• MATERIALS AND METHODS
• Plant materials and pathogens
• Inoculation of Pathogens
• RNA isolation and semi-quantitative RT-PCR analysis
• RESULTS AND DISCUSSION
• 적요
• ACKNOWLEDGEMENTS

 Grape (Vi t is sp.) is the most economically important fruit crop worldwide, however, cultivated grapevine is sub-ject to a number of bacterial, fungal, and viral diseases(Wang et al., 2011). Grape anthracnose, caused by Elsinoe ampelina Shear, reduces the productivity and fruit quality in European grapes (Vitis vinifera), particularly, and V. vinifera hybrids growing in warm and humid conditions (Mirica, 1994). Young and green succulent shoots are most suscepti-ble to this disease, and often growing points of shoots are G killed. Symptoms on berry consist of whitish-gray lesions with a dark margin (Magarey et al., 1993).

 The grapevine crown gall disease causes a serious eco-nomic loss by the significant inferior growth and reduction of fruit productivity in the majority of grape-growing regions in the whole world (Burr et al., 1998). Damage caused by galls frequently surpasses that of the initial injury (Young et al., 2001). The major symptom of crown gall in grapevines is white, fleshy, and callus-like overgrowth on vine trunks(Lehoczky, 1968). The various defense related genes expressed in the leaves of ‘Tamnara’ grapevine inoculated with  Rhizobium vitis or treated with salicylic acid and Korean wild grape (Choi et al., 2008; 2010; 2011).

 The spreads of damage caused by E. ampelina and R. vitis in grapevines have demanded for the development of a dis-ease control system such as breeding resistant cultivars. The identification of genes expressed during disease resistance could provide informative sources in developing new culti-vars and in understanding disease resistance responses in plants. This study aimed to screen the differential expression of genes related with defense response in Korean wild grapevine, V. flexuosa by inoculation of E. ampelina and R. vitis and to provide information and resources in disease resistant grape breeding programs.

MATERIALS AND METHODS

Plant materials and pathogens

 Leaves of V. flexuosa VISKO001, which was maintained in a grapevine germplasm collection field of Yeungnam University, Gyeongsan, were used for gene expression anal-ysis by pathogen inoculation. The pathogens used in this study were virulent strain of E. ampelina (EA-1), which was isolated from infected leaves by Dr. W.K. Kim, National Academy of Agricultural Science, RDA, Korea and R. vitis strain Cheonan 493 gifted from Prof. J.S. Cha, Chungbuk National University, Korea.

Inoculation of Pathogens

 Several colonies of the E. ampelina were incubated in a shaking incubator (140 rpm) at 28℃ for 10 d. The cultures were harvested by centrifugation, ground in a homogenizer in sterile distilled water, then poured onto V-8 juice agar medium (20% (v/v) V-8 juice, 2% (w/v) agar) and incubated at 28℃ under a near ultraviolet lamp for 2 d, to produce spores of the pathogen (Yun et al., 2003). Spores of the E. ampelina were collected by scraping-off the plates with ster-ile distilled water. The concentration was adjusted to 105 spores/ml, then sprayed onto leaves. Leaves inoculated with a spore suspension were incubated in a moist box at 28℃ for 48 h.

 After incubating the pathogen in Fries medium at 28℃ for 21 d, cell-free culture filtrates (CFCF) of E. ampelina were collected from the supernatant by centrifugation and steril-ized by ultra-filtration (0.2 µm pore diameter). Leaves were injured slightly with a pencil tip and 30 µl of E. ampelina culture filtrate were dropped onto the wounded portion of leaves.

 A single colony for bacteria was grown in YEP medium(yeast extract 1 g, beef extract 5 g, peptone 5 g, sucrose 5 g, MgSO₄ 0.5 g/L, pH7.2) at 28℃ in a shaking incubator and then they spun down by centrifugation and resuspended with sterile water. Leaves were injured slightly with a pencil tip and 200 µl of R. vitis cell suspension were dropped onto the wounded portion of leaves. Leaves were harvested at the indicated time points (0, 6, 24, and 48 h) after inoculation, immediately frozen in liquid nitrogen, and then stored at −80℃ for future use.

RNA isolation and semi-quantitative RT-PCR analysis

 Total RNAs were extracted from grapevine leaves using the modified pine tree method (Chang et al., 1993). The dif-ferential expressions of genes were confirmed by semi-quantitative RT-PCR using 16 gene specific primer pairs(Table 1). From the total RNA (1 µg), first-strand cDNA was synthesized using the PrimeScript^TM  1^st strand cDNA synthesis kit (Takara Bio Inc., Japan) and subsequently used as the template for PCR. The actin gene primers were used as an internal control in this study. The PCR reaction was performed as follows; an initial 5 min of denaturation at 94℃ ; 35 cycles at 94℃ for 45 sec, 55℃ for 45 sec, and 72℃ for 1 min; and final 7 min incubation at 72℃. The PCR products were identified by 1% (W/V) agarose gel electrophoresis with 0.5X TBE running buffer.

Table 1. Sequences of primers used for RT-PCR analysis in this study.

RESULTS AND DISCUSSION

 The expression levels of selected 16 genes in V. flexuosa grapevines inoculated with E. ampelina and R. vitis were confirmed using semi-quantitative RT-PCR. The expres-sions of plant defense-related genes such as thaumatin-like protein (TLP), glutathione peroxidase (GPX), glutathione S-transferase (GST), chalcone synthesis (CHS), protein kinase regulator (14-3-3), and â-glucanase-like protein (Glu), and signal transduction-related genes such as leucine reach-repeat (LRR) and lipoxygenase (LOX), and cell wall modi-fication-related gene such as polygalacturonase-inhibiting protein (PGIP) were induced by spore suspension inocula-tion of E. ampelina (Table 2). While WRKY transcription factor 10 (WRKY), fatty acid elongase (FAE), tonoplast intrinsic protein (TIP), hypothetical protein, and meiosis 5(Mei5) were also up-regulated, and proline-rich protein(PRPs) were down-regulated by both spore inoculation and CF treatment of E. ampelina. (Table 2 and Fig. 1, 3).

Table 2. Expression of genes responsive to spore suspension and culture filtrates of E. ampelina and R. vitis in V. flexuosa.

Fig. 1. Semiquantitative RT-PCR analysis of 16 defense-related gene expressions in V. flexuosa against inoculation of spore and treatment of culture filtrates (CF) of E. ampelina. Up-regulated genes (A) and down-regulated genes (B) by pathogen inoculation and culture filtrate (CF) treatment.

Fig. 2. Semi-quantitative RT-PCR analysis of 16 defense-related gene expressions in V. flexuosa inoculated with R. vitis. Up-regulated genes (A) and down-regulated genes (B) by pathogen inoculation.

Fig. 3. Venn diagrams of DEGs in Vitis flexuosa responsive to spore suspension (spore) and culture filtrates (CF) of Elsinoe ampelina, and R. vitis. A, up-regulated genes; B, down-regulated genes.

 Among 15 up-regulated genes, LRR, LOX, TLP, and GST were particularly up-regulated by spore inoculation than CF treatment of E. ampelina. On the contrary, CHS and TIP were highly up-regulated by CF treatment than spore inocu-lation of E. ampelina.

 It was reported that chitinase, stilbene synthase, protein/sugar kinase and transcriptional factor genes were found uniquely expressed in anthracnose tolerant upon  E. ampelina infection in Florida hybrid bunch grape cultivars(Vasanthiaiah et al., 2010).  Chitinase and stilbene synthase genes were reported to be involved in regulation of fungal growth and development in grapevines (Hammerschmidt, 1999; Jayasankar et al., 2000). In this study, chitinase-like protein was induced slightly by spore inoculation, while down-regulated 48 h after by CF treatment of E. ampelina. It suggests that spores in suspension secrete various signal molecules to induce defense mechanism in plants compared to CF from E. ampelina during attacking them.

 In this study, WRKY protein genes and genes related with cell wall modifications, such as PGIP, were up-regulated by both spore inoculation and CF treatment of E. ampelina. It is consistent with reports that WRKY transcription factor 10(WRKY) and PGIP were regulated by pathogen attack, fun-gal elicitors, and salicylic acid (Eulgem et al., 2000; Maleck et al., 2000; Rushton et al.,  1996; Schenk et al., 2000).

Genes of LRR, CLP, LOX, TLP, GPX, 14-3-3, GST, PGIP, FAE, TIP, Glu, Mei5, and WRKY were up-regulated, and genes of CHS and PRPs were down-regulated by R. vitis inoculation in  V. flexuosa grapevines (Table 2 and Fig. 2, 3). TLP gene and active oxygen species-related genes such as GPX and GST were highly activated, while other genes were slightly activated by inoculation of R. vitis in V. flexuosa grapevines. 

Table 3. Specifically expressed genes in V. flexuosa against inoculation of spore and treatment of culture filtrates (CF) of E. ampelina, and inoculation of R. vitis.

 In ‘Tamnara’ grapevine leaves, genes involved in plant defense responses such as TLP, CHS, and LOX were induced by both R. vitis inoculation and SA treatment (Choi et al., 2008). Choi et al. (2008) also reported that the acti-vated genes by R. vitisinoculation might be mediated by jas-monic acid (JA) or ethylene. LOX, lipid transferase, and ones related with secondary metabolisms which were involved in JA biosynthesis were found to be responsive to wound and R. vitis attack in grapevines (Creelman and Mul-let, 1997; Dong, 1998; Lin et al., 2007). Genes of JA-depen-dent responses such as CLP, LOX, TLP, GPX were highly induced in V. flexuosa vines which were inoculated with R. vitis in this study.

In this study, 16 genes related with SA-, AOS-, JA-depen-dent defense responses in V. flexuosa were screened for their differential expression against bacterial and fungal pathogen attacks. Most of genes tested in this study were induced by pathogen inoculations or CF treatment of pathogen. Analy-sis results of their differential expression of defense-related genes in V. flexuosa grapevines native to Korea could provide very valuable resources in molecular breeding program of dis-ease-resistant grapes and important information in elucidating the mechanism of resistance to diseases in grapevines. Sequences of genes with specific expression to each pathogen could be valuable in develop molecular markers based on SNPS/IndDels in disease resistant grape breeding programs.

ACKNOWLEDGEMENTS

 This work was supported by a grant from the Next-Gener-ation BioGreen 21 program (No. PJ008213), Rural Devel-opment Administration, Republic of Korea, and by the Technology Innovation Program (Industrial Strategic Tech-nology Development Program, 10033630) funded by the Ministry of Knowledge Economy (MKE, Korea).