Transient expression of glyoxal oxidase from the Chinese wild grape Vitis pseudoreticulata can suppress powdery mildew in a susceptible genotype

Vitis pseudoreticulata glyoxal oxidase (VpGLOX) was previously isolated from the Chinese wild vine V. pseudoreticulata accession “Baihe-35-1” during a screen for genes that are upregulated in response to infection with grapevine powdery mildew (Erysiphe necator, PM). In the present study, a possible function of VpGLOX for defense against PM was investigated using Agrobacterium-mediated transient expression. After optimizing agro-infiltration, VpGLOX was transiently overexpressed in leaves of either PM-susceptible (accession “6-12-2”) or PM-resistant (accession “6-12-6”) plants. The efficiency of transfection was verified using a β-glucuronidase (GUS) reporter and was found to comprise most leaf areas regardless of the initial leaf position. Upon infection with E. necator, clear differences were observed with respect to hyphal development between agro-infiltrated leaves and control groups of both, the susceptible and the resistant, genotypes. The expression of VpGLOX was followed by real-time polymerase chain reaction in both genotypes. Whereas in the susceptible host (“6-12-2”) expression was found to increase only in transfected leaves and remained transient, in the resistant host (“6-12-6”), a second peak appeared later in transfected leaves, probably representing the response of the endogenous VpGLOX. The data support the interpretation that VpGLOX is sufficient to confer resistance to E. necator.     

Expression and functional analysis of two genes encoding transcription factors, VpWRKY1 and VpWRKY2, isolated from Chinese wild Vitis pseudoreticulata

In this study, two WRKY genes were isolated from Erysiphe necator-resistant Chinese wild Vitis pseudoreticulata W. T. Wang ‘Baihe-35-1’, and designated as VpWRKY1 (GenBank accession no. GQ884198) and VpWRKY2 (GenBank accession no. GU565706). Nuclear localization of the two proteins was demonstrated in onion epidermal cells, while trans-activation function was confirmed in the leaves of ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was induced rapidly by salicylic acid treatment in ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was also induced rapidly by E. necator infection in 11 grapevine genotypes; the maximum induction of VpWRKY1 was greater in E. necator-resistant grapevine genotypes than in susceptible ones post E. necator inoculation. Furthermore, ectopic expression of VpWRKY1 or VpWRKY2 in Arabidopsis enhanced resistance to powdery mildew Erysiphe cichoracearum, and enhanced salt tolerance of transgenic plants. VpWRKY2 also enhanced cold tolerance of transgenic plants. In addition, the two proteins were shown to regulate the expression of some defense marker genes in Arabidopsis and grapevine. The data suggest that VpWRKY1 and VpWRKY2 may underlie the resistance in transgenic grapevine to E. necator and tolerance to salt and cold stresses.     

<Emphasis Type="Italic">VpUR9</Emphasis>, a novel RING-type ubiquitin ligase gene from <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>, is involved in powdery mildew response in transgenic <Emphasis Type="Italic">V. vinifera</Emphasis> plants

Ubiquitination plays important roles in disease resistance in plants. We report the identification and functional characterization of the RING-type ubiquitin ligase gene VpUR9 from Chinese wild Vitis pseudoreticulata accession Baihe-35-1. VpUR9, encodes 164 amino acids and possesses a RING conserved motif. It is homologously cloned from the cDNA library of the high powdery mildew (Erysiphe necator [Schw.] Burr) resistant V. pseudoreticulata accession Baihe-35-1 inoculated with E. necator. The gene is induced in response to powdery mildew and salicylic acid. VpUR9 fused with FLAG-tag controlled by 35S promoter was transformed into 15 regenerated V. vinifera L. cv. Red Globe lines via Agrobacterium tumefaciens-mediated transformation. Twelve of these lines were confirmed by Western blot of FLAG-tag. As a result, the powdery mildew-resistance of Red Globe transformed with VpUR9 was repressed. Furthermore, the expression of some disease-resistant related genes (NPR1, PR1, PR10 and PAL) of the transgenic Red Globe declined compared with wild type grapes when inoculated with powdery mildew or salicylic acid. When treated with jasmonic acid methyl ester, its PR1 gene expression decreased, while the expressions of NPR1, PR10 and PAL all increased, contrasting with the wild type grape.     

Transient silencing of VvCSN5 enhances powdery mildew resistance in grapevine (Vitis vinifera)

As one of the most economically important fruit crops in the world, the grapevine (Vitis vinifera) suffers significant yield losses from various pathogens including powdery mildew caused by Erysiphe necator. In contrast, several wild Chinese grapevines, including Vitis pseudoreticulata accession Baihe-35-1, are highly resistant to powdery mildew pathogens. Here, we identified a grapevine gene CSN5 (COP9 signalosome complex subunit 5), designated VvCSN5, that was differentially expressed between the resistant ‘Baihe-35-1’ and susceptible ‘Thompson Seedless’ during powdery mildew isolate Erysiphe necator NAFU1 infection. Moreover, transient silencing of VvCSN5 in ‘Thompson Seedless’ leaves enhanced resistance to En NAFU1. This resistance manifested in cell wall callose deposition at attempted infection sites and hypersensitive response-like cell death of penetrated epidermal cells. Several defense-related marker genes (VvPR1, VvPR3, VvPAD4, and VvRBOHD) had higher basal expression levels in VvCSN5-silenced leaves. In addition, we found the structure and activity of CSN5 promoters in ‘Thompson Seedless’ and ‘Baihe-35-1’ were different, which may have been behind their different resistances to powdery mildew infection. Taken together, these results implied that grapevine CSN5 plays an important role in the response to powdery mildew infection.

     

Screening proteins interacting with VpPR10.1 of Chinese wild grapevine using the yeast two-hybrid system

Among the 17 plant pathogenesis-related (PR) protein families, only PR10 family is intracellular and cytosolic. PR proteins are expressed in response to pathogen challenge and abiotic stresses in higher plants. However, the molecular mechanisms of their actions remain poorly understood. In a previous work, we isolated a PR10 gene from Erysiphe necator-resistant Chinese wild Vitis sp. (Baihe-35-1) and it was designated as VpPR10.1. In this study, yeast two-hybrid method was used to screen proteins interacting with VpPR10.1 proteins. Twenty-one ESTs were isolated and sequenced. All sequences were compared using BLASTx to identify presumptive orthologs. Several proteins associated with VpPR10.1 protein were screened, including CNR8, UFGT6, HSP, DEAD-box, Trx h2, Grx C9 and GLOX. These proteins are closely related to defensive action of plants against pathogens and abiotic stresses. Our results reveal that VpPR10.1 gene may be involved in hormone signaling, programmed cell death and defense responses of grapevine.     

Glyoxal oxidases: their nature and properties

H₂O₂ has been found to be required for the activity of the main microbial enzymes responsible for lignin oxidative cleavage, peroxidases. Along with other small radicals, it is implicated in the early attack of plant biomass by fungi. Among the few extracellular H₂O₂-generating enzymes known are the glyoxal oxidases (GLOX). GLOX is a copper-containing enzyme, sharing high similarity at the level of active site structure and chemistry with galactose oxidase. Genes encoding GLOX enzymes are widely distributed among wood-degrading fungi especially white-rot degraders, plant pathogenic and symbiotic fungi. GLOX has also been identified in plants. Although widely distributed, only few examples of characterized GLOX exist. The first characterized fungal GLOX was isolated from Phanerochaete chrysosporium. The GLOX from Utilago maydis has a role in filamentous growth and pathogenicity. More recently, two other glyoxal oxidases from the fungus Pycnoporus cinnabarinus were also characterized. In plants, GLOX from Vitis pseudoreticulata was found to be implicated in grapevine defence mechanisms. Fungal GLOX were found to be activated by peroxidases in vitro suggesting a synergistic and regulatory relationship between these enzymes. The substrates oxidized by GLOX are mainly aldehydes generated during lignin and carbohydrates degradation. The reactions catalysed by this enzyme such as the oxidation of toxic molecules and the production of valuable compounds (organic acids) makes GLOX a promising target for biotechnological applications. This aspect on GLOX remains new and needs to be investigated.     

A Pathogenesis Related Protein,VpPR-10.1, from Vitis pseudoreticulata: An Insight of Its Mode of Antifungal Activity

Previously, VpPR-10.1 was isolated and characterized from a cDNA library of a fungus-resistant accession of Chinese wild grape (Vitis pseudoreticulata). We found that expression of VpPR-10.1 is affected by the fungal pathogen Erysiphe necator. To investigate the biochemical basis of the nuclease activity of VpPR-10.1 and its role in antifungal resistance, we generated recombinant VpPR-10.1 as well as site-directed mutations targeting three conserved amino acid residues among plant PR-10 s: Lys55, Glu149, and Tyr151. We showed that wild-type recombinant VpPR-10.1 exhibits both RNase and DNase activities. Mutant VpPR10.1-Y151H essentially retained all these activities. In contrast, VpPR10.1-K55N, where Lys55 in the P-loop region is mutated to Asn, and VpPR10.1-E149G, where Glu149 is mutated to Gly, lost their nuclease activity, indicating that both residues play a critical role in catalyzing RNA and DNA degradation. Furthermore, VpPR10.1 and VpPR10.1-Y151H inhibited the growth of the cultured fungal pathogen Alternaria alternate. Through transient expression in grapevine, we also demonstrated that VpPR10.1-K55N and VpPR10.1-E149G compromised resistance to E. necator. Finally, we further found that VpPR-10.1 can lead to programmed cell death and DNA degradation when incubated with tobacco BY-2 suspension cells. We show here that Lys55 and Glu149, but not Tyr151, are required for the RNase, DNase and antifungal activities of VpPR-10.1. The strong correlation between the level of VpPR-10.1 nuclease activity and its antifungal property indicates that the former is the biochemical basis for the latter. Taken together, our experiments revealed that VpPR-10.1 is critical in mediating fungal resistance in grape, potentially playing a dual role by degrading pathogen RNA and inducing programmed death of host cells.     

cDNA Clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli

Powdery mildew, caused by Uncinula necator Burr, is one of the most seriously damaging diseases of grapevine all over the world. To gain the novel gene and investigate the resistance mechanism in Chinese Wild Vitis pseudoreticulata clone Baihe-35-1, mRNA differential display was employed to study the differential expression of the resistant gene to the disease of it when inoculated by Uncinula necator under natural field conditions, 5′ RACE and 3′ RACE have been used to clone the whole cDNA sequences of VpAPX, the novel gene related to Ascorbate Peroxidase which involved in resistant to the disease, is composed of specific sequence 1077 bp and has an open reading frame of 750 bp coding for 250 amino acid residues with a molecular weight of 27.566 kDa. The VpAPX gene was obtained by polymerase chain reaction (PCR) with the special primers synthesized according to the sequences of cDNA, and further cloned it into the pGEM-T easy vector. The cloned VpAPX gene was cut out again with two restriction enzymes and was inserted into the prokaryotic expression vector pGEX-4T-1, then transferred into E. coli BL21. As result, GST-VpAPX fusion protein was successfully expressed by induction of IPTG and purified by GST affinity resin. After injecting rabbit, the polyclonal antibodies were produced. Western blot analyses showed that the antibody reacted specifically to GST-VpAPX fusion protein and the titer for this antibody is 10⁵. This research made the foundation to transform the VpAPX gene into grape plants for follow research in processing. Ling Lin, Xiping Wang: These two authors contributed equally to this work.     

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the ?148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the ?148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.     

Changes in protein abundance during powdery mildew infection of leaf tissues of Cabernet Sauvignon grapevine (Vitis vinifera L.)

A comparative analysis of differentially expressed proteins in a susceptible grapevine (Vitis vinifera ‘Cabernet Sauvignon’) during the infection of Erysiphe necator, the causal pathogen of grapevine powdery mildew (PM), was conducted using iTRAQ. The quantitative labeling analysis revealed 63 proteins that significantly changed in abundance at 24, 36, 48, and 72 h post inoculation with powdery mildew conidiospores. The functional classification of the PM‐responsive proteins showed that they are involved in photosynthesis, metabolism, disease/defense, protein destination, and protein synthesis. A number of the proteins induced in grapevine in response to E. necator are associated with the plant defense response, suggesting that PM‐susceptible Cabernet Sauvignon is able to initiate a basal defense but unable to restrict fungal growth or slow down disease progression.