene Indices Clustering Tools (TGICL). At each step, ESTs of less than 100 bp length were removed. To obtain more relevant functional annotation results, Blast2GO was used (Götz et al., 2008). The functional annotation was performed using the BLAST search in PESTAS system, InterPro Scan, SignalP, PSORT II, and TMHMM-based algorisms. The BLAST search and InterPro Scan were performed setting an E-value less than 1e−5 and 0.01, respectively. Simple sequence repeats (SSRs) in the cDNA of ‘Tamnara’ grapevines were detected using Tandem Repeat Finder program with basic parameters (Benson, 1999). A worldwide web server interface http://atc3.biomass.mssm.edu/ trf.html has been established for automated use of the program.

Fig. 1. Distribution of nucleotide (A) and amino acid (B) length for 6776 unigenes induced by R. vitis-inoculated and SA-treated ‘Tamnara’ grapevines.

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2.5. Whole genome mapping of defense-related genes The sim4 is efficiently aligning the transcribed and spliced DNA sequence with a genomic sequence containing ESTs, allowing for introns in the genomic sequence and a relatively small number of sequencing errors (Florea et al., 1998). For aligning ESTs from ‘Tamnara’ grapevines with a genomic DNA sequence of ‘Pinot Noir’ (Vitis vinifera L.) from genome of NCBI database (Accession Nos. NC 012007–NC 012025), sim4 was used. The threshold was set by identity >90%, and length coverage >90% in this study. This program can be obtained by anonymous ftp from http://globin.cse.psu.edu/ or over the worldwide web from http://globin.cse.psu.edu/.

3. Results

500 bp or longer than 2000 bp (Fig. 1A, Table 1). The average length of the proteins predicted from the cDNAs was 296 amino acids (Fig. 1B). There were 6446 proteins (95.2%) with 101–500 amino acid residues, 96 ones (1.4%) with <100 residues and 234 ones (3.4%) with >500 residues. As shown in Table 1, about 6397 and 4835 of ESTs were annotated with BLASTX and InterPro Scan, respectively. Among 4821 ESTs annotated under both databases, 1576 of ESTs were annotated only with BLASTX, and 14 with Interpro Scan. Based on an E-value cutoff of ≤1e−5 , 0.01 of BLASTX and Interpro Scan, 94% (6411) of the ‘Tamnara’ grapevine unigenes were significantly similar to the database. Approximately 20% (1355) of these protein homologues were annotated as no hit, hypothetical, or expressed proteins, while the remaining ones (5421) corresponded to proteins with putatively known functions in Blast2GO analysis. Approximately, 80% of the 6776 ESTs were annotated on the GenBank database.

3.1. Sequencing and assembly 3.2. Functional annotation of ESTs from ‘Tamnara’ grapevines The unique ESTs from the cDNA library were summarized in Table 1. A total of 13,191 high-quality EST sequences (about 96.1%) were generated from the 13,728 ESTs. The ESTs were assembled into about 6776 unique sequences including 1915 contig and 4861 singleton ESTs. The percentage of unique sequences was 49.4%, with 50.6% of transcript redundancy in the ESTs after infection by R. vitis (Table 1). The average lengths of the EST and contigs were 818 and 883 bp, respectively; 5658 (83.5%) transcripts had 501–1000 nucleotides, and 340 (5%) genes were either shorter than

BLASTX and InterPro Scan were conducted against the GenBank database to assign putative identity to the Vitis unigene set. Annotated function of ESTs from R. vitis-inoculated ‘Tamnara’ grapevines was assigned by mapping unigenes on gene ontology (GO) structure using the TGICL. Unigenes with assigned putative functions were classified into three ontologies, namely, molecular function, biological process, and cellular component by controlled GO vocabulary. In total, 6776 unigenes including 1915 contigs and 4861

Fig. 2. Distribution of ‘Tamnara’ grapevine unigenes with putative functions assigned through GO annotation at level 2. (A) Molecular function; (B) biological process; (C) cellular component. Assignments were based on the data available at TGICL.

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Fig. 3. Distribution of ‘Tamnara’ grapevine unigenes with putative functions assigned through GO annotation. (A) Biological process; (B) molecular function; (C) cellular component. Assignments were based on the data available at TGICL at level 3.

singletons were annotated to one or more ontologies, with multiple assignments into different functional categories based on GO (Ashburner et al., 2000). A total of 5062 clusters to 11 main molecular functional categories, about 8112 clusters to 19 main biological process categories and 10,935 clusters to 11 main cellular component categories were assigned at level 2 (Fig. 2). Of the biological process categories, cellular (31.3%) and metabolic process (30.7%) were contained the most clusters. Majority of the assignments were to the cellular metabolic, primary metabolic, and macromolecule metabolic processes. Response to stimulus was one of the most abundant assignments under the biological process categories, which contained responses to abiotic stimulus, stress, and

chemical stimulus, suggesting that a significant portion of the ESTs were produced from tissues subjected to biotic stresses by R. vitis inoculation in ‘Tamnara’ grapevines (Figs. 2A and 3A). The molecular functional categories of catalytic activity (39.9%) and binding proteins (45.5%) contained the most clusters. The sub-categories of transferase activity, hydrolase activity, ion binding, and protein binding proteins were most abundant in the catalytic activity and binding protein category, respectively (Figs. 2B and 3B). The largest groups mapped into cellular component ontologies were assigned into the cell (21.1%) and cell part (20.7%) categories, with the subgroup of intracellular, intracellular part, and intracellular organelle (Figs. 2C and 3C).

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Table 2 The most redundant 35 ESTs induced by R. vitis inoculation in ‘Tamnara’ grapevine leaves. Cluster ID EPP163JWAA

No. of hits/cluster

Accession ID

E-value

Blast2GO annotation

12C000002 12C000003 12C000725 12C000722 12C000007 12C000735 12C000009 12C000005 12C000726 12C000730 12C000724 12S000051 12C000748 12S000171 12C000731 12C000738 12C001387 12C000733 12C000747 12C000745 12C000746 12C000782 12C000753 12C000752 12S000565 12C000008 12C000757 12C000762 12C000761 12C000754 12C000756 12C000744 12C000771 12C000770

310/4 152/8 139/2 95/1 93/1 86/2 75/1 74/1 65/1 56/3 51/2 50/50 48/2 48/48 47/1 43/1 42/40 40/2 38/2 38/1 37/1 35/3 35/3 34/1 31/31 29/2 27/1 26/1 26/1 26/1 25/2 25/1 24/2 24/1

CAO21606 CAN64291 CAN78872 CAO71299 CAN73057 P51118/100 CAO69160/ CAN71283 CAO49140 CAN74445 CAO38909 CAO17917 CAO15830 CAN75566 ABO21382 ABM67586 CAO61870 CAO47624 CAN71620 CAO48253 CAO48206 CAO65723 CAN79041 CAO43062 CAO66169 CAO66917 NP 001053957 CAN77841 CAN60921 CAO64440 CAN78901 ABC86744 CAN79984 CAO47393

0 8.01e−92 0 3.43e−40 3.19e−154 0 0 1.8e−69 4.97e−73 0 0 2.49e−46 0 1.74e−132 2.97e−88 0 1.49e−17 8.22e−41 9.87e−130 0 0 1.86e−45 0 0 3.26e−147 3.14e−153 0 1.04e−155 2.96e−154 1.57e−81 0 5.68e−21 2.84e−84 0

Chloroplast RuBP carboxylase oxygenase activase large protein isoform RuBP carboxylase oxygenase small subunit At3g26650 mlj15 5 Histone h1 Light harvesting chlorophyll a/b binding protein Glutamine synthetase Chloroplast RuBP carboxylase oxygenase activase small protein isoform Ubiquitin extension protein At3g26650 mlj15 5 Fructose bisphosphate aldolase Vtc2-like protein PSI type III chlorophyll a/b binding protein Aspartyl protease family protein Chlorophyll a/b binding protein Fructose-bisphosphate aldolase CHS PSII protein Glycine-rich protein Chloroplast light harvesting complex II protein Fructose bisphosphate aldolase Ferredoxin-NADP reductase Af104392 1extensin-like protein Desaturase-like protein At5g28840 f7p1 20 Lhca2 protein Chlorophyll a/b binding protein Polyubiquitin Hypothetical protein Cyclase family protein ␤-1,3-Glucanase Phosphoribulokinase precursor Abscisic stress ripening protein Chloroplast chlorophyll a/b binding protein 4 Hydroxypyruvate reductase

3.3. Highly expressed genes, protein domains, and enzymes of ESTs derived from ‘Tamnara’ grapevines A total of 6776 unigenes of the 13,728 ESTs from cDNA library of R. vitis-inoculated ‘Tamnara’ grapevine leaves were assembled and annotated using Blast2GO analysis (Götz et al., 2008). Most of the highly represented ESTs were photosynthesis- and carbon fixation-related genes such as ribulose-1,5-bisphosphate

(RuBP) carboxylase oxygenase small chain precursor, light harvesting chlorophyll a/b binding protein, and fructose bisphosphate aldolase by Blast2GO analysis. Type 2 metallothionine, chalcone synthase (CHS), and endo-1,3-␤-glucanase genes were also highly annotated to defense response against R. vitis inoculation and SA treatment (Table 2). Most of the highly expressed EST contigs consisted of 1–4 clusters, but hits and clusters 12S000051, 12S000171, 12C001387, and 12S000565 of EPP163JWAA series were similar in

Table 3 The first 25 highly expressed domains induced by R. vitis inoculation in ‘Tamnara’ grapevine leaves. Cluster ID EPP163JWAA

InterPro Scan ID

Description

No. of hits

12C000372 12C000373 12C000433 12C000802 12C000972 12C001242 12C001338 12C001483 12C001753 12S000258 12S000821 12S001737 12S001909 12S003333 12S003399 12S003591 12S003922 12S004061 12S005737 12S005975 12S008366 12S008876 12S009095 12S009463 12S009917

IPR001344 IPR011009 IPR001611 IPR000504 IPR011046 IPR016024 IPR001128 IPR016040 IPR001841 IPR002885 IPR017442 IPR002110 IPR002213 IPR013210 IPR000608 IPR001993 IPR001810 IPR001471 IPR015655 IPR006447 IPR002182 IPR002085 IPR002198 IPR012336 IPR001087

Chlorophyll a/b binding protein Protein kinase-like LRR RNA recognition motif, RNP-1 WD40 repeat-like Armadillo-type fold Cytochrome P450 NAD(P)-binding Zn finger, RING-type Pentatricopeptide repeat Serine/threonine protein kinase-related Ankyrin UDP-glucuronosyl/UDP-glucosyltransferase LRR, N-terminal Ubiquitin-conjugating enzyme, E2 Mitochondrial substrate carrier Cyclin-like F-box PR transcriptional factor and ERF, DNA-binding Protein phosphatase 2C MYB-like DNA-binding region, SHAQKYF class NB-ARC Alcohol dehydrogenase superfamily, Zn-containing Short-chain dehydrogenase/reductase Thioredoxin-like fold Lipase

188 135 63 58 57 48 43 40 36 36 34 33 32 30 30 29 28 27 25 24 24 23 23 23 22

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Table 4 The most abundant EC number analyzed with UniprotKB in R. vitis-inoculated ‘Tamnara’ grapevine leaves. EC no.

Enzyme

Organism

No. of hits

Unique enzyme

4.1.1.39 2.7.11.1 6.3.2.4.1.2.13 1.2.1.13 4.2.1.1 3.6.1.-

RuBP carboxylase oxygenase small chain Shaggy-related protein kinase iota Ubiquitin carrier protein Fructose-bisphosphate aldolase Glyceraldehyde-3-phosphate dehydrogenase B, chloroplast precursor Carbonic anhydrase Putative SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 3-like 1 Glutamine synthetase cytosolic isozyme 2 Peptidyl-prolyl cis–trans isomerase ATP synthase subunit ␤ CHS ADP-ribosylation factor 1 Ferredoxin-NADP reductase GDP-mannose 3,5-epimerase 1 Serine/threonine protein phosphatase Serine hydroxymethyltransferase Probable xyloglucan glycosyltransferase 9 Phosphoribulokinase Malate dehydrogenase Mg-protoporphyrin IX monomethyl ester cyclase, chloroplast precursor S-Adenosylmethionine synthetase Transketolase, chloroplast precursor Sucrose synthase GST ␤-1,3-Glucanase Cell division protease ftsH homolog 9, chloroplast precursor 3-Ketoacyl-CoA synthase 21 Peroxiredoxin Q, chloroplast precursor Putative L-ascorbate peroxidase 6

V. vinifera A. thaliana V. vinifera V. vinifera Pisum sativum Populus trichocarpa A. thaliana

322 162 104 101 92 74 70

8 90 63 9 7 5 50

63 60 52 44 40 37 35 31 31 31 30 30 30 28 28 27 27 26 26 26 26 25

8 28 16 4 2 1 3 23 4 26 4 3 5 4 3 3 13 11 11 8 5 6

6.3.1.2 5.2.1.8 3.6.3.14 2.3.1.74 3.6.5.2 1.18.1.2 5.1.3.18 3.1.3.16 2.1.2.1 2.4.1.2.7.1.19 1.1.1.37 1.14.13.81 2.5.1.6 2.2.1.1 2.4.1.13 2.5.1.18 3.2.1.39 3.4.24.2.3.1.1.11.1.15 1.11.1.11

their number (Table 2). These EST clones were highly homologous among the aligned sequence, but low level of sequence coverage formed large number of clusters. The cluster 12S000051 was annotated photosystem (PS) I type III chlorophyll a/b binding protein, and the 50 of counted EST contigs composed of 50 clusters. However, their aligned sequences of approximately 200 bp in length were correlated with the conserved domain region of the reference sequence of the BLAST search. Under domain analysis with InterPro Scan, chlorophyll a/b binding protein domain was the most abundantly and highly represented ESTs (Table 3). In the protein

V. vinifera V. vinifera V. vinifera V. vinifera Medicago truncatula V. vinifera Oryza sativa subsp. Japonica V. vinifera V. vinifera O. sativa subsp. Japonica V. vinifera V. vinifera Gossypium hirsutum V. vinifera Solanum tuberosum Coffea canephora Persea americana A. thaliana A. thaliana A. thaliana Populus jackii A. thaliana

domain analysis with InterPro Scan, resistance and defense-related domains, such as leucine rich repeat (LRR), cytochrome P450, Zn finger, and serine/threonine protein kinase-related, were highly expressed in ESTs generated from R. vitis-inoculated and SA-treated ‘Tamnara’ grapevine leaves. Thirty highly represented enzymes are shown in Table 4. Most of the highly redundant enzymes were also related to photosynthesis such as RuBP carboxylase small chain and shaggy-related protein kinase iota. Highly annotated defense response-related enzymes such as CHS, serine/threonine protein phosphatase, putative glucan endo-1,3-␤-glucosidase 11 precursor cell, and putative l-ascorbate peroxidase 6 were significantly homologous to with those of V. vinifera L. and Arabidopsis thaliana L. Enzymes including defense-related such as LRR, cytochrome P450, Zn finger, pathogenesis-related (PR) transcriptional factor and ERF, DNA binding, and MYB-liked DNA binding region were highly annotated (Table 4). Proteins of secretory organs (35%), plasma membrane (30%), endoplasmic reticulum (20%), vacuole (11%), and others (1%) were predicted to be deduced in ‘Tamnara’ grapevine ESTs using the bio-informatics tools of SignalP, TMHMM, and PSORT II for the subcellular localization of putative proteins (Fig. 4). 3.4. Defense-related ESTs from R. vitis-inoculated and SA-treated ‘Tamnara’ grapevine leaves

Fig. 4. Distribution of predicted subcellular localization of 6776 unigene products analyzed with PSORT II in ‘Tamnara’ grapevines induced by R. vitis inoculation.

The cDNA library showed diverse transcripts following R. vitis inoculation and SA treatment. Out of 6776 unigenes, the 426 ones (6.3%) responsive to R. vitis and SA were categorized into 7 defense-related groups encoding proteins functioning in defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell wall fortification, and transcription factors related defense responses (Table 5). Several PR proteins are known to be highly expressed during R. vitis invasions. Among them, proteins related with plant defense responses and wound responsive ones such as ␤-1,3-glucanase, chitinase, thaumatin-like protein, PR-10, and proline-rich protein were shown to be induced in this library. The expression of signal transduction-related genes such

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Table 5 Defense-related genes expressed in R. vitis-inoculated and SA-treated ‘Tamnara’ grapevines. Gene

Homology

No. of hits

Glucan endo-␤-glucosidase 3 expressed ␤-1,3-Glucanase Chitinase Thaumatin-like protein Wound responsive protein Hypersensitive-induced response protein CHS PAL

99 9 2 14 9 8 5 46 6

LOX NBS-LRR type disease resistance protein

32 17 15

CAT Glutathione peroxidase Methionine sulfoxide reductase GST Ferritin subunit precursor

57 13 8 6 27 3

Cyanogenic glucosidase Chytochrome P450 Serine carboxypeptidase Flavanone 3-hydroxylase Methyltransferase Cytochrome b5

75 1 25 21 17 7 4

Hydroxyproline-rich glycoprotein Extensin-like protein Cinnamyl alcohol dehydrogenase

32 7 17 8

Drought-induced protein 1 Heat shock

44 2 42

WRKY MYB

87 25 62

Defense

Signal transduction

AOS

Secondary metabolism

Cell wall fortification

Abiotic stress

Transcription factor

as, lipoxygenase (LOX) and LRR containing disease resistance genes, active oxygen species (AOS)-related genes, such as catalase (CAT), glutathione peroxidase, methionine sulfoxide reductase, and glutathione S-transferase (GST), were also induced following R. vitis inoculation and SA treatment. Cell wall fortification-related genes such as hydroxyproline-rich glycoprotein, extension-like protein, and cinnamyl alcohol dehydrogenase, and abiotic stress genes such as drought-induced protein 1, and heat shock proteins, and defense-related transcription factors such as WRKY and MYB were most abundant (Table 5). 3.5. RNA slot blot analysis To determine expression of selected defense-related genes in response to mechanical wounding and R. vitis inoculation, selected ESTs were used as probes for RNA slot blot analysis at multiple time points (Fig. 5). PR-genes such as ␤-1,3-glucanase showed the similar expression pattern at 0.5–72 h by R. vitis inoculation and wound treatment. Transcripts of proline-rich protein increased the expression at 0.5, 3, and 24–72 h after R. vitis inoculation. Chalcone synthase and LOX were induced 1–6 h after R. vitis inoculation, but not significantly by wounding. 3.6. Mapping to V. vinifera L. chromosome of defense-related ESTs and detection of SSRs in ‘Tamnara’ grapevines A total of 199 ESTs related with defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell wall

Fig. 5. RNA slot blot hybridization analysis with (A) proline-rich protein, (B) CHS (chalcone synthase), (C) ␤-1,3-G (␤-1,3-glucanase), and (D) LOX (lipoxygenase) as a respective probe in ‘Tamnara’ grapevine cultivars. C, control; R, R. vitis inoculation; W, wound.

fortification, and transcription factors were aligned against V. vinifera whole genome sequence by sim4 analysis (Fig. 6). Genome sequence of ‘Pinot Noir’ grapevine in GenBank (chromosome 1–19, reference assembly based on 8× WGS), whole genome shotgun sequence, and Accession Nos. NC 012007–NC 012025) were used for reference sequence, unigenes indiscriminately mapped chloromosomes 1–19. GLP1–12, MHD98, and MHD145, known to control of powdery mildew, located the surrounding of Run1 (Barker et al., 2005; Donald et al., 2002) were located on 13.6, 15.3, and 16.7 Mbp of the chromosome 12, respectively. About 700 SSR regions were found in the ‘Tamnara’ grapevine cDNAs. Among them, approximately 100 SSRs of 2–5 nt were detected in the cDNA of the ‘Tamnara’ grapevine ESTs, along with information on repeat size and composition were sorted (Supplementary Table 1). 4. Discussion By use of PESTAS, among 13,728 ESTs from the ‘Tamnara’ grapevine cDNA library upon R. vitis inoculation and SA treatment, 6776 unigenes were obtained. Induction of the defense-, signal transduction-, AOS-, secondary metabolism-, cell wall fortification, and various abiotic stress transcription factors-related genes suggested that defense responses were activated successfully by R. vitis inoculation and SA treatment. AOS-related genes by R. vitis inoculation and SA treatment contributes to the structural reinforcement of plant cell wall (Alvarez et al., 1998), the regulation of benzoic acid-2 hydroxylase enzyme activity (Léon et al., 1995), and the activation of some protection mechanisms (Levine et al., 1994). Anand et al. (2008) reported that SA as well as SA-mediated defense genes were involved in crown gall disease resistance mechanism in SA-mediated NPR1 gene-silenced tobacco plants forming large tumors following R. vitis inoculation. In this study, induction of SA-dependant PR genes and LOX genes suggested that SA signaling was involved in defense responses in R. vitis-inoculated ‘Tamnara’ grapevines. Abiotic stress genes, heat shock proteins and WRKY and MYB transcription factors have been reported to have important functions in target genes expression by wound, pathogen attack, fungal elicitors, and SA (Eulgem et al., 2000; Lee and Vierling, 2000; Sugimoto et al., 2000). Wound responsive ESTs are also considered to have important function in defense response in R.

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Fig. 6. Sequence-based mapping of defense-related ESTs induced by R. vitis inoculation in ‘Tamnara’ grapevines onto V. vinifera L. pseudochromosome sequence (V. vinifera L. chromosomes 1–19, reference assembly (based on 8× WGS), whole genome shotgun sequence, Accession Nos. NC 012007–NC 012025). Resistant gene analog markers GLP1-12, MHD98, and MHD145 were underlined on the chromosome 12.

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vitis-inoculated and SA-treated ‘Tamnara’ grapevines. As overlapping of pathogen and wound-related genes might be the interactions among the wounding, abiotic stress, and hormonal responses in A. thaliana L. (Cheong et al., 2002), wound-inducible genes seem to be caused not only by wounding but also by R. vitis inoculation. Of the annotated ESTs, 199 ones involved in defense responses were mapped to the genome of V. vinifera L. with the markers, known to phenotypic loci of disease resistance. GLP1–12, MHD98, and MHD145 are known to be linked to resistance against powdery mildew with the SSR markers, VVIV67 and UDV-015 (Akkurt et al., 2007; Barker et al., 2005; Donald et al., 2002). rgVrip064, a resistance gene analog marker for downy mildew resistance, was identified with three SSR markers, VMC8d11, VrZag62, and UDV-082 (Di Gaspero and Cipriani, 2002; Zyprian et al., 2005). The STS-AA6 marker was reported in the same region of PdR1, Pierce’s disease resistance locus in grapevines (Krivanek et al., 2006). Approximately, 700 SSR candidates were detected in the cDNA of ‘Tamnara’ grapevines using tandem repeat finder. EST-SSRs being applicable to studies at several taxonomic levels, a large number of SSRs (approximately 1000) to be available from an expanded EST database of 45,000 had many potential applications in mapping and identity research (Scott et al., 2000). If EST-derived SSRs are developed as gene-based markers, they are useful for selection of disease resistance hybrid lines or newly developed grape line. The EST collections generated in this study provide useful information for characterizing genes involved in defense responses against crown gall disease. Studies for differential expression screening using microarray analyses of genes targeted specifically to the defense responses will provide useful data in understanding of defenses in grapevines. Therefore, we are planning to carry out further expression analysis for these clones using cDNA microarray, to obtain new information about disease resistance pathway in grapevines. Discovery of specifically expressed genes against R. vitis or new promising candidate genes with potential roles in defense response will be useful for breeding grapevines resistant to diseases by using them as selection markers in crossing and as genes improving characteristics in transformation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jplph.2010.02.005. References Akkurt M, Welter L, Maul E, Töpfer R, Zyprian E. Development of SCAR markers linked to powdery mildew (Uncinulla necator) resistance in grapevine (Vitis vinifera L. and Vitis sp.). Mol Breed 2007;19:103–11. Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C. Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 1998;92:773–84. Anand A, Uppalapati SR, Ryu CM, Allen SN, Kang L, Tang Y, et al. Salicylic acid and systemic acquired resistance play a role in attenuating crown gall

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