Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death‐like defence response by interacting with VpVDAC3

As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage‐dependent anion channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8’ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1’, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defence response to P. viticola in grapevine.     

Historical Introgression of the Downy Mildew Resistance Gene Rpv12 from the Asian Species Vitis amurensis into Grapevine Varieties

The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance–a hypersensitive response in leaves challenged with P. viticola–was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12⁺ haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12⁺ haplotype is shared by 15 varieties, the most ancestral of which are the century-old ‘Zarja severa’ and ‘Michurinets’. Before this knowledge, the chromosome segment around Rpv12⁺ became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12⁺ has an additive effect with Rpv3⁺ to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3⁺ plants.     

Induction of defence mechanisms in grapevine leaves by emodin- and anthraquinone-rich plant extracts and their conferred resistance to downy mildew

The ability of two plant extracts, Rheum palmatum root extract (RPRE) and Frangula alnus bark extract (FABE), to protect Vitis vinifera leaves from Plasmopara viticola infection was evaluated. These natural products are toxic to the pathogen and induce defence reactions in a susceptible cultivar of V. vinifera (V. vinifera cv. Chasselas), including stilbenic phytoalexin accumulation, enhanced peroxidase (EC 1.11.1.7) activity, and a hypersensitive reaction. Inhibition of the first stage of biotrophic hyphal development of P. Viticola by the two plant extracts was observed. HPLC–DAD–MS analysis showed that these two natural extracts contain many phenolic compounds belonging to the anthraquinone family, such as rhein, frangulin A, emodin, aloe-emodin, chrysophanol, and physcion. Emodin alone is able to impair P. viticola development and to stimulate viniferins and the accumulation of pterostilbene.     

Influence of constitutive phenolic compounds on the response of grapevine (Vitis vinifera L.) leaves to infection by Plasmopara viticola

Flavonols and hydroxycinnamic acids are known to contribute to plant resistance against pathogens, but there are few reports on the implication of flavonols in the resistance of grapevine against Plasmopara viticola, and none on the involvement of hydroxycinnamic acids. In order to analyze the effect of flavonols on P. viticola infection, variable amounts of flavonols were induced by different light conditions in otherwise phenologically identical leaves. Differences in content of leaf hydroxycinnamic acids were induced at the same time. A non-invasive monitoring of flavonols and hydroxycinnamic acids was performed with Dualex leaf-clip optical sensors. Whatever the light condition, there were no significant changes in flavonol or in hydroxycinnamic acid contents for control and inoculated leaves during the development of P. viticola until 6 days after inoculation. The violet-blue autofluorescence of stilbenes, the main phytoalexins of grapevine that accumulate in inoculated leaves, was used as an indicator of infection by P. viticola. The implication of leaf constitutive flavonols and hydroxycinnamic acids in the defence of Vitis vinifera against P. viticola could be investigated in vivo thanks to this indicator. The increase in stilbene violet-blue autofluorescence started earlier for leaves with low flavonol content than for leaves with higher content, suggesting that constitutive flavonols are able to slow down the infection by P. viticola. On the contrary, constitutive hydroxycinnamic acids did not seem to play a role in defence against P. viticola. The non-destructive nature of the methods used alleviates the major problem of destructive experiments: the large variability in leaf phenolic contents.     

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.     

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.

     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Effect of UV-C on peroxidase isoenzymes in axillary bud cultures ofVitis species differing in fungal resistance toPlasmopara viticola

The effect of shortwave (250 nm) UV radiation (UV-C) on the level of peroxidase activity and peroxidase isoenzyme patterns in leaves of resistant ([Vitis vinifera x Viris riparia] x Vitis rupestris andVitis rupestris) and susceptible (Vitis vinifera) grapevine species toPlasmopara viticola (downy mildew) was studied. The results show that although UV-C did not produce significant changes in peroxidase activity in susceptible species, and only minor changes in resistant species, treatment with UV-light induces an acidic isoperoxidase (isoperoxidase A₁), capable of oxidising 4-hydroxystilbenes in resistant species. It was named HSPrx 2. Since peroxidase is apparently the enzyme responsible for ε-viniferin synthesis from resveratrol in grapevines, a close relationship between this peroxidase isoenzyme and ε-viniferin synthesis which occurs in grapevine leaves after UV-C treatment must be expected.     

Construction of a reference linkage map of Vitis amurensis and genetic mapping of Rpv8, a locus conferring resistance to grapevine downy mildew

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the major threats to grapevine. All traditional cultivars of grapevine (Vitis vinifera) are susceptible to downy mildew, the control of which requires regular application of fungicides. In contrast, many sources of resistance to P. viticola have been described in the Vitis wild species, among which is V. amurensis Rupr. (Vitaceae), a species originating from East Asia. A genetic linkage map of V. amurensis, based on 122 simple sequence repeat and 6 resistance gene analogue markers, was established using S1 progeny. This map covers 975?cM on 19 linkage groups, which represent 82% of the physical coverage of the V. vinifera reference genetic map. To measure the general level of resistance, the sporulation of P. viticola and the necrosis produced in response to infection, five quantitative and semi-quantitative parameters were scored 6?days post-inoculation on the S1 progeny. A quantitative trait locus (QTL) analysis allowed us to identify on linkage group 14 a major QTL controlling the resistance to downy mildew found in V. amurensis, which explained up to 86.3% of the total phenotypic variance. This QTL was named ??Resistance to Plasmopara viticola 8?? (Rpv8).     

Up-regulated RxLR effector genes of Plasmopara viticola in synchronized host-free stages and infected leaves of hosts with different susceptibility

Fast recognition of host signals and early activation of infection mechanisms in Plasmopara viticola are decisive for successful infestation of Vitis vinifera. To better understand interactive processes at the first front line of combat between the pathogen and its host, a specific pre-infective stage was generated in a host-free system. Zoospore encystment was triggered within minutes after treatment with CaCl₂. Subsequently, high rates of germ tube formation occurred in a synchronized manner. This method was employed to compare development-related gene expression in strains of different virulence. Soon after germination, spores showed strong up-regulation of two effector genes, PvRxLR18 and PvRxLR28, particularly in the high virulence strain. On infected grapevine leaf-discs of cultivars with different susceptibility, a similar up-regulation was found at 6 hours post inoculation (hpi). This effect was much more evident in the high virulence than in the low virulence strain and was significantly higher on leaves of the tolerant cultivar Regent than on Müller-Thurgau. In addition, PvRxLR67 was up-regulated 24 hpi in the high virulence strain indicating that different effectors are active in later infection stages. Differences in the expression pattern of RxLR effector genes between the two strains corroborated with infection symptoms visible by sporulation.     

Genetic linkage maps of two interspecific grape crosses (Vitis spp.) used to localize quantitative trait loci for downy mildew resistance

Two populations (Pop) segregating quantitatively for resistance to downy mildew (DM), caused by Plasmopara viticola, were used to construct genetic maps and to carry out quantitative trait locus (QTL) analysis. Pop1 comprised of 174 F₁ individuals from a cross of ‘Moscato Bianco’, a susceptible Vitis vinifera cultivar, and a resistant individual of Vitis riparia. Pop2 consisted of 94 progeny from a cross of two interspecific hybrids, ‘VRH3082 1-42’ and ‘SK77 5/3’, with resistance traits inherited from Vitis rotundifolia and Vitis amurensis, respectively. Resistance of progeny was measured in field and greenhouse conditions by visual evaluation of disease symptoms on leaves. Linkage maps of 1037.2 and 651 cM were built essentially with simple sequence repeat markers and were enriched with gene-derived single-strand conformational polymorphism and single-nucleotide polymorphism markers. Simple interval mapping and Kruskall–Wallis analysis detected a stable QTL involved in field resistance to DM on linkage group (LG) 7 of the Pop1 integrated map co-localized with a putative Caffeoyl-CoA O-methyltransferase-derived marker. Additional QTLs were detected on LGs 8, 12 and 17. We were able to identify genetic factors correlated with resistance to P. viticola with lower statistical significance on LGs 1, 6 and 7 of the Pop2 map. Finally, no common QTLs were found between the two crosses analyzed. A search of the grapevine genome sequence revealed either homologues to non-host-, host- or defense-signalling genes within the QTL intervals. These positional candidate genes may provide new information about chromosomal regions hosting phenotypic loci.     

Histological study of the responses of two Vitis vinifera cultivars (resistant and susceptible) to Plasmopara viticola infections

Leaves of Vitis vinifera L. cvs. Chasselas (susceptible) and Solaris (resistant) were inoculated with Plasmopara viticola. Samples were then examined by scanning electron microscopy, light and epifluorescence microscopy. On the resistant cv. Solaris, stomatal deposits, identified as callose, were visible around the germinating zoospores 7 h after inoculation. Twenty-four hours after inoculation, infected stomata exhibited secretions that enveloped the zoospores. At this time, infected stomata were surrounded by necrotic tissues. At 120 h after inoculation, undefined material was deposited on the cuticle in the necrotic areas. Stomata in the vicinity of the infection sites contained callose deposits in and around the stomatal openings, but no necrotic zones were observed. On the sensitive cv. Chasselas neither secretion nor callose deposits were observed. Sporangiophores emerged about 96 h after inoculation and were fully developed 24 h later. Sporulation through small stomata-like apertures present all along the primary vein was also observed on the upper leaf surface. The role of stomatal callose deposits in the defense reactions of the Solaris grapevine cultivar against P. viticola is discussed.     

Prevalence,geographic distribution and phylogenetic relationships among cryptic species of Plasmopara viticola in grape‐producing regions of Georgia and Florida,USA

Previous phylogenetic studies of the grape downy mildew pathogen, Plasmopara viticola, revealed five cryptic species in eastern North America that differed in their host range and geographic distribution. Preliminary comparative studies also documented differences in temperature responses during infection between certain cryptic species, indicating the biological relevance of knowing which cryptic species of the pathogen are present in a given region. However, limited information is available regarding the presence, prevalence and dynamics of cryptic species of P. viticola in the southeastern United States. Here, 301 P. viticola isolates obtained from cultivated grape species in five distinct grape‐growing regions of Georgia and Florida were subjected to cleaved amplified polymorphic sequence analysis and multilocus sequencing (internal transcribed spacer region of the rDNA, actin and β‐tubulin) to identify cryptic species and infer phylogenetic relationships. Three cryptic species, P. viticola clade aestivalis (Pva), clade vinifera (Pvv) and clade vulpina (Pvu), were identified in Georgia, whereas two, Pva and Pvv, were found in Florida; all three cryptic species are reported here for the first time in Georgia, whereas Pva is reported for the first time in Florida. Pva was the most prevalent cryptic species (72.1% of isolates) and was distributed widely from the North Georgia Mountains to Mid‐Florida, whereas Pvv (27.2%) and Pvu (0.7%) were found only in the Coastal Plain region of the two states. Interestingly, Pvu was obtained from French American hybrid Blanc du Bois and could be subcultured on Vitis vinifera Chardonnay, suggesting a broader host range than only the wild species Vitis vulpina reported previously.