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Journal of Plant Biotechnology 2016; 43(1): 37-48

Published online March 31, 2016

https://doi.org/10.5010/JPB.2016.43.1.37

© The Korean Society of Plant Biotechnology

Transcriptional profiles of Rhizobium vitis-inoculated and salicylic acid-treated ‘Tamnara’ grapevines based on microarray analysis

Youn Jung Choi1, and Hae Keun Yun2,*

1National Institute for Horticultural and Herbal Science, Wanju 55365, Korea,
2Department of Horticulture and Life Science, Yeungnam University, Gyeonsan 38541, Korea

Correspondence to : e-mail: haekeun@ynu.ac.kr

Received: 3 March 2016; Revised: 3 March 2016; Accepted: 9 March 2016

The transcriptional profiles of ‘Tamnara’ grapevine (Vitis labruscana L.) to Rhizobium vitis were determined using 12,000 gene oligonucleotide microarray chips constructed with 6,776 unigenes based on the EST sequencing. Among them, 95 clones were up-regulated more than three times and 90 were down-regulated more than 5-times in the R. vitis-inoculated grapevines relative to the control vines. Treatment of salicylic acid showed that 337 clones were upregulated and 52 clones were down regulated in grapevines. Microarray analysis, reverse transcription-polymer chain reaction, and slot blot hybridization analysis revealed that 5, 14, and 64 clones were up-regulated and 10, 12, and 61 clones were down-regulated in wounded, salicylic acid- treated, and R. vitis-inoculated ‘Tamnara’ grapevine leaves, respectively. The expression patterns of β-1,3-glucanase, proline-rich protein, and lipoxygenase genes of ‘Tamnara’ moderately resistant to R. vitis were similar to those of resistant ‘Concord’ and ‘Delaware’ grapevines. However, chalcone synthase genes in ‘Tamnara’ grapevines showed similar expression patterns to susceptible grapevines ‘Neomuscat’ and ‘Rizamat’. Further expression studies with various clones for each gene should be conducted to elucidate their roles in resistant responses against pathogens or other stimuli in grapevines. These results could provide better resources for understanding the mechanism of defense responses against crown gall disease and clues for identifying new genes that may play a role in defense against R. vitis in grapevines.

Keywords Grape, Gene expression, RT-PCR, Slot blot

Grape (Vitis sp.) is attacted to a number of bacterial, fungal, and viral diseases like other plants (Pearson and Goheen 1998). Crown gall is a major disease responsible for severe reduction of yield and poor quality of fruit in grape production regions throughout the world, including Korea (Burr et al. 1998; Park et al. 2000). Chemical or biological attempts to control crown gall disease conducted to date have failed, with the exception of Agrobacterium radiobacter strain K84 of the biological control (Anand et al. 2008; Burr and Otten 1999). However, A. radiobacter strain K84 is only effective at controlling crown gall caused by nopaline-type strains of A. tumefaciens and A. rhizogenes, while it has no effect on crown galls induced by Rhizobium vitis in grapevines (Kerr 1980). Since effective agro-chemicals to control crown gall have not been released, development of novel grapevine cultivars resistant to crown gall is critical and will be a useful tool in protection of grapevines from disease (Burr et al. 1988; Park et al. 2000; Stover et al. 1997).

There has been continuous study of genes related to disease resistance and defense response of grapevines to fungal, bacterial, and viral pathogens, including crown gall disease through comparative genomics, transcriptomics and the genome wide identification analysis for useful genes and molecular markers (Burr et al. 1998 and 2003; Choi et al. 2008; Hur et al. 2015). However, development of disease resistant grapes based on molecular biology has been limited because of the relatively low amount of genetic and molecular information available regarding genotypes resistant to certain diseases. Although the entire genome of Vitis vinifera ‘Pinot Noir’ has been sequenced (The France-Italian Public Consortium for Grapevine 2007) and annotated, the functions of many genes must be still investigated.

To develop new grape cultivars resistant to diseases, systematic monitoring of the mechanism of plant response and defense against pathogen attacks and more detailed functional studies of the selected differentially expressed genes (DEGs) are required. Microarray analysis, which can screen the expression patterns of many genes simultaneously in a single analysis, is considered a foundational technology capable of high-throughput and high-speed transcriptional profiling. Accordingly, this technique has various applications including unique gene identification and diagnostics of certain diseases (Schulze and Downward 2001; Stears 2003).

In this study, the gene expression patterns in response to R. vitis inoculation were investigated in R. vitis-inoculated, salicylic acid (SA)-treated, wounded, and untreated control leaves of ‘Tamnara’ grapevine which was bred in Korea (Park et al. 2004). Using microarray gene expression profiling, 6,776 unigenes of expressed sequence tag (EST)-based sequence were analyzed in grapevine leaves. The detailed expression patterns of selected up- and down-regulated genes in the microarray were confirmed by slot blot hybridization and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Expression of DEGs was also analyzed in grapevines resistant and susceptible to crown gall in response to R. vitis inoculation.

Plant materials and treatments

Grapevines of ‘Tamnara’ (moderately resistant to crown gall disease), ‘Delaware’ and ‘Concord’ (resistant to crown gall disease), and ‘Neomuscat’ and ‘Rizamat’ (susceptible to crown gall disease) were grown in a greenhouse at 25°C~30°C under natural light, then inoculated with R. vitis Cheonan 493 (Yun et al. 2003). Leaves were harvested at 1, 3, 6, 12, 24, 48, and 72 h after wound, SA treatment, and R. vitis inoculation, immediately frozen in liquid nitrogen, and stored at -80°C until used for RNA extraction. All samples harvested from each treatment were used for RNA extraction, analysis of differential expression of cDNA, and RT-PCR analysis.

Microarray chip construction

A total of 6,776 unigenes were obtained from the ‘Tamnara’ grapevine cDNA library constructed after R. vitis inoculation and SA treatment. Microarray chips were constructed with 35?40 nt of unigene based oligonucleotide. Gene specific oligonucleotides were arrayed onto a slide glass with an average of two replications and oligonucleotide microarray techniques were employed to detect R. vitis-responsive genes in ‘Tamnara’ grapevines.

RNA isolation and microarray hybridization

Total RNA was isolated from R. vitis-inoculated, SA-treated, and control grapevine leaves using the modified pine tree method of removing polysaccharides and phenolic compounds (Chang et al. 1993) with RNA extraction buffer consisting of 2% cetyltrimethylammonium bromide, 2% polyvinylpyrrolidone, 100 mM Tris-HCl (pH 8.0), 25 mM ethylenediaminetetraacetic acid, 2 M NaCl, 0.5 g·L-1 spermidine, and 2% β-mercaptoethanol. To determine the SA and R. vitis-responsive genes, the oligonucleotide microarrays were hybridized with probes prepared from the total RNA of SA-treated, R. vitis- inoculated, and control leaves.

The MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit (Ambion, Woodward Austin, TX, USA) is based on the RNA amplification protocol developed in the laboratory of James Eberwine (Van Gelder et al. 1990). Microarray hybridization was performed with 5 μg of a labeled target sample per one CustomArray™ using a 12K microarray hybridized and scanned PMT 500-700, pixel size of 5, focus position 130. Analyses were conducted using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA, USA). After data extraction, backgrounds for individual samples were calculated. One-way analysis of variance (ANOVA) and a t-test were applied to determine differentially expressed sets of genes across three experimental groups. Statistical significances were adjusted by Benjamini- Hochberg FDR multiple-testing correction (Benjamini and Hochberg 1995). Complete linkage hierarchical clustering based on the Euclidean distances of samples was performed using the normalized significant genes. The patterns of expressed changes were analyzed for groups using the Avadis Prophetic Ver. 3.3 software (StrandGenomics, Bangalore, India, http://avadis.Strandgenomics.com/).

Semiquantitative RT-PCR analysis

Semiquantitative RT-PCR analysis was conducted using 95 up-regulated genes and 90 down-regulated genes. cDNAs was synthesized using a ReverTra-plus-™ High Fidelity RT-PCR Kit (Toyobo, PCR-501, Japan). PCR amplification was conducted by subjecting the samples to 94°C for 2 min, followed by 30 cycles of 98°C for 10 s, 58°C for 30 s and 68°C for 1 min using KOD-Plus taq polymerase (Toyobo, KOD- 201, Japan). PCR amplification was conducted using primers specific for each gene and actin primers as an internal control under appropriate conditions.

RNA slot blot hybridization analysis

Total RNA (5 μg) isolated from the leaves of grapevines was used for the RNA slot blot hybridization analysis. The RNA mixtures were denatured at 65°C for 10 min, then blotted onto membranes using the Bio-Dot SF (BioRad). RNA samples were transferred and immobilized to Hybond-N+ nylon membrane with UV-crosslinker. Hybridization, washing, detection, and exposure on X-ray film were performed as previously described.

Hierarchical clustering of the ESTs derived from ‘Tamnara’ grapevines

To obtain molecular profiles of responses to R. vitis in grapevines, the differential transcriptional profiles of R. vitis-inoculated and SA-treated grapevines were investigated using microarray analysis with 12,000 gene oligonucleotides. Samples for each treatment were harvested at multiple time points to screen for a large number of biochemical changes expected to occur after R. vitis-inoculation and SA-treatment. Genes showing induced and suppressed expression patterns in grapevines inoculated with R. vitis or treated with SA compared to untreated controls were selected as up- and down-regulated and used to validate expression analysis (Fig. 1).

Fig. 1.

Venn diagrams showing the numbers of overlapped and unique genes induced (A) and suppressed (B) more than twice in the level of their expressions by R. vitis inoculation and salicylic acid (SA) treatment. Results were based on the mean inductions of three replicates


As shown in the Venn diagram, 337 and 27 genes were induced more than two times by SA treatment and R. vitis inoculation, respectively (Fig. 1A). Among ten genes commonly up-regulated under both treatments, several were well-known defense-related genes, including proline-rich protein 1, leuco-anthocyanidin dioxgenase, cytosolic heat shock 70 protein, and sarcosine oxidase family protein, while the functions of others were unknown. Similarly, among the ESTs of ‘Tamnara’ grapevines, 499 genes from R. vitis- inoculated and 52 genes from SA-treated samples were down-regulated by more than two times, respectively (Fig. 1B). Additionally, 35 genes were suppressed by both treatments, which include genes encoding heavy metal-associated domain- containing protein, Zn (C3HC4-type RING) finger family protein, putative WRKY transcription factors 4 and 30, MAP kinase-like protein, and hypothetical proteins. It was reported that several transcripts such as subtilisin-like protease, phenylalanine ammonia lyase (PAL), S-adenosylmethionine synthase, WD-repeat protein like, and J2P, were up-regulated in ‘Regent’ grapevine against the mildew (Figueiredo et al. 2013). Camparative analysis between resistant and susceptible grape cultivars to Pierce’s disease showed that significant differences in transcripts including some of the PR proteins such as β-1,3-glucanase and chitinases and proline-rich proteins were shown at the stem tissues infected with Xylella fastidiosa (Lin et al. 2007). In this study, it was shown that expressions of various defense-related genes were differentially regulated in the grapevines by R. vitis infection and SA-treatment.

R. vitis and SA responsive DEGs with microarray analysis

Overall, 95 up-regulated cDNA clones showed expression that was up-regulate/d by more than 3-times, while 90 down-regulated clones showed decreases in expression of more than 5-times in R. vitis-inoculated grapevines relative to untreated samples (Tables 1 and 2). Arabidopsis thaliana seed imbibition 2 (ATSI2) hydrolyzing O-glycosyl compound gene and btb and taz domain protein related genes were highly up-regulated. Conversely, cell wall protein, expansin, and endosperm specific protein genes were significantly suppressed. Additionally, the transcriptional levels of defense, signal transduction, active oxygen related genes such as methallothionine-like protein, LRR domains, and PR protein, as well as transcription factors such as MYB transcription factors were highly activated. These results were consistent with those of ESTs from R. vitis inoculated ‘Tamnara’ grapevines (Choi et al. 2010).

Table 1 . Genes in ‘Tamnara’ grapevines up-regulated in response to R. vitis inoculation and SA treatment

Gene No. in EPP163JWAA seriesNo. of slot blot, RT-PCRPutative functionRatio of signal intensity

R. vitisSA
12C000256B9U1ATSI 2 hydrolyzing O-glycosyl compounds yabby15 protein15.9±0.49-14.2±0.44
12S013353F4U2Btb and taz domain protein11.2±0.85-4.2±0.27
12C000750F2U3Metallothionein-like protein10.3±0.63-6.0±0.79
12C000845D7U4LOX9.3±0.52-11.1±0.66
12C001644B6U5Dark inducible 10 hydrolyzing O-glycosyl compounds7.6±0.46-7.8±0.36
12S000483H3U6No hit7.6±0.70-3.5±0.81
12S008848H1U7Hypothetical protein7.4±0.19-3.1±0.71
12S003419A7U8Glycosyl hydrolase family 1 protein7.0±0.15-4.8±0.25
12S007850G6U9CHS6.8±0.94-4.5±0.72
12S003388A3U10Glycosyl hydrolase family 1 protein6.5±0.07-4.7±0.21
12S009383F5U11Aspartyl protease family protein6.4±0.57-4.2±0.63
12S005565B2U12Glucose-6-phosphate translocator6.3±0.18-5.5±0.31
12S002570B10U13Limonoid udp-lucosyltransferase6.2±0.26-3.6±0.39
12S002915B3U14Cytochrome P4506.2±0.17-4.2±0.32
12S009437A12U15ATSI 2 hydrolyzing O-glycosyl compounds6.0±0.36-7.3±0.16
12S006649C4U16Isoamylase isoform 36.0±0.12-5.2±0.44
12S000876D2U17Cytochrome p4506.0±0.43-4.9±0.33
12S010715G3U18Cytochrome p4505.9±0.69-3.4±0.58
12S012744E11U19Proline-rich cell wall protein5.9±0.49-2.9±0.53
12S005981A5U20Organic cation transporter5.8±0.19-5.0±0.22
12S002410D4U21Thaumatin-like protein5.8±0.60-6.9±0.57
12S009693D11U22Starch phosphorylase5.8±0.44-4.2±0.34
12C000042G2U23Myb transcription factor5.7±0.54-4.1±0.70
12S013702A6U24Aldehyde dehydrogenase5.7±0.16-6.0±0.27
12C001360C1U25Protein5.6±0.33-6.0±0.38
12S011414E4U26GST5.6±0.11-3.9±0.56
12S000490A2U27Mate efflux family expressed5.5±0.15-3.5±0.10
12S012305B12U28Hypothetical protein5.5±0.25-4.2±0.37
12S006861C5U29Reductase 15.3±0.25-2.7±0.30
12S006594A11U30Molybdenum cofactor sulfurase4.9±0.22-6.2±0.28
12S007535A10U31Glyoxalase i4.6±0.13-2.3±0.22
12S005609D8U32Expansin-like protein4.6±0.14-3.7±0.46
12C000943D1U33Ef-1 a4.5±0.40-2.7±0.26
12S011005C3U34Alkaline a galactosidase4.4±0.35-4.7±0.33
12S000980D10U35Fatty acid hydroperoxide lyase4.4±0.29-3.6±0.39
12S002559B7U36Af303396_1udp-glucosyltransferase hra254.4±0.28-2.9±0.16
12S010472B11U37Tpa:gid1-like gibberellin receptor4.4±0.33-3.0±0.06
12S006784F8U38Glucose acyltransferase4.3±0.50-3.7±0.35
12S011826F12U39Ethephon-induced protein4.3±0.49-3.1±0.50
12S005921C7U40Fibrillin4.3±0.25-2.9±0.28
12S009053E6U41Condensation domain-containing protein4.2±0.46-3.0±0.11
12C000227E3U42Basic helix-loop-helixfamily protein4.2±0.14-2.7±0.44
12C000359E1U43Sucrose synthase4.1±0.21-3.6±0.22
12S010980A9U44Pyrroline-5-carboxylate synthetase4.1±0.44-5.7±0.34
12S002233E12U45Glyceraldehyde-3-phosphate dehydrogenase4.1±0.48-3.5±0.28
12C001111G5U46Chalcone-flavanone isomerase family expressed4.1±0.63-2.9±0.54
12S011777H10U47Abscisic acid responsive elements-binding factor4.0±0.92-2.3±0.15
12S009704A4U48Hypothetical protein4.0±0.14-3.0±0.16
12C001087H11U49Methionine gamma-lyase3.9±0.30-2.6±0.92
12S002496H2U50Protein kinase3.8±0.33-3.0±0.60

Table 2 . Genes in ‘Tamnara’ grapevines down-regulated in response to R. vitis inoculation and SA treatment

Gene No. in EPP163JWAA SeriesNo. of slot blot, RT-PCRPutative functionRatio of signal intensity

R. vitisSA
12C001593D8D1Cell wall protein-50.2±0.8736.1±0.56
12S000426A2D2Expansin-45.6±0.2032.6±0.32
12S005616A10D3At5g25460 f18g18_200-33.9±0.4732.6±0.33
12C000040F5D4Cytochrome C oxidase polypeptide vc-21.7±0.9416.0±0.78
12C000966E7D5Endosperm specific-21.7±0.8216.1±0.65
12C000783B1D6Protease inhibitor seed storage lipid transfer protein-21.6±0.4015.2±0.41
12C000998E4D7Protein-18.1±0.8317.4±0.59
12S003758F1D8En/Spm-like transposon protein-17.9±0.9215.1±0.48
12C001094F12D9Meiosis 5-17.8±1.0913.8±0.89
12S013145A9D10Proline-rich protein apg isolog-16.4±0.2011.3±0.37
12S010548B12D11DNA heat shock N-terminal domain-containing protein-15.9±0.3912.3±0.49
12C000058G6D12Acid phosphatase-15.4±1.429.4±0.57
12C000034A6D13Expansin-14.9±0.2717.3±0.19
12C001244B6D14Heavy metal-associated domain-containing protein-14.7±0.315.5±0.40
12C000247A5D15SA-induced fragment 1 protein-13.8±0.2113.4±0.27
12S011527F2D16Cold induced-13.6±0.845.1±0.17
12C000203A4D17Rho GDP-dissociation inhibitor 1-13.4±0.267.8±0.20
12C001631E2D18Proline rich protein 2-13.1±0.7711.1±0.26
12S009428B10D19Hypothetical protein-13.1±0.4812.5±0.15
12C000933A7D20Fatty acid elongase-12.8±0.309.7±0.21
12S005808A1D21Proline-rich protein-10.8±0.0614.1±0.20
12C000939D5D22β-Ketoacyl-synthase-10.7±0.254.5±0.52
12C001125D10D23Xyloglucan endotransglycosylase-10.6±0.6410.8±0.43
12S011610D9D24Hypothetical protein-10.6±0.237.8±0.56
12S008346G7D25No hit-10.0±1.3512.0±0.57
12S008268F11D26Tonoplast membrane integral protein4-4-9.9±0.765.2±0.48
12S011177D3D27Hypothetical protein-9.7±0.497.5±0.19
12C000816C6D28Chloroplast chlorophyll a/b binding protein-9.7±0.4814.7±0.22
12C000797A8D29Chloroplast chlorophyll a/b binding protein-9.6±0.3010.5±0.34
12S005860E5D30CCHC-type integrase-9.5±0.283.6±0.65
12C000927G1D31Protein binding protein-8.8±0.745.8±0.51
12S008817C3D32LRR protein-8.5±0.156.2±0.39
12C001369D4D33Transferase family protein-8.4±0.2410.3±0.52
12S001577C1D34No hit-8.4±0.183.1±0.34
12S003449A3D35Mee60 (maternal effect embryo arrest 60)-8.3±0.2111.9±0.21
12C001193B9D36Yabby15 protein-8.1±0.286.8±0.36
12S009302G2D37Aspartyl protease family protein-8.0±0.745.7±0.73
12S011056B11D38At3g15630 msj11_3-7.9±0.253.0±0.25
12C001299E1D39Endo-β-glucanase precursor-7.8±0.464.7±0.46
12S013533H2D40Expansin-like protein a-7.7±0.422.2±0.66
12S013059G5D41Thaumatin-like protein-7.7±1.017.0±0.62
12C000786F7D42Tonoplast intrinsic protein-7.6±0.635.6±0.49
12S006978C10D43WRKY transcription factor 10-7.5±0.395.3±0.32
12C000038B8D44Hypothetical protein-7.5±0.324.8±0.18
12C001095G10D45α Tubulin 1-7.3±1.163.9±0.58
12S010793F10D46At5g44130 mln1_5-7.2±0.605.8±0.62
12C000621E8D47Chitinase-like protein-7.2±0.585.5±0.29
12C000406G3D48Glutamine synthetase-7.2±0.764.9±0.66
12C000508B7D49Nucleoid DNA-binding protein cnd41-7.1±0.186.7±0.35
12S009795C5D50Calmodulin-like protein-7.0±0.295.2±0.35

As shown in the Tables 1 and 2, some genes showed antagonistic expression patterns between induced genes of R. vitis-inoculated and SA-treated grapevines. The genes activated by R. vitis inoculation might be mediated by jasmonic acid (JA) or ethylene. The expression of 95 up-regulated genes was suppressed by R. vitis inoculation in SA-treated grapevine leaves. In contrast, the expression of 90 genes down-regulated by R. vitis inoculation increased in response to SA treatment. When R. vitis attacks the vines, highly induced JA-dependent responses might suppress the gene expression involved SA-dependent defense pathway due to pathway crosstalk by R. vitis. Antagonisms between JA or ethylene and SA signaling have been extensively studied (Dong 1998; Kunkel and Brooks 2002). A large number of genes, includingc lipoxygenase (LOX), lipid transferase, and genes related to secondary metabolisms (Creelman and Mullet 1997; Lin et al. 2007) that were involved in JA biosynthesis, have been shown to respond to wounding and R. vitis attack in grapevines. The crosstalk among a number of signaling molecules appears to be related to controlling defense systems in response to pathogen attacks in grapevines as in other plants (Shah 2003).

Genes in response to R. vitis, SA, and wound in ‘Tamnara’ grapevines

Transcriptional profiling in response to R. vitis with the 6,776 unigenes obtained from the R. vitis-inoculated and SA-treated ‘Tamnara’ grapevine cDNA library was conducted by use of gene specific oligonucleotide microarray chips. cDNA contigs responsive to R. vitis inoculation and SA treatment could be categorized into seven groups encoding proteins involved in defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell wall fortification, and transcription factors (Table 3). The expression level of 95 up-regulated and 90 down-regulated ESTs was confirmed by semiquantitative RT-PCR (Fig. 2 and 3) and RNA slot blot hybridization analysis (Fig. 5 and 6). Confirmation using the three expression profiling methods revealed that 5, 14, and 64 cDNAs were up-regulated by wound, SA-treatment, and R. vitis inoculation, while 10, 12, and 61 were down-regulated by each treatment, respectively (Table 4).

Table 3 . Defense-related cDNA responsive to R. vitis inoculation and SA treatment in ‘Tamnara’ grapevines

??Gene??Putative function/homologyRatio of signal intensity

R. vitisSA
Defense-relatedβ-1,3-Glucanase-3.4±0.36-1.4±0.38
Chitinase-4.4±0.25-1.2±0.26
Basic endochitinase precursor2.3±0.37-1.7±0.32
Chitinase III-2.6±0.51-1.4±0.66
Thaumatin-like protein5.8±0.60-1.3±0.24
Chalcone synthase6.8±0.941.5±1.11

Signal transductionLOX9.3±0.52-1.2±0.69
NBS LRR-containing protein3.5±0.731.2±0.81

Active oxygen relatedCatalase2.5±0.71-1.1±0.70
Glutathione peroxidase3.2±0.191.1±0.42
Glutathione S-transferase5.6±0.111.4±0.56
Ascorbate peroxidase1.8±0.251.1±0.23

Secondary metabolitesCytochrome P4506.0±0.43-4.9±0.33
Cytochrome C oxidase subunit Vb1.8±0.211.1±0.33

Abiotic stress-relatedSmall heat shock protein3.5±0.291.3±0.35
Heat shock protein3.7±0.181.6±0.32

Cell wall fortificationProline-rich cell wall protein5.9±0.492.0±0.43

Transcription factorsWRKY transcription factor1.7±0.21-1.1±0.26
MYB transcription factor5.8±0.541.4±0.84

Fig. 2.

Semiquantitative RT-PCR analysis of 95 highly up-regulated genes in ‘Tamnara’ grapevines. cDNA samples were amplified with a ReverTra-plus™-High Fidelity RT-PCR Kit. MAU: up-regulated in microarray analysis; C, control; W, wound; S, SA treatment; R, R. vitis inoculation; M, size marker


Fig. 3.

Semiquantitative RT-PCR analysis of 90 highly down-regulated genes in ‘Tamnara’ grapevines. cDNA samples were amplified with a ReverTra-plus™-High Fidelity RT-PCR Kit. MAD: down-regulated in microarray analysis; C, control; W, wound; S, SA treatment; R, R. vitis inoculation; M, size marker


Fig. 4.

Venn diagram of DEGs upregulated (A) and down-regulated (B) in ‘Tamnara’ grapevines responsive to R. vitis inoculation, wound, and SA treatment


Fig. 5.

RNA slot blot hybridization analysis with (A) β-1,3-glucanase, (B) CHS, (C) LOX, and (D) proline-rich protein as a probe in several grapevine cultivars. C, control; R, R. vitis inoculation; W, wound


Fig. 6.

RNA slot blot hybridization analysis with (A) ATSI 2 and (B) organic cation transporter as a probe in several grapevine cultivars. C, control; R, R. vitis inoculation; W, wound


Table 4 . Genes specifically expressed in response to wound, SA treatment, and R. vitis inoculation with microarray, RT-PCR, and slot blot hybridization analyses in ‘Tamnara’ grapevines

A. Up-regulated genes

?Confirming methodWoundSAR. vitis
Microarray and slot blot783782
Microarray and RT-PCR73273
Microarray, slotblot, and RT-PCR51464

B. Down-regulated genes

?Confirming methodWoundSAR. vitis
Microarray and slot blot706471
Microarray and RT-PCR141674
Microarray, slotblot, and RT-PCR101261

As shown in the Venn diagram (Fig. 4A), 50 cDNAs, including ATSI protein 2 hydrolyzing O-glycosyl compounds yabby15 protein, CHS, cytochrome P450, thaumatin-like protein, GST, and LRR containing protein, were specifically activated by R. vitis inoculation in ‘Tamnara’ grapevine leaves. However, no cDNA was shown to be up-regulated by wound or SA treatment in grapevines. Five genes, LOX, aspartyl protease family protein, heavy metal-associated domain- containing protein, tyrosine aminotransferase, and one with unknown function, were commonly induced by R. vitis inoculation, wound, and SA treatments. Similarly, 1, 4, and 48 cDNAs were down-regulated by wound, SA treatment and R. vitis inoculation, respectively (Fig. 4B). Expansin, cytochrome C oxidase polypeptide vc, seed storage lipid transfer protein, meiosis 5, SA-induced fragment 1 protein, WRKY transcription factor 10, chitinase-like protein, and Zn finger (gata type) family proteins were specifically down-regulated by R. vitis inoculation. Four genes such as lipid transfer protein, extensin-like protein, and two proteins with unknown function were commonly inhibited under both R. vitis inoculation and SA treatment (Table 5). More detailed functional studies should be conducted to determine if they are involved in defense mechanisms. It has been reported that PR genes were activated by endo- and exogenous SA treatments and pathogen attacks in many plants (Malamy 1990). In this study, genes involved in plant defense responses such as thaumatin-like protein, chalcone synthase (CHS), and LOX were induced by R. vitis inoculation and SA treatment.

Table 5 . DEGs responsive to R. vitis inoculation, wound, and SA treatment in ‘Tamnara’ grapevines

Up-regulatedDown-regulated
R. vitis?ATSI protein 2 hydrolyzing O-glycosyl compounds?Expansin
?yabby15 protein?Cytochrome C oxidase polypeptide vc
?CHS?Seed storage lipid transfer protein
?Cytochrome P450?Meiosis 5
?Thaumatin-like protein?Proline-rich protein apg isolog
?GST?SA-induced fragment 1 protein
?Sucrose synthase?WRKY transcription factor 10
?Small heat shock protein?Chitinase-like protein
?LRR containing protein?Zn finger (gata type) family protein

Wound-?Fasciclin-like arabinogalactan protein 14

SA-?Chloroplast chlorophyll a/b binding protein
?β-Glucanase-like protein
?Pollen-specific protein

R. vitis and SA?Btb and taz domain protein?Tonoplast membrane integral protein4-4
?Limonoid UDP-glucosyltransferase?Histone h3
?Alkaline α galactosidase

R. vitis and Wound-?Cell wall protein
?Glucose-methanol-cholineoxidoreductase family protein

R. vitis, SA, and Wound?LOX
?Aspartyl protease family protein?Lipid transfer protein
?Heavy metal-associated domain-containing protein?Extensin-like protein
?Tyrosine aminotransferase

In this study, some genes such as β-1,3-glucanase and chitinase III responded similarly to R. vitis inoculation and SA treatment. Using the GeneFishing and RACE technology, the full-length cDNA of several PR genes including β-1,3 glucanase, PR 10, and thaumatin-like proteins expressed specifically by R. vitis inoculation and SA treatment were cloned from the grapevine leaves (Choi et al. 2008). Cheong et al. (2002) reported that various genes such as LOX, catalase (CAT), glutathione S-transferase (GST), cytochrome P450, and WRKY, as well as MYB transcription factors were activated by wound stress in grapevines. A number of components in their signaling pathways appear to share the response against pathogen infection and wounding stress (Cheong et al. 2002; Maleck and Dietrich 1999). Accordingly, further functional analysis is required to determine the expression dynamics of selected genes in response to mechanical wounding, R. vitis inoculation, and SA treatment.

Semiquantitative RT-PCR and slot blot hybridization analysis of cDNA

The expression level of 95 up-regulated and 90 down-regulated ESTs was confirmed by semiquantitative RT-PCR (Fig. 2 and 3) and RNA slot blot hybridization analysis (Fig. 5 and 6). Confirmation using the three expression profiling methods revealed that 5, 14, and 64 cDNAs were up-regulated by wound, SA treatment, and R. vitis inoculation, while 10, 12, and 61 were down-regulated by these treatments, respectively (Table 4).

As shown in the Venn diagram (Fig. 4A), 50 cDNA samples, including ATSI protein 2 hydrolyzing O-glycosyl compounds yabby15 protein, CHS, cytochrome P450, thaumatin-like protein, GST, and LRR containing protein, were specifically activated by R. vitis inoculation in ‘Tamnara’ grapevine leaves. However, no cDNA was shown to be up-regulated by wound or SA treatment in grapevines. Five genes, LOX, aspartyl protease family protein, heavy metal-associated domain-containing protein, tyrosine aminotransferase, and one with unknown function, were commonly induced by R. vitis inoculation, wound, and SA treatments. Similarly, 1, 4, and 48 cDNAs were down-regulated by wound, SA treatment and R. vitis inoculation, respectively (Fig. 4B). Expansin, cytochrome C oxidase polypeptide vc, seed storage lipid transfer protein, meiosis 5, SA-induced fragment 1 protein, WRKY transcription factor 10, chitinase-like protein, and Zn finger (gata type) family proteins were specifically down-regulated by R. vitis inoculation. Four genes were commonly inhibited under both R. vitis inoculation and SA treatment, lipid transfer protein, extensin-like protein, and two proteins with unknown function (Table 5). The function of these genes is not clear yet, accordingly, more detailed functional studies should be conducted to determine if they are involved in defense mechanisms.

Comparative analysis of defense-related gene expression in R. vitis-inoculated grapevines

To investigate the expression of selected genes, RNA slot blot hybridization was performed using ‘Delaware’ and ‘Concond’, ‘Neomuscat’ and ‘Rizamat’, and ‘Tamnara’ grapevine leaves harvested at several time courses. When β-1,3-glucanase, CHS, LOX, proline-rich cell wall protein, ATSI 2, and organic cation transporter genes were used as probes for the hybridization analyses, differential expression was observed between resistant and susceptible cultivars in response to R. vitis inoculation and wound treatment (Fig. 5 and 6). The expression patterns of β-1,3-glucanase, proline-rich protein, and LOX genes in ‘Tamnara’ grapevines were similar to their expressions in ‘Concord’ and ‘Delaware’ which are resistant to crown gall disease. Although proline-rich cell wall protein genes were highly expressed within 12 to 72 h in ‘Concord’, ‘Delaware’, and ‘Tamnara’ grapevines inoculated with R. vitis, they showed low initial expression level, and increased level from 30 min to 12 h after R. vitis inoculation in ‘Neomuscat’ and ‘Rizamat’ grapevines, which are susceptible to crown gall. Conversely, the CHS gene in ‘Tamnara’ grapevines was expressed at levels similar to those of ‘Neomuscat’ and ‘Rizamat’ grapevines. Although transcripts of CHS increased at 48 to 72 h after R. vitis inoculation and wound treatment, they showed low expression at 0.5 to 12 h after R. vitis inoculation in ‘Neomuscat’, ‘Rizamat’, and ‘Tamnara’ grapevines. In the case of comparative analysis by Lin et al. (2002), PR genes were expressed in both resistant and susceptible grapevine cultivars in response to X. fastidiosa inoculation. These findings suggest that susceptible cultivars have a host defense response mechanism that responds to X. fastidiosa inoculation, but they may fail to protect themselves from pathogen infections.

Fig. 7.

Slot blot hybridization analysis of 95 highly up-regulated genes in ‘Tamnara’ grapevines. cDNA probes were synthesized with a ReverTra-plus™-High Fidelity RT-PCR Kit. C, control; W, wound; S, SA treatment; R, R. vitis inoculation; VACT, actin cloned from ‘Tamnara’ grapevines


Fig. 8.

Slot blot hybridization analysis of 90 highly down-regulated genes in ‘Tamnara’ grapevines. cDNA probes were synthesized with a ReverTra-plus™-High Fidelity RT-PCR Kit. C, control; W, wound; S, SA treatment; R, R. vitis inoculation; VACT, actin cloned from ‘Tamnara’ grapevines


ATSI2 and organic cation transporter genes were highly activated by R. vitis inoculation in ‘Tamnara’ grapevines as confirmed by microarray, RT-PCR, and slot blot hybridization analyses (Fig. 6). ATSI 2, formerly raffinose synthase, is a key enzyme that transfers sucrose into the raffinose oligosaccharide, an oligosaccharide commonly found in plant seeds and other tissues (Nishizawa et al. 2008). This gene is known to be activated by abiotic stressors such as frost, drought, and salt (Kandler and Hopf 1984; Keller and Pharr 1996; Peterbauer and Richter 2001; Peterbauer et al. 2000). The results of the present study suggest that expression of the ATSI2 gene was also related to the defense responses to R. vitis infection in grapevines.

To understand the resistant responses to disease in grapevines, it is important to monitor the specific expression of genes in response pathogen attacks or signal molecules accumulated by pathogens in vines. Among various strategies to screen for specific gene expression, microarray analysis can be used to analyze variations in the expression of thousands of genes simultaneously (Meyers et al. 2004; Schulze and Downward 2001; Stears 2003). In grapevines, microarray analysis has been used in investigations of transcriptomes related to berry development (Deluc et al. 2007; Terrier et al. 2005; Waters et al. 2006), water and salinity stress (Cramer et al. 2007), ultraviolet-B radiation (Pontin et al. 2010), and virus (Espinoza et al. 2007) and fungal infection (Figueiredo et al. 2008).

In this study, the gene expression patterns in response to R. vitis bacterium causing crown gall in grapevines were investigated in R. vitis-inoculated, salicylic acid (SA)-treated, wounded, and untreated ‘Tamnara’ grapevines. Microarray analysis using 12,000 gene oligonucleotides of microarray chips constructed with 6,776 unigenes based on EST sequencing revealed that 95 clones were up-regulated by more than 3 times and 90 were down-regulated by more than 5 times in R. vitis-inoculated ‘Tamnara’ grapevines than in untreated vines. Among these, 5 to 61 up-regulated genes, and 10 to 61 clones showed different expression levels in response to wound, SA, and R. vitis in grapevines upon RT-PCR and slot blot hybridization analysis. Some genes, such as β-1,3-glucanase, proline-rich protein, and LOX, were induced in moderately resistant cultivars, while others, such as CHS, were expressed in moderately resistant and susceptible grapevines in response to R. vitis. Further expression studies with various clones per each gene should be conducted to elucidate their roles in resistant responses to pathogens or other stimuli in grapevines. Identification and characterization of the putative genes involved in defense response will be useful for breeding grapes resistant to crown gall. These results could provide a better understanding of the mechanisms of defenses against crown gall disease and clues for identifying new genes that may play a role in defense responses to infection of R. vitis.

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Article

Research Article

Journal of Plant Biotechnology 2016; 43(1): 37-48

Published online March 31, 2016 https://doi.org/10.5010/JPB.2016.43.1.37

Copyright © The Korean Society of Plant Biotechnology.

Transcriptional profiles of Rhizobium vitis-inoculated and salicylic acid-treated ‘Tamnara’ grapevines based on microarray analysis

Youn Jung Choi1, and Hae Keun Yun2,*

1National Institute for Horticultural and Herbal Science, Wanju 55365, Korea,
2Department of Horticulture and Life Science, Yeungnam University, Gyeonsan 38541, Korea

Correspondence to:e-mail: haekeun@ynu.ac.kr

Received: 3 March 2016; Revised: 3 March 2016; Accepted: 9 March 2016

Abstract

The transcriptional profiles of ‘Tamnara’ grapevine (Vitis labruscana L.) to Rhizobium vitis were determined using 12,000 gene oligonucleotide microarray chips constructed with 6,776 unigenes based on the EST sequencing. Among them, 95 clones were up-regulated more than three times and 90 were down-regulated more than 5-times in the R. vitis-inoculated grapevines relative to the control vines. Treatment of salicylic acid showed that 337 clones were upregulated and 52 clones were down regulated in grapevines. Microarray analysis, reverse transcription-polymer chain reaction, and slot blot hybridization analysis revealed that 5, 14, and 64 clones were up-regulated and 10, 12, and 61 clones were down-regulated in wounded, salicylic acid- treated, and R. vitis-inoculated ‘Tamnara’ grapevine leaves, respectively. The expression patterns of β-1,3-glucanase, proline-rich protein, and lipoxygenase genes of ‘Tamnara’ moderately resistant to R. vitis were similar to those of resistant ‘Concord’ and ‘Delaware’ grapevines. However, chalcone synthase genes in ‘Tamnara’ grapevines showed similar expression patterns to susceptible grapevines ‘Neomuscat’ and ‘Rizamat’. Further expression studies with various clones for each gene should be conducted to elucidate their roles in resistant responses against pathogens or other stimuli in grapevines. These results could provide better resources for understanding the mechanism of defense responses against crown gall disease and clues for identifying new genes that may play a role in defense against R. vitis in grapevines.

Keywords: Grape, Gene expression, RT-PCR, Slot blot

Introduction

Grape (Vitis sp.) is attacted to a number of bacterial, fungal, and viral diseases like other plants (Pearson and Goheen 1998). Crown gall is a major disease responsible for severe reduction of yield and poor quality of fruit in grape production regions throughout the world, including Korea (Burr et al. 1998; Park et al. 2000). Chemical or biological attempts to control crown gall disease conducted to date have failed, with the exception of Agrobacterium radiobacter strain K84 of the biological control (Anand et al. 2008; Burr and Otten 1999). However, A. radiobacter strain K84 is only effective at controlling crown gall caused by nopaline-type strains of A. tumefaciens and A. rhizogenes, while it has no effect on crown galls induced by Rhizobium vitis in grapevines (Kerr 1980). Since effective agro-chemicals to control crown gall have not been released, development of novel grapevine cultivars resistant to crown gall is critical and will be a useful tool in protection of grapevines from disease (Burr et al. 1988; Park et al. 2000; Stover et al. 1997).

There has been continuous study of genes related to disease resistance and defense response of grapevines to fungal, bacterial, and viral pathogens, including crown gall disease through comparative genomics, transcriptomics and the genome wide identification analysis for useful genes and molecular markers (Burr et al. 1998 and 2003; Choi et al. 2008; Hur et al. 2015). However, development of disease resistant grapes based on molecular biology has been limited because of the relatively low amount of genetic and molecular information available regarding genotypes resistant to certain diseases. Although the entire genome of Vitis vinifera ‘Pinot Noir’ has been sequenced (The France-Italian Public Consortium for Grapevine 2007) and annotated, the functions of many genes must be still investigated.

To develop new grape cultivars resistant to diseases, systematic monitoring of the mechanism of plant response and defense against pathogen attacks and more detailed functional studies of the selected differentially expressed genes (DEGs) are required. Microarray analysis, which can screen the expression patterns of many genes simultaneously in a single analysis, is considered a foundational technology capable of high-throughput and high-speed transcriptional profiling. Accordingly, this technique has various applications including unique gene identification and diagnostics of certain diseases (Schulze and Downward 2001; Stears 2003).

In this study, the gene expression patterns in response to R. vitis inoculation were investigated in R. vitis-inoculated, salicylic acid (SA)-treated, wounded, and untreated control leaves of ‘Tamnara’ grapevine which was bred in Korea (Park et al. 2004). Using microarray gene expression profiling, 6,776 unigenes of expressed sequence tag (EST)-based sequence were analyzed in grapevine leaves. The detailed expression patterns of selected up- and down-regulated genes in the microarray were confirmed by slot blot hybridization and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Expression of DEGs was also analyzed in grapevines resistant and susceptible to crown gall in response to R. vitis inoculation.

Materials and Methods

Plant materials and treatments

Grapevines of ‘Tamnara’ (moderately resistant to crown gall disease), ‘Delaware’ and ‘Concord’ (resistant to crown gall disease), and ‘Neomuscat’ and ‘Rizamat’ (susceptible to crown gall disease) were grown in a greenhouse at 25°C~30°C under natural light, then inoculated with R. vitis Cheonan 493 (Yun et al. 2003). Leaves were harvested at 1, 3, 6, 12, 24, 48, and 72 h after wound, SA treatment, and R. vitis inoculation, immediately frozen in liquid nitrogen, and stored at -80°C until used for RNA extraction. All samples harvested from each treatment were used for RNA extraction, analysis of differential expression of cDNA, and RT-PCR analysis.

Microarray chip construction

A total of 6,776 unigenes were obtained from the ‘Tamnara’ grapevine cDNA library constructed after R. vitis inoculation and SA treatment. Microarray chips were constructed with 35?40 nt of unigene based oligonucleotide. Gene specific oligonucleotides were arrayed onto a slide glass with an average of two replications and oligonucleotide microarray techniques were employed to detect R. vitis-responsive genes in ‘Tamnara’ grapevines.

RNA isolation and microarray hybridization

Total RNA was isolated from R. vitis-inoculated, SA-treated, and control grapevine leaves using the modified pine tree method of removing polysaccharides and phenolic compounds (Chang et al. 1993) with RNA extraction buffer consisting of 2% cetyltrimethylammonium bromide, 2% polyvinylpyrrolidone, 100 mM Tris-HCl (pH 8.0), 25 mM ethylenediaminetetraacetic acid, 2 M NaCl, 0.5 g·L-1 spermidine, and 2% β-mercaptoethanol. To determine the SA and R. vitis-responsive genes, the oligonucleotide microarrays were hybridized with probes prepared from the total RNA of SA-treated, R. vitis- inoculated, and control leaves.

The MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit (Ambion, Woodward Austin, TX, USA) is based on the RNA amplification protocol developed in the laboratory of James Eberwine (Van Gelder et al. 1990). Microarray hybridization was performed with 5 μg of a labeled target sample per one CustomArray™ using a 12K microarray hybridized and scanned PMT 500-700, pixel size of 5, focus position 130. Analyses were conducted using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA, USA). After data extraction, backgrounds for individual samples were calculated. One-way analysis of variance (ANOVA) and a t-test were applied to determine differentially expressed sets of genes across three experimental groups. Statistical significances were adjusted by Benjamini- Hochberg FDR multiple-testing correction (Benjamini and Hochberg 1995). Complete linkage hierarchical clustering based on the Euclidean distances of samples was performed using the normalized significant genes. The patterns of expressed changes were analyzed for groups using the Avadis Prophetic Ver. 3.3 software (StrandGenomics, Bangalore, India, http://avadis.Strandgenomics.com/).

Semiquantitative RT-PCR analysis

Semiquantitative RT-PCR analysis was conducted using 95 up-regulated genes and 90 down-regulated genes. cDNAs was synthesized using a ReverTra-plus-™ High Fidelity RT-PCR Kit (Toyobo, PCR-501, Japan). PCR amplification was conducted by subjecting the samples to 94°C for 2 min, followed by 30 cycles of 98°C for 10 s, 58°C for 30 s and 68°C for 1 min using KOD-Plus taq polymerase (Toyobo, KOD- 201, Japan). PCR amplification was conducted using primers specific for each gene and actin primers as an internal control under appropriate conditions.

RNA slot blot hybridization analysis

Total RNA (5 μg) isolated from the leaves of grapevines was used for the RNA slot blot hybridization analysis. The RNA mixtures were denatured at 65°C for 10 min, then blotted onto membranes using the Bio-Dot SF (BioRad). RNA samples were transferred and immobilized to Hybond-N+ nylon membrane with UV-crosslinker. Hybridization, washing, detection, and exposure on X-ray film were performed as previously described.

Results and Discussion

Hierarchical clustering of the ESTs derived from ‘Tamnara’ grapevines

To obtain molecular profiles of responses to R. vitis in grapevines, the differential transcriptional profiles of R. vitis-inoculated and SA-treated grapevines were investigated using microarray analysis with 12,000 gene oligonucleotides. Samples for each treatment were harvested at multiple time points to screen for a large number of biochemical changes expected to occur after R. vitis-inoculation and SA-treatment. Genes showing induced and suppressed expression patterns in grapevines inoculated with R. vitis or treated with SA compared to untreated controls were selected as up- and down-regulated and used to validate expression analysis (Fig. 1).

Figure 1.

Venn diagrams showing the numbers of overlapped and unique genes induced (A) and suppressed (B) more than twice in the level of their expressions by R. vitis inoculation and salicylic acid (SA) treatment. Results were based on the mean inductions of three replicates


As shown in the Venn diagram, 337 and 27 genes were induced more than two times by SA treatment and R. vitis inoculation, respectively (Fig. 1A). Among ten genes commonly up-regulated under both treatments, several were well-known defense-related genes, including proline-rich protein 1, leuco-anthocyanidin dioxgenase, cytosolic heat shock 70 protein, and sarcosine oxidase family protein, while the functions of others were unknown. Similarly, among the ESTs of ‘Tamnara’ grapevines, 499 genes from R. vitis- inoculated and 52 genes from SA-treated samples were down-regulated by more than two times, respectively (Fig. 1B). Additionally, 35 genes were suppressed by both treatments, which include genes encoding heavy metal-associated domain- containing protein, Zn (C3HC4-type RING) finger family protein, putative WRKY transcription factors 4 and 30, MAP kinase-like protein, and hypothetical proteins. It was reported that several transcripts such as subtilisin-like protease, phenylalanine ammonia lyase (PAL), S-adenosylmethionine synthase, WD-repeat protein like, and J2P, were up-regulated in ‘Regent’ grapevine against the mildew (Figueiredo et al. 2013). Camparative analysis between resistant and susceptible grape cultivars to Pierce’s disease showed that significant differences in transcripts including some of the PR proteins such as β-1,3-glucanase and chitinases and proline-rich proteins were shown at the stem tissues infected with Xylella fastidiosa (Lin et al. 2007). In this study, it was shown that expressions of various defense-related genes were differentially regulated in the grapevines by R. vitis infection and SA-treatment.

R. vitis and SA responsive DEGs with microarray analysis

Overall, 95 up-regulated cDNA clones showed expression that was up-regulate/d by more than 3-times, while 90 down-regulated clones showed decreases in expression of more than 5-times in R. vitis-inoculated grapevines relative to untreated samples (Tables 1 and 2). Arabidopsis thaliana seed imbibition 2 (ATSI2) hydrolyzing O-glycosyl compound gene and btb and taz domain protein related genes were highly up-regulated. Conversely, cell wall protein, expansin, and endosperm specific protein genes were significantly suppressed. Additionally, the transcriptional levels of defense, signal transduction, active oxygen related genes such as methallothionine-like protein, LRR domains, and PR protein, as well as transcription factors such as MYB transcription factors were highly activated. These results were consistent with those of ESTs from R. vitis inoculated ‘Tamnara’ grapevines (Choi et al. 2010).

Table 1 . Genes in ‘Tamnara’ grapevines up-regulated in response to R. vitis inoculation and SA treatment.

Gene No. in EPP163JWAA seriesNo. of slot blot, RT-PCRPutative functionRatio of signal intensity

R. vitisSA
12C000256B9U1ATSI 2 hydrolyzing O-glycosyl compounds yabby15 protein15.9±0.49-14.2±0.44
12S013353F4U2Btb and taz domain protein11.2±0.85-4.2±0.27
12C000750F2U3Metallothionein-like protein10.3±0.63-6.0±0.79
12C000845D7U4LOX9.3±0.52-11.1±0.66
12C001644B6U5Dark inducible 10 hydrolyzing O-glycosyl compounds7.6±0.46-7.8±0.36
12S000483H3U6No hit7.6±0.70-3.5±0.81
12S008848H1U7Hypothetical protein7.4±0.19-3.1±0.71
12S003419A7U8Glycosyl hydrolase family 1 protein7.0±0.15-4.8±0.25
12S007850G6U9CHS6.8±0.94-4.5±0.72
12S003388A3U10Glycosyl hydrolase family 1 protein6.5±0.07-4.7±0.21
12S009383F5U11Aspartyl protease family protein6.4±0.57-4.2±0.63
12S005565B2U12Glucose-6-phosphate translocator6.3±0.18-5.5±0.31
12S002570B10U13Limonoid udp-lucosyltransferase6.2±0.26-3.6±0.39
12S002915B3U14Cytochrome P4506.2±0.17-4.2±0.32
12S009437A12U15ATSI 2 hydrolyzing O-glycosyl compounds6.0±0.36-7.3±0.16
12S006649C4U16Isoamylase isoform 36.0±0.12-5.2±0.44
12S000876D2U17Cytochrome p4506.0±0.43-4.9±0.33
12S010715G3U18Cytochrome p4505.9±0.69-3.4±0.58
12S012744E11U19Proline-rich cell wall protein5.9±0.49-2.9±0.53
12S005981A5U20Organic cation transporter5.8±0.19-5.0±0.22
12S002410D4U21Thaumatin-like protein5.8±0.60-6.9±0.57
12S009693D11U22Starch phosphorylase5.8±0.44-4.2±0.34
12C000042G2U23Myb transcription factor5.7±0.54-4.1±0.70
12S013702A6U24Aldehyde dehydrogenase5.7±0.16-6.0±0.27
12C001360C1U25Protein5.6±0.33-6.0±0.38
12S011414E4U26GST5.6±0.11-3.9±0.56
12S000490A2U27Mate efflux family expressed5.5±0.15-3.5±0.10
12S012305B12U28Hypothetical protein5.5±0.25-4.2±0.37
12S006861C5U29Reductase 15.3±0.25-2.7±0.30
12S006594A11U30Molybdenum cofactor sulfurase4.9±0.22-6.2±0.28
12S007535A10U31Glyoxalase i4.6±0.13-2.3±0.22
12S005609D8U32Expansin-like protein4.6±0.14-3.7±0.46
12C000943D1U33Ef-1 a4.5±0.40-2.7±0.26
12S011005C3U34Alkaline a galactosidase4.4±0.35-4.7±0.33
12S000980D10U35Fatty acid hydroperoxide lyase4.4±0.29-3.6±0.39
12S002559B7U36Af303396_1udp-glucosyltransferase hra254.4±0.28-2.9±0.16
12S010472B11U37Tpa:gid1-like gibberellin receptor4.4±0.33-3.0±0.06
12S006784F8U38Glucose acyltransferase4.3±0.50-3.7±0.35
12S011826F12U39Ethephon-induced protein4.3±0.49-3.1±0.50
12S005921C7U40Fibrillin4.3±0.25-2.9±0.28
12S009053E6U41Condensation domain-containing protein4.2±0.46-3.0±0.11
12C000227E3U42Basic helix-loop-helixfamily protein4.2±0.14-2.7±0.44
12C000359E1U43Sucrose synthase4.1±0.21-3.6±0.22
12S010980A9U44Pyrroline-5-carboxylate synthetase4.1±0.44-5.7±0.34
12S002233E12U45Glyceraldehyde-3-phosphate dehydrogenase4.1±0.48-3.5±0.28
12C001111G5U46Chalcone-flavanone isomerase family expressed4.1±0.63-2.9±0.54
12S011777H10U47Abscisic acid responsive elements-binding factor4.0±0.92-2.3±0.15
12S009704A4U48Hypothetical protein4.0±0.14-3.0±0.16
12C001087H11U49Methionine gamma-lyase3.9±0.30-2.6±0.92
12S002496H2U50Protein kinase3.8±0.33-3.0±0.60

Table 2 . Genes in ‘Tamnara’ grapevines down-regulated in response to R. vitis inoculation and SA treatment.

Gene No. in EPP163JWAA SeriesNo. of slot blot, RT-PCRPutative functionRatio of signal intensity

R. vitisSA
12C001593D8D1Cell wall protein-50.2±0.8736.1±0.56
12S000426A2D2Expansin-45.6±0.2032.6±0.32
12S005616A10D3At5g25460 f18g18_200-33.9±0.4732.6±0.33
12C000040F5D4Cytochrome C oxidase polypeptide vc-21.7±0.9416.0±0.78
12C000966E7D5Endosperm specific-21.7±0.8216.1±0.65
12C000783B1D6Protease inhibitor seed storage lipid transfer protein-21.6±0.4015.2±0.41
12C000998E4D7Protein-18.1±0.8317.4±0.59
12S003758F1D8En/Spm-like transposon protein-17.9±0.9215.1±0.48
12C001094F12D9Meiosis 5-17.8±1.0913.8±0.89
12S013145A9D10Proline-rich protein apg isolog-16.4±0.2011.3±0.37
12S010548B12D11DNA heat shock N-terminal domain-containing protein-15.9±0.3912.3±0.49
12C000058G6D12Acid phosphatase-15.4±1.429.4±0.57
12C000034A6D13Expansin-14.9±0.2717.3±0.19
12C001244B6D14Heavy metal-associated domain-containing protein-14.7±0.315.5±0.40
12C000247A5D15SA-induced fragment 1 protein-13.8±0.2113.4±0.27
12S011527F2D16Cold induced-13.6±0.845.1±0.17
12C000203A4D17Rho GDP-dissociation inhibitor 1-13.4±0.267.8±0.20
12C001631E2D18Proline rich protein 2-13.1±0.7711.1±0.26
12S009428B10D19Hypothetical protein-13.1±0.4812.5±0.15
12C000933A7D20Fatty acid elongase-12.8±0.309.7±0.21
12S005808A1D21Proline-rich protein-10.8±0.0614.1±0.20
12C000939D5D22β-Ketoacyl-synthase-10.7±0.254.5±0.52
12C001125D10D23Xyloglucan endotransglycosylase-10.6±0.6410.8±0.43
12S011610D9D24Hypothetical protein-10.6±0.237.8±0.56
12S008346G7D25No hit-10.0±1.3512.0±0.57
12S008268F11D26Tonoplast membrane integral protein4-4-9.9±0.765.2±0.48
12S011177D3D27Hypothetical protein-9.7±0.497.5±0.19
12C000816C6D28Chloroplast chlorophyll a/b binding protein-9.7±0.4814.7±0.22
12C000797A8D29Chloroplast chlorophyll a/b binding protein-9.6±0.3010.5±0.34
12S005860E5D30CCHC-type integrase-9.5±0.283.6±0.65
12C000927G1D31Protein binding protein-8.8±0.745.8±0.51
12S008817C3D32LRR protein-8.5±0.156.2±0.39
12C001369D4D33Transferase family protein-8.4±0.2410.3±0.52
12S001577C1D34No hit-8.4±0.183.1±0.34
12S003449A3D35Mee60 (maternal effect embryo arrest 60)-8.3±0.2111.9±0.21
12C001193B9D36Yabby15 protein-8.1±0.286.8±0.36
12S009302G2D37Aspartyl protease family protein-8.0±0.745.7±0.73
12S011056B11D38At3g15630 msj11_3-7.9±0.253.0±0.25
12C001299E1D39Endo-β-glucanase precursor-7.8±0.464.7±0.46
12S013533H2D40Expansin-like protein a-7.7±0.422.2±0.66
12S013059G5D41Thaumatin-like protein-7.7±1.017.0±0.62
12C000786F7D42Tonoplast intrinsic protein-7.6±0.635.6±0.49
12S006978C10D43WRKY transcription factor 10-7.5±0.395.3±0.32
12C000038B8D44Hypothetical protein-7.5±0.324.8±0.18
12C001095G10D45α Tubulin 1-7.3±1.163.9±0.58
12S010793F10D46At5g44130 mln1_5-7.2±0.605.8±0.62
12C000621E8D47Chitinase-like protein-7.2±0.585.5±0.29
12C000406G3D48Glutamine synthetase-7.2±0.764.9±0.66
12C000508B7D49Nucleoid DNA-binding protein cnd41-7.1±0.186.7±0.35
12S009795C5D50Calmodulin-like protein-7.0±0.295.2±0.35

As shown in the Tables 1 and 2, some genes showed antagonistic expression patterns between induced genes of R. vitis-inoculated and SA-treated grapevines. The genes activated by R. vitis inoculation might be mediated by jasmonic acid (JA) or ethylene. The expression of 95 up-regulated genes was suppressed by R. vitis inoculation in SA-treated grapevine leaves. In contrast, the expression of 90 genes down-regulated by R. vitis inoculation increased in response to SA treatment. When R. vitis attacks the vines, highly induced JA-dependent responses might suppress the gene expression involved SA-dependent defense pathway due to pathway crosstalk by R. vitis. Antagonisms between JA or ethylene and SA signaling have been extensively studied (Dong 1998; Kunkel and Brooks 2002). A large number of genes, includingc lipoxygenase (LOX), lipid transferase, and genes related to secondary metabolisms (Creelman and Mullet 1997; Lin et al. 2007) that were involved in JA biosynthesis, have been shown to respond to wounding and R. vitis attack in grapevines. The crosstalk among a number of signaling molecules appears to be related to controlling defense systems in response to pathogen attacks in grapevines as in other plants (Shah 2003).

Genes in response to R. vitis, SA, and wound in ‘Tamnara’ grapevines

Transcriptional profiling in response to R. vitis with the 6,776 unigenes obtained from the R. vitis-inoculated and SA-treated ‘Tamnara’ grapevine cDNA library was conducted by use of gene specific oligonucleotide microarray chips. cDNA contigs responsive to R. vitis inoculation and SA treatment could be categorized into seven groups encoding proteins involved in defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell wall fortification, and transcription factors (Table 3). The expression level of 95 up-regulated and 90 down-regulated ESTs was confirmed by semiquantitative RT-PCR (Fig. 2 and 3) and RNA slot blot hybridization analysis (Fig. 5 and 6). Confirmation using the three expression profiling methods revealed that 5, 14, and 64 cDNAs were up-regulated by wound, SA-treatment, and R. vitis inoculation, while 10, 12, and 61 were down-regulated by each treatment, respectively (Table 4).

Table 3 . Defense-related cDNA responsive to R. vitis inoculation and SA treatment in ‘Tamnara’ grapevines.

??Gene??Putative function/homologyRatio of signal intensity

R. vitisSA
Defense-relatedβ-1,3-Glucanase-3.4±0.36-1.4±0.38
Chitinase-4.4±0.25-1.2±0.26
Basic endochitinase precursor2.3±0.37-1.7±0.32
Chitinase III-2.6±0.51-1.4±0.66
Thaumatin-like protein5.8±0.60-1.3±0.24
Chalcone synthase6.8±0.941.5±1.11

Signal transductionLOX9.3±0.52-1.2±0.69
NBS LRR-containing protein3.5±0.731.2±0.81

Active oxygen relatedCatalase2.5±0.71-1.1±0.70
Glutathione peroxidase3.2±0.191.1±0.42
Glutathione S-transferase5.6±0.111.4±0.56
Ascorbate peroxidase1.8±0.251.1±0.23

Secondary metabolitesCytochrome P4506.0±0.43-4.9±0.33
Cytochrome C oxidase subunit Vb1.8±0.211.1±0.33

Abiotic stress-relatedSmall heat shock protein3.5±0.291.3±0.35
Heat shock protein3.7±0.181.6±0.32

Cell wall fortificationProline-rich cell wall protein5.9±0.492.0±0.43

Transcription factorsWRKY transcription factor1.7±0.21-1.1±0.26
MYB transcription factor5.8±0.541.4±0.84

Figure 2.

Semiquantitative RT-PCR analysis of 95 highly up-regulated genes in ‘Tamnara’ grapevines. cDNA samples were amplified with a ReverTra-plus™-High Fidelity RT-PCR Kit. MAU: up-regulated in microarray analysis; C, control; W, wound; S, SA treatment; R, R. vitis inoculation; M, size marker


Figure 3.

Semiquantitative RT-PCR analysis of 90 highly down-regulated genes in ‘Tamnara’ grapevines. cDNA samples were amplified with a ReverTra-plus™-High Fidelity RT-PCR Kit. MAD: down-regulated in microarray analysis; C, control; W, wound; S, SA treatment; R, R. vitis inoculation; M, size marker


Figure 4.

Venn diagram of DEGs upregulated (A) and down-regulated (B) in ‘Tamnara’ grapevines responsive to R. vitis inoculation, wound, and SA treatment


Figure 5.

RNA slot blot hybridization analysis with (A) β-1,3-glucanase, (B) CHS, (C) LOX, and (D) proline-rich protein as a probe in several grapevine cultivars. C, control; R, R. vitis inoculation; W, wound


Figure 6.

RNA slot blot hybridization analysis with (A) ATSI 2 and (B) organic cation transporter as a probe in several grapevine cultivars. C, control; R, R. vitis inoculation; W, wound


Table 4 . Genes specifically expressed in response to wound, SA treatment, and R. vitis inoculation with microarray, RT-PCR, and slot blot hybridization analyses in ‘Tamnara’ grapevines.

A. Up-regulated genes

?Confirming methodWoundSAR. vitis
Microarray and slot blot783782
Microarray and RT-PCR73273
Microarray, slotblot, and RT-PCR51464

B. Down-regulated genes

?Confirming methodWoundSAR. vitis
Microarray and slot blot706471
Microarray and RT-PCR141674
Microarray, slotblot, and RT-PCR101261

As shown in the Venn diagram (Fig. 4A), 50 cDNAs, including ATSI protein 2 hydrolyzing O-glycosyl compounds yabby15 protein, CHS, cytochrome P450, thaumatin-like protein, GST, and LRR containing protein, were specifically activated by R. vitis inoculation in ‘Tamnara’ grapevine leaves. However, no cDNA was shown to be up-regulated by wound or SA treatment in grapevines. Five genes, LOX, aspartyl protease family protein, heavy metal-associated domain- containing protein, tyrosine aminotransferase, and one with unknown function, were commonly induced by R. vitis inoculation, wound, and SA treatments. Similarly, 1, 4, and 48 cDNAs were down-regulated by wound, SA treatment and R. vitis inoculation, respectively (Fig. 4B). Expansin, cytochrome C oxidase polypeptide vc, seed storage lipid transfer protein, meiosis 5, SA-induced fragment 1 protein, WRKY transcription factor 10, chitinase-like protein, and Zn finger (gata type) family proteins were specifically down-regulated by R. vitis inoculation. Four genes such as lipid transfer protein, extensin-like protein, and two proteins with unknown function were commonly inhibited under both R. vitis inoculation and SA treatment (Table 5). More detailed functional studies should be conducted to determine if they are involved in defense mechanisms. It has been reported that PR genes were activated by endo- and exogenous SA treatments and pathogen attacks in many plants (Malamy 1990). In this study, genes involved in plant defense responses such as thaumatin-like protein, chalcone synthase (CHS), and LOX were induced by R. vitis inoculation and SA treatment.

Table 5 . DEGs responsive to R. vitis inoculation, wound, and SA treatment in ‘Tamnara’ grapevines.

Up-regulatedDown-regulated
R. vitis?ATSI protein 2 hydrolyzing O-glycosyl compounds?Expansin
?yabby15 protein?Cytochrome C oxidase polypeptide vc
?CHS?Seed storage lipid transfer protein
?Cytochrome P450?Meiosis 5
?Thaumatin-like protein?Proline-rich protein apg isolog
?GST?SA-induced fragment 1 protein
?Sucrose synthase?WRKY transcription factor 10
?Small heat shock protein?Chitinase-like protein
?LRR containing protein?Zn finger (gata type) family protein

Wound-?Fasciclin-like arabinogalactan protein 14

SA-?Chloroplast chlorophyll a/b binding protein
?β-Glucanase-like protein
?Pollen-specific protein

R. vitis and SA?Btb and taz domain protein?Tonoplast membrane integral protein4-4
?Limonoid UDP-glucosyltransferase?Histone h3
?Alkaline α galactosidase

R. vitis and Wound-?Cell wall protein
?Glucose-methanol-cholineoxidoreductase family protein

R. vitis, SA, and Wound?LOX
?Aspartyl protease family protein?Lipid transfer protein
?Heavy metal-associated domain-containing protein?Extensin-like protein
?Tyrosine aminotransferase

In this study, some genes such as β-1,3-glucanase and chitinase III responded similarly to R. vitis inoculation and SA treatment. Using the GeneFishing and RACE technology, the full-length cDNA of several PR genes including β-1,3 glucanase, PR 10, and thaumatin-like proteins expressed specifically by R. vitis inoculation and SA treatment were cloned from the grapevine leaves (Choi et al. 2008). Cheong et al. (2002) reported that various genes such as LOX, catalase (CAT), glutathione S-transferase (GST), cytochrome P450, and WRKY, as well as MYB transcription factors were activated by wound stress in grapevines. A number of components in their signaling pathways appear to share the response against pathogen infection and wounding stress (Cheong et al. 2002; Maleck and Dietrich 1999). Accordingly, further functional analysis is required to determine the expression dynamics of selected genes in response to mechanical wounding, R. vitis inoculation, and SA treatment.

Semiquantitative RT-PCR and slot blot hybridization analysis of cDNA

The expression level of 95 up-regulated and 90 down-regulated ESTs was confirmed by semiquantitative RT-PCR (Fig. 2 and 3) and RNA slot blot hybridization analysis (Fig. 5 and 6). Confirmation using the three expression profiling methods revealed that 5, 14, and 64 cDNAs were up-regulated by wound, SA treatment, and R. vitis inoculation, while 10, 12, and 61 were down-regulated by these treatments, respectively (Table 4).

As shown in the Venn diagram (Fig. 4A), 50 cDNA samples, including ATSI protein 2 hydrolyzing O-glycosyl compounds yabby15 protein, CHS, cytochrome P450, thaumatin-like protein, GST, and LRR containing protein, were specifically activated by R. vitis inoculation in ‘Tamnara’ grapevine leaves. However, no cDNA was shown to be up-regulated by wound or SA treatment in grapevines. Five genes, LOX, aspartyl protease family protein, heavy metal-associated domain-containing protein, tyrosine aminotransferase, and one with unknown function, were commonly induced by R. vitis inoculation, wound, and SA treatments. Similarly, 1, 4, and 48 cDNAs were down-regulated by wound, SA treatment and R. vitis inoculation, respectively (Fig. 4B). Expansin, cytochrome C oxidase polypeptide vc, seed storage lipid transfer protein, meiosis 5, SA-induced fragment 1 protein, WRKY transcription factor 10, chitinase-like protein, and Zn finger (gata type) family proteins were specifically down-regulated by R. vitis inoculation. Four genes were commonly inhibited under both R. vitis inoculation and SA treatment, lipid transfer protein, extensin-like protein, and two proteins with unknown function (Table 5). The function of these genes is not clear yet, accordingly, more detailed functional studies should be conducted to determine if they are involved in defense mechanisms.

Comparative analysis of defense-related gene expression in R. vitis-inoculated grapevines

To investigate the expression of selected genes, RNA slot blot hybridization was performed using ‘Delaware’ and ‘Concond’, ‘Neomuscat’ and ‘Rizamat’, and ‘Tamnara’ grapevine leaves harvested at several time courses. When β-1,3-glucanase, CHS, LOX, proline-rich cell wall protein, ATSI 2, and organic cation transporter genes were used as probes for the hybridization analyses, differential expression was observed between resistant and susceptible cultivars in response to R. vitis inoculation and wound treatment (Fig. 5 and 6). The expression patterns of β-1,3-glucanase, proline-rich protein, and LOX genes in ‘Tamnara’ grapevines were similar to their expressions in ‘Concord’ and ‘Delaware’ which are resistant to crown gall disease. Although proline-rich cell wall protein genes were highly expressed within 12 to 72 h in ‘Concord’, ‘Delaware’, and ‘Tamnara’ grapevines inoculated with R. vitis, they showed low initial expression level, and increased level from 30 min to 12 h after R. vitis inoculation in ‘Neomuscat’ and ‘Rizamat’ grapevines, which are susceptible to crown gall. Conversely, the CHS gene in ‘Tamnara’ grapevines was expressed at levels similar to those of ‘Neomuscat’ and ‘Rizamat’ grapevines. Although transcripts of CHS increased at 48 to 72 h after R. vitis inoculation and wound treatment, they showed low expression at 0.5 to 12 h after R. vitis inoculation in ‘Neomuscat’, ‘Rizamat’, and ‘Tamnara’ grapevines. In the case of comparative analysis by Lin et al. (2002), PR genes were expressed in both resistant and susceptible grapevine cultivars in response to X. fastidiosa inoculation. These findings suggest that susceptible cultivars have a host defense response mechanism that responds to X. fastidiosa inoculation, but they may fail to protect themselves from pathogen infections.

Figure 7.

Slot blot hybridization analysis of 95 highly up-regulated genes in ‘Tamnara’ grapevines. cDNA probes were synthesized with a ReverTra-plus™-High Fidelity RT-PCR Kit. C, control; W, wound; S, SA treatment; R, R. vitis inoculation; VACT, actin cloned from ‘Tamnara’ grapevines


Figure 8.

Slot blot hybridization analysis of 90 highly down-regulated genes in ‘Tamnara’ grapevines. cDNA probes were synthesized with a ReverTra-plus™-High Fidelity RT-PCR Kit. C, control; W, wound; S, SA treatment; R, R. vitis inoculation; VACT, actin cloned from ‘Tamnara’ grapevines


ATSI2 and organic cation transporter genes were highly activated by R. vitis inoculation in ‘Tamnara’ grapevines as confirmed by microarray, RT-PCR, and slot blot hybridization analyses (Fig. 6). ATSI 2, formerly raffinose synthase, is a key enzyme that transfers sucrose into the raffinose oligosaccharide, an oligosaccharide commonly found in plant seeds and other tissues (Nishizawa et al. 2008). This gene is known to be activated by abiotic stressors such as frost, drought, and salt (Kandler and Hopf 1984; Keller and Pharr 1996; Peterbauer and Richter 2001; Peterbauer et al. 2000). The results of the present study suggest that expression of the ATSI2 gene was also related to the defense responses to R. vitis infection in grapevines.

To understand the resistant responses to disease in grapevines, it is important to monitor the specific expression of genes in response pathogen attacks or signal molecules accumulated by pathogens in vines. Among various strategies to screen for specific gene expression, microarray analysis can be used to analyze variations in the expression of thousands of genes simultaneously (Meyers et al. 2004; Schulze and Downward 2001; Stears 2003). In grapevines, microarray analysis has been used in investigations of transcriptomes related to berry development (Deluc et al. 2007; Terrier et al. 2005; Waters et al. 2006), water and salinity stress (Cramer et al. 2007), ultraviolet-B radiation (Pontin et al. 2010), and virus (Espinoza et al. 2007) and fungal infection (Figueiredo et al. 2008).

In this study, the gene expression patterns in response to R. vitis bacterium causing crown gall in grapevines were investigated in R. vitis-inoculated, salicylic acid (SA)-treated, wounded, and untreated ‘Tamnara’ grapevines. Microarray analysis using 12,000 gene oligonucleotides of microarray chips constructed with 6,776 unigenes based on EST sequencing revealed that 95 clones were up-regulated by more than 3 times and 90 were down-regulated by more than 5 times in R. vitis-inoculated ‘Tamnara’ grapevines than in untreated vines. Among these, 5 to 61 up-regulated genes, and 10 to 61 clones showed different expression levels in response to wound, SA, and R. vitis in grapevines upon RT-PCR and slot blot hybridization analysis. Some genes, such as β-1,3-glucanase, proline-rich protein, and LOX, were induced in moderately resistant cultivars, while others, such as CHS, were expressed in moderately resistant and susceptible grapevines in response to R. vitis. Further expression studies with various clones per each gene should be conducted to elucidate their roles in resistant responses to pathogens or other stimuli in grapevines. Identification and characterization of the putative genes involved in defense response will be useful for breeding grapes resistant to crown gall. These results could provide a better understanding of the mechanisms of defenses against crown gall disease and clues for identifying new genes that may play a role in defense responses to infection of R. vitis.

Fig 1.

Figure 1.

Venn diagrams showing the numbers of overlapped and unique genes induced (A) and suppressed (B) more than twice in the level of their expressions by R. vitis inoculation and salicylic acid (SA) treatment. Results were based on the mean inductions of three replicates

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 2.

Figure 2.

Semiquantitative RT-PCR analysis of 95 highly up-regulated genes in ‘Tamnara’ grapevines. cDNA samples were amplified with a ReverTra-plus™-High Fidelity RT-PCR Kit. MAU: up-regulated in microarray analysis; C, control; W, wound; S, SA treatment; R, R. vitis inoculation; M, size marker

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 3.

Figure 3.

Semiquantitative RT-PCR analysis of 90 highly down-regulated genes in ‘Tamnara’ grapevines. cDNA samples were amplified with a ReverTra-plus™-High Fidelity RT-PCR Kit. MAD: down-regulated in microarray analysis; C, control; W, wound; S, SA treatment; R, R. vitis inoculation; M, size marker

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 4.

Figure 4.

Venn diagram of DEGs upregulated (A) and down-regulated (B) in ‘Tamnara’ grapevines responsive to R. vitis inoculation, wound, and SA treatment

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 5.

Figure 5.

RNA slot blot hybridization analysis with (A) β-1,3-glucanase, (B) CHS, (C) LOX, and (D) proline-rich protein as a probe in several grapevine cultivars. C, control; R, R. vitis inoculation; W, wound

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 6.

Figure 6.

RNA slot blot hybridization analysis with (A) ATSI 2 and (B) organic cation transporter as a probe in several grapevine cultivars. C, control; R, R. vitis inoculation; W, wound

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 7.

Figure 7.

Slot blot hybridization analysis of 95 highly up-regulated genes in ‘Tamnara’ grapevines. cDNA probes were synthesized with a ReverTra-plus™-High Fidelity RT-PCR Kit. C, control; W, wound; S, SA treatment; R, R. vitis inoculation; VACT, actin cloned from ‘Tamnara’ grapevines

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Fig 8.

Figure 8.

Slot blot hybridization analysis of 90 highly down-regulated genes in ‘Tamnara’ grapevines. cDNA probes were synthesized with a ReverTra-plus™-High Fidelity RT-PCR Kit. C, control; W, wound; S, SA treatment; R, R. vitis inoculation; VACT, actin cloned from ‘Tamnara’ grapevines

Journal of Plant Biotechnology 2016; 43: 37-48https://doi.org/10.5010/JPB.2016.43.1.37

Table 1 . Genes in ‘Tamnara’ grapevines up-regulated in response to R. vitis inoculation and SA treatment.

Gene No. in EPP163JWAA seriesNo. of slot blot, RT-PCRPutative functionRatio of signal intensity

R. vitisSA
12C000256B9U1ATSI 2 hydrolyzing O-glycosyl compounds yabby15 protein15.9±0.49-14.2±0.44
12S013353F4U2Btb and taz domain protein11.2±0.85-4.2±0.27
12C000750F2U3Metallothionein-like protein10.3±0.63-6.0±0.79
12C000845D7U4LOX9.3±0.52-11.1±0.66
12C001644B6U5Dark inducible 10 hydrolyzing O-glycosyl compounds7.6±0.46-7.8±0.36
12S000483H3U6No hit7.6±0.70-3.5±0.81
12S008848H1U7Hypothetical protein7.4±0.19-3.1±0.71
12S003419A7U8Glycosyl hydrolase family 1 protein7.0±0.15-4.8±0.25
12S007850G6U9CHS6.8±0.94-4.5±0.72
12S003388A3U10Glycosyl hydrolase family 1 protein6.5±0.07-4.7±0.21
12S009383F5U11Aspartyl protease family protein6.4±0.57-4.2±0.63
12S005565B2U12Glucose-6-phosphate translocator6.3±0.18-5.5±0.31
12S002570B10U13Limonoid udp-lucosyltransferase6.2±0.26-3.6±0.39
12S002915B3U14Cytochrome P4506.2±0.17-4.2±0.32
12S009437A12U15ATSI 2 hydrolyzing O-glycosyl compounds6.0±0.36-7.3±0.16
12S006649C4U16Isoamylase isoform 36.0±0.12-5.2±0.44
12S000876D2U17Cytochrome p4506.0±0.43-4.9±0.33
12S010715G3U18Cytochrome p4505.9±0.69-3.4±0.58
12S012744E11U19Proline-rich cell wall protein5.9±0.49-2.9±0.53
12S005981A5U20Organic cation transporter5.8±0.19-5.0±0.22
12S002410D4U21Thaumatin-like protein5.8±0.60-6.9±0.57
12S009693D11U22Starch phosphorylase5.8±0.44-4.2±0.34
12C000042G2U23Myb transcription factor5.7±0.54-4.1±0.70
12S013702A6U24Aldehyde dehydrogenase5.7±0.16-6.0±0.27
12C001360C1U25Protein5.6±0.33-6.0±0.38
12S011414E4U26GST5.6±0.11-3.9±0.56
12S000490A2U27Mate efflux family expressed5.5±0.15-3.5±0.10
12S012305B12U28Hypothetical protein5.5±0.25-4.2±0.37
12S006861C5U29Reductase 15.3±0.25-2.7±0.30
12S006594A11U30Molybdenum cofactor sulfurase4.9±0.22-6.2±0.28
12S007535A10U31Glyoxalase i4.6±0.13-2.3±0.22
12S005609D8U32Expansin-like protein4.6±0.14-3.7±0.46
12C000943D1U33Ef-1 a4.5±0.40-2.7±0.26
12S011005C3U34Alkaline a galactosidase4.4±0.35-4.7±0.33
12S000980D10U35Fatty acid hydroperoxide lyase4.4±0.29-3.6±0.39
12S002559B7U36Af303396_1udp-glucosyltransferase hra254.4±0.28-2.9±0.16
12S010472B11U37Tpa:gid1-like gibberellin receptor4.4±0.33-3.0±0.06
12S006784F8U38Glucose acyltransferase4.3±0.50-3.7±0.35
12S011826F12U39Ethephon-induced protein4.3±0.49-3.1±0.50
12S005921C7U40Fibrillin4.3±0.25-2.9±0.28
12S009053E6U41Condensation domain-containing protein4.2±0.46-3.0±0.11
12C000227E3U42Basic helix-loop-helixfamily protein4.2±0.14-2.7±0.44
12C000359E1U43Sucrose synthase4.1±0.21-3.6±0.22
12S010980A9U44Pyrroline-5-carboxylate synthetase4.1±0.44-5.7±0.34
12S002233E12U45Glyceraldehyde-3-phosphate dehydrogenase4.1±0.48-3.5±0.28
12C001111G5U46Chalcone-flavanone isomerase family expressed4.1±0.63-2.9±0.54
12S011777H10U47Abscisic acid responsive elements-binding factor4.0±0.92-2.3±0.15
12S009704A4U48Hypothetical protein4.0±0.14-3.0±0.16
12C001087H11U49Methionine gamma-lyase3.9±0.30-2.6±0.92
12S002496H2U50Protein kinase3.8±0.33-3.0±0.60

Table 2 . Genes in ‘Tamnara’ grapevines down-regulated in response to R. vitis inoculation and SA treatment.

Gene No. in EPP163JWAA SeriesNo. of slot blot, RT-PCRPutative functionRatio of signal intensity

R. vitisSA
12C001593D8D1Cell wall protein-50.2±0.8736.1±0.56
12S000426A2D2Expansin-45.6±0.2032.6±0.32
12S005616A10D3At5g25460 f18g18_200-33.9±0.4732.6±0.33
12C000040F5D4Cytochrome C oxidase polypeptide vc-21.7±0.9416.0±0.78
12C000966E7D5Endosperm specific-21.7±0.8216.1±0.65
12C000783B1D6Protease inhibitor seed storage lipid transfer protein-21.6±0.4015.2±0.41
12C000998E4D7Protein-18.1±0.8317.4±0.59
12S003758F1D8En/Spm-like transposon protein-17.9±0.9215.1±0.48
12C001094F12D9Meiosis 5-17.8±1.0913.8±0.89
12S013145A9D10Proline-rich protein apg isolog-16.4±0.2011.3±0.37
12S010548B12D11DNA heat shock N-terminal domain-containing protein-15.9±0.3912.3±0.49
12C000058G6D12Acid phosphatase-15.4±1.429.4±0.57
12C000034A6D13Expansin-14.9±0.2717.3±0.19
12C001244B6D14Heavy metal-associated domain-containing protein-14.7±0.315.5±0.40
12C000247A5D15SA-induced fragment 1 protein-13.8±0.2113.4±0.27
12S011527F2D16Cold induced-13.6±0.845.1±0.17
12C000203A4D17Rho GDP-dissociation inhibitor 1-13.4±0.267.8±0.20
12C001631E2D18Proline rich protein 2-13.1±0.7711.1±0.26
12S009428B10D19Hypothetical protein-13.1±0.4812.5±0.15
12C000933A7D20Fatty acid elongase-12.8±0.309.7±0.21
12S005808A1D21Proline-rich protein-10.8±0.0614.1±0.20
12C000939D5D22β-Ketoacyl-synthase-10.7±0.254.5±0.52
12C001125D10D23Xyloglucan endotransglycosylase-10.6±0.6410.8±0.43
12S011610D9D24Hypothetical protein-10.6±0.237.8±0.56
12S008346G7D25No hit-10.0±1.3512.0±0.57
12S008268F11D26Tonoplast membrane integral protein4-4-9.9±0.765.2±0.48
12S011177D3D27Hypothetical protein-9.7±0.497.5±0.19
12C000816C6D28Chloroplast chlorophyll a/b binding protein-9.7±0.4814.7±0.22
12C000797A8D29Chloroplast chlorophyll a/b binding protein-9.6±0.3010.5±0.34
12S005860E5D30CCHC-type integrase-9.5±0.283.6±0.65
12C000927G1D31Protein binding protein-8.8±0.745.8±0.51
12S008817C3D32LRR protein-8.5±0.156.2±0.39
12C001369D4D33Transferase family protein-8.4±0.2410.3±0.52
12S001577C1D34No hit-8.4±0.183.1±0.34
12S003449A3D35Mee60 (maternal effect embryo arrest 60)-8.3±0.2111.9±0.21
12C001193B9D36Yabby15 protein-8.1±0.286.8±0.36
12S009302G2D37Aspartyl protease family protein-8.0±0.745.7±0.73
12S011056B11D38At3g15630 msj11_3-7.9±0.253.0±0.25
12C001299E1D39Endo-β-glucanase precursor-7.8±0.464.7±0.46
12S013533H2D40Expansin-like protein a-7.7±0.422.2±0.66
12S013059G5D41Thaumatin-like protein-7.7±1.017.0±0.62
12C000786F7D42Tonoplast intrinsic protein-7.6±0.635.6±0.49
12S006978C10D43WRKY transcription factor 10-7.5±0.395.3±0.32
12C000038B8D44Hypothetical protein-7.5±0.324.8±0.18
12C001095G10D45α Tubulin 1-7.3±1.163.9±0.58
12S010793F10D46At5g44130 mln1_5-7.2±0.605.8±0.62
12C000621E8D47Chitinase-like protein-7.2±0.585.5±0.29
12C000406G3D48Glutamine synthetase-7.2±0.764.9±0.66
12C000508B7D49Nucleoid DNA-binding protein cnd41-7.1±0.186.7±0.35
12S009795C5D50Calmodulin-like protein-7.0±0.295.2±0.35

Table 3 . Defense-related cDNA responsive to R. vitis inoculation and SA treatment in ‘Tamnara’ grapevines.

??Gene??Putative function/homologyRatio of signal intensity

R. vitisSA
Defense-relatedβ-1,3-Glucanase-3.4±0.36-1.4±0.38
Chitinase-4.4±0.25-1.2±0.26
Basic endochitinase precursor2.3±0.37-1.7±0.32
Chitinase III-2.6±0.51-1.4±0.66
Thaumatin-like protein5.8±0.60-1.3±0.24
Chalcone synthase6.8±0.941.5±1.11

Signal transductionLOX9.3±0.52-1.2±0.69
NBS LRR-containing protein3.5±0.731.2±0.81

Active oxygen relatedCatalase2.5±0.71-1.1±0.70
Glutathione peroxidase3.2±0.191.1±0.42
Glutathione S-transferase5.6±0.111.4±0.56
Ascorbate peroxidase1.8±0.251.1±0.23

Secondary metabolitesCytochrome P4506.0±0.43-4.9±0.33
Cytochrome C oxidase subunit Vb1.8±0.211.1±0.33

Abiotic stress-relatedSmall heat shock protein3.5±0.291.3±0.35
Heat shock protein3.7±0.181.6±0.32

Cell wall fortificationProline-rich cell wall protein5.9±0.492.0±0.43

Transcription factorsWRKY transcription factor1.7±0.21-1.1±0.26
MYB transcription factor5.8±0.541.4±0.84

Table 4 . Genes specifically expressed in response to wound, SA treatment, and R. vitis inoculation with microarray, RT-PCR, and slot blot hybridization analyses in ‘Tamnara’ grapevines.

A. Up-regulated genes

?Confirming methodWoundSAR. vitis
Microarray and slot blot783782
Microarray and RT-PCR73273
Microarray, slotblot, and RT-PCR51464

B. Down-regulated genes

?Confirming methodWoundSAR. vitis
Microarray and slot blot706471
Microarray and RT-PCR141674
Microarray, slotblot, and RT-PCR101261

Table 5 . DEGs responsive to R. vitis inoculation, wound, and SA treatment in ‘Tamnara’ grapevines.

Up-regulatedDown-regulated
R. vitis?ATSI protein 2 hydrolyzing O-glycosyl compounds?Expansin
?yabby15 protein?Cytochrome C oxidase polypeptide vc
?CHS?Seed storage lipid transfer protein
?Cytochrome P450?Meiosis 5
?Thaumatin-like protein?Proline-rich protein apg isolog
?GST?SA-induced fragment 1 protein
?Sucrose synthase?WRKY transcription factor 10
?Small heat shock protein?Chitinase-like protein
?LRR containing protein?Zn finger (gata type) family protein

Wound-?Fasciclin-like arabinogalactan protein 14

SA-?Chloroplast chlorophyll a/b binding protein
?β-Glucanase-like protein
?Pollen-specific protein

R. vitis and SA?Btb and taz domain protein?Tonoplast membrane integral protein4-4
?Limonoid UDP-glucosyltransferase?Histone h3
?Alkaline α galactosidase

R. vitis and Wound-?Cell wall protein
?Glucose-methanol-cholineoxidoreductase family protein

R. vitis, SA, and Wound?LOX
?Aspartyl protease family protein?Lipid transfer protein
?Heavy metal-associated domain-containing protein?Extensin-like protein
?Tyrosine aminotransferase

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