Plasmopara viticola effector PvCRN11 induces disease resistance to downy mildew in grapevine.

The downy mildew of grapevine (Vitis vinifera L.) is caused by Plasmopara viticola and is a major production problem in most grape-growing regions. The vast majority of effectors act as virulence factors and sabotage plant immunity. Here, we describe in detail one of the putative P. viticola Crinkler (CRN) effector genes, PvCRN11, which is highly transcribed during the infection stages in the downy mildew-susceptible grapevine V. vinifera cv. 'Pinot Noir' and V. vinifera cv. 'Thompson Seedless'. Cell death-inducing activity analyses reveal that PvCRN11 was able to induce spot cell death in the leaves of Nicotiana benthamiana but did not induce cell death in the leaves of the downy mildew-resistant V. riparia accession 'Beaumont' or of the downy mildew-susceptible 'Thompson Seedless'. Unexpectedly, stable expression of PvCRN11 inhibited the colonization of P. viticola in grapevine and Phytophthora capsici in Arabidopsis. Both transgenic grapevine and Arabidopsis constitutively expressing PvCRN11 promoted plant immunity. PvCRN11 is localized in the nucleus and cytoplasm, whereas PvCRN11-induced plant immunity is nucleus-independent. The purified protein PvCRN11Opt initiated significant plant immunity extracellularly, leading to enhanced accumulations of reactive oxygen species, activation of MAPK and up-regulation of the defense-related genes PR1 and PR2. Furthermore, PvCRN11Opt induces BAK1-dependent immunity in the apoplast, whereas PvCRN11 overexpression in intracellular induces BAK1-independent immunity. In conclusion, the PvCRN11 protein triggers resistance against P. viticola in grapevine, suggesting a potential for the use of PvCRN11 in grape production as a protectant against downy mildew.

Plasmopara viticola RxLR effector PvAvh77 triggers cell death and governs immunity responses in grapevine.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola, is one of the most significant production challenges for the grape and wine industry. P. viticola injects a plethora of effectors into its host cells to disrupt immune processes, but the mechanisms by which these effectors act at the molecular level have not been well characterized. Herein, we show that a candidate P. viticola avirulence homolog (Avh) RxLR effector gene, designated PvAvh77, was strongly up-regulated during the initial stages of P. viticola infection in Vitis vinifera. Further experiments demonstrated that PvAvh77 could trigger non-specific cell death when expressed in the wild grapevine Vitis riparia and in tobacco (Nicotiana benthamiana and Nicotiana tabacum). In addition, a truncated form of PvAvh77, designated PvAvh77-M2, was more active in inducing cell death in N. benthamiana and V. riparia than full-length PvAvh77. Ectopic expression of PvAvh77 in V. vinifera 'Thompson Seedless' leaves neutralized host immunity and enhanced colonization by P. viticola, and the immune-inhibiting activity of PvAvh77 on susceptible Eurasian grapevine depended on its nuclear localization. Using a yeast signal sequence trap approach, we showed that the signal peptide of PvAvh77 is functional in yeast. Moreover, PvAvh77 with a signal peptide stimulated plant immune responses in the apoplast. Notably, application of exogenous purified PvAvh77-M2 effectively initiated defence responses in grapevine extracellularly, as evidenced by increased accumulation of salicylic acid and H2O2, and reduced infection of inoculated P. viticola. In summary, we identified a novel effector, PvAvh77, from P. viticola, which has the potential to serve as an inducer of plant immunity.

The cytosolic iron-sulphur cluster assembly mechanism in grapevine is one target of a virulent Crinkler effector from Plasmopara viticola.

Grapevine downy mildew is one of the most devastating diseases in grape production worldwide, but its pathogenesis remains largely unknown. A thorough understanding of the interaction between grapevine and the causal agent, Plasmopara viticola, is helpful to develop alternative disease control measures. Effector proteins that could be secreted to the interaction interface by pathogens are responsible for the susceptibility of host plants. In this study, a Crinkler effector, named PvCRN17, which is from P. viticola and showed virulent effects towards Nicotiana benthamiana previously, was further investigated. Consistently, PvCRN17 showed a virulent effect on grapevine plants. Protein-protein interaction experiments identified grapevine VAE7L1 (Vitis protein ASYMMETRIC LEAVES 1/2 ENHANCER 7-Like 1) as one target of PvCRN17. VAE7L1 was found to interact with VvCIA1 and VvAE7, thus it may function in the cytosolic iron-sulphur cluster assembly (CIA) pathway. Transient expression of VAE7L1 in Vitis riparia and N. benthamiana leaves enhanced the host resistance to oomycete pathogens. Downstream of the CIA pathway in grapevine, three iron-sulphur (Fe-S) proteins showed an enhancing effect on the disease resistance of N. benthamiana. Competitive co-immunoprecipitation assay showed PvCRN17 could compete with VvCIA1 to bind with VAE7L1 and VvAE7. Moreover, PvCRN17 and VAE7L1 were colocalized at the plasma membrane of the plant cell. To conclude, after intruding into the grapevine cell, PvCRN17 would compete with VCIA1 to bind with VAE7L1 and VAE7, demolishing the CIA Fe-S cluster transfer complex, interrupting the maturation of Fe-S proteins, to suppress Fe-S proteins-mediated defence responses.

An RxLR effector from Plasmopara viticola suppresses plant immunity in grapevine by targeting and stabilizing VpBPA1.

Grapevine downy mildew, caused by Plasmopara viticola, is one of the most devastating diseases in viticulture. Plasmopara viticola secretes RxLR effectors to modulate immune responses in grapevine. Here, we report an RxLR effector RxLR50253 from P. viticola that can interfere with plant immune response and thus promote pathogen colonization. RxLR50253 was induced at an early stage of P. viticola infection and could suppress elicitor (INF1 and Bax)-triggered cell death. RxLR50253 promote pathogen colonization in both tobacco and grapevine leaves. VpBPA1 was found to be the host target of RxLR50253 by yeast two-hybrid screening, and interaction between RxLR50253 and VpBPA1 was confirmed by multiple in vivo and in vitro assays. Further analysis revealed that VpBPA1 promoted pathogen colonization and decreased H2 O2 accumulation in transgenic tobacco and grapevine, while there was enhanced resistance and H2 O2 accumulation in NbBPA1-silenced Nicotiana benthamiana leaves. Moreover, transient expression of VpBPA1 in NbBPA1-silenced N. benthamiana leaves could reduce the accumulation of H2 O2 . Experiments in vivo demonstrated that RxLR50253 inhibits degradation of VpBPA1. Taken together, our findings showed that RxLR50253 targets and stabilizes VpBPA1 to attenuate plant immunity through decreasing H2 O2 accumulation during pathogen infection.

Grafting with rootstocks promotes phenolic compound accumulation in grape berry skin during development based on integrative multi-omics analysis.

In viticulture, grafting has been practiced widely and influences grape development as well as berry and wine quality. However, there is limited understanding of the effects of rootstocks on grape phenolic compounds, which are located primarily in the berry skin and contribute to certain sensory attributes of wine. In this study, scion-rootstock interactions were investigated at the green-berry stage and the veraison stage when grapevines were hetero-grafted with three commonly used rootstock genotypes (5BB, 101-14MG, and SO4). Physiological investigations showed that hetero-grafts, especially CS/5BB, contained higher concentrations of total proanthocyanidins (PAs) and various PA components in berry skins compared with the auto-grafted grapevines. Further metabolomics analysis identified 105 differentially accumulated flavonoid compounds, the majority of which, including anthocyanins, PAs, and flavonols, were significantly increased in the berry skins of hetero-grafted grapevines compared with auto-grafted controls. In addition, transcriptomic analysis of the same samples identified several thousand differentially expressed genes between hetero-grafted and auto-grafted vines. The three rootstocks not only increased the transcript levels of stilbene, anthocyanin, PA, and flavonol synthesis genes but also affected the expression of numerous transcription factor genes. Taken together, our results suggest that hetero-grafting can promote phenolic compound accumulation in grape berry skin during development. These findings provide new insights for improving the application value of grafting by enhancing the accumulation of nutritious phenolic components in grape.

Ultrasonic Particle Manipulation in Glass Capillaries: A Concise Review.

Ultrasonic particle manipulation (UPM), a non-contact and label-free method that uses ultrasonic waves to manipulate micro- or nano-scale particles, has recently gained significant attention in the microfluidics community. Moreover, glass is optically transparent and has dimensional stability, distinct acoustic impedance to water and a high acoustic quality factor, making it an excellent material for constructing chambers for ultrasonic resonators. Over the past several decades, glass capillaries are increasingly designed for a variety of UPMs, e.g., patterning, focusing, trapping and transporting of micron or submicron particles. Herein, we review established and emerging glass capillary-transducer devices, describing their underlying mechanisms of operation, with special emphasis on the application of glass capillaries with fluid channels of various cross-sections (i.e., rectangular, square and circular) on UPM. We believe that this review will provide a superior guidance for the design of glass capillary-based UPM devices for acoustic tweezers-based research.

Glyoxalase I-4 functions downstream of NAC72 to modulate downy mildew resistance in grapevine.

Glyoxalase I (GLYI) is part of the glyoxalase system; its major function is the detoxification of α-ketoaldehydes, including the potent and cytotoxic methylglyoxal (MG). Methylglyoxal disrupts mitochondrial respiration and increases production of reactive oxygen species (ROS), which also increase during pathogen infection of plant tissues; however, there have been few studies relating the glyoxalase system to the plant pathogen response. We used the promoter of VvGLYI-4 to screen the upstream transcription factors and report a NAC (NAM/ATAF/CUC) domain-containing transcription factor VvNAC72 in grapevine, which is localized to the nucleus. Our results show that VvNAC72 expression is induced by downy mildew, Plasmopara viticola, while the transcript level of VvGLYI-4 decreases. Further analysis revealed that VvNAC72 can bind directly to the promoter region of VvGLYI-4 via the CACGTG element, leading to inhibition of VvGLYI-4 transcription. Stable overexpression of VvNAC72 in grapevine and tobacco showed a decreased expression level of VvGLYI-4 and increased content of MG and ROS, as well as stronger resistance to pathogen stress. Taken together, these results demonstrate that grapevine VvNAC72 negatively modulates detoxification of MG through repression of VvGLYI-4, and finally enhances resistance to downy mildew, at least in part, via the modulation of MG-associated ROS homeostasis through a salicylic acid-mediated defense pathway.

Proteomic analysis of early-stage incompatible and compatible interactions between grapevine and P. viticola.

Wild grapevines can show strong resistance to the downy mildew pathogen P. viticola, but the associated mechanisms are poorly described, especially at early stages of infection. Here, we performed comparative proteomic analyses of grapevine leaves from the resistant genotype V. davidii "LiuBa-8" (LB) and susceptible V. vinifera "Pinot Noir" (PN) 12 h after inoculation with P. viticola. By employing the iTRAQ technique, a total of 444 and 349 differentially expressed proteins (DEPs) were identified in LB and PN, respectively. The majority of these DEPs were related to photosynthesis, respiration, cell wall modification, protein metabolism, stress, and redox homeostasis. Compared with PN, LB showed fewer downregulated proteins associated with photosynthesis and more upregulated proteins associated with metabolism. At least a subset of PR proteins (PR10.2 and PR10.3) was upregulated upon inoculation in both genotypes, whereas HSP (HSP70.2 and HSP90.6) and cell wall-related XTH and BXL1 proteins were specifically upregulated in LB and PN, respectively. In the incompatible interaction, ROS signaling was evident by the accumulation of H2O2, and multiple APX and GST proteins were upregulated. These DEPs may play crucial roles in the grapevine response to downy mildew. Our results provide new insights into molecular events associated with downy mildew resistance in grapevine, which may be exploited to develop novel protection strategies against this disease.

Characterization of CRN-Like Genes From Plasmopara viticola: Searching for the Most Virulent Ones.

Grapevine downy mildew is an insurmountable disease that endangers grapevine production and the wine industry worldwide. The causal agent of the disease is the obligate biotrophic oomycete Plasmopara viticola, for which the pathogenic mechanism remains largely unknown. Crinkling and necrosis proteins (CRN) are an ancient class of effectors utilized by pathogens, including oomycetes, that interfere with host plant defense reactions. In this study, 27 CRN-like genes were cloned from the P. viticola isolate YL genome, hereafter referred to as PvCRN genes, and characterized in silico and in planta. PvCRN genes in 'YL' share high sequence identities with their ortholog genes in the other three previously sequenced P. viticola isolates. Sequence divergence among the genes in the PvCRN family indicates that different PvCRN genes have different roles. Phylogenetic analysis of the PvCRN and the CRN proteins encoded by genes in the P. halstedii genome suggests that various functions might have been acquired by the CRN superfamily through independent evolution of Plasmopara species. When transiently expressed in plant cells, the PvCRN protein family shows multiple subcellular localizations. None of the cloned PvCRN proteins induced hypersensitive response (HR)-like cell death on the downy mildew-resistant grapevine Vitis riparia. This was in accordance with the result that most PvCRN proteins, except PvCRN11, failed to induce necrosis in Nicotiana benthamiana. Pattern-triggered immunity (PTI) induced by INF1 was hampered by several PvCRN proteins. In addition, 15 PvCRN proteins prevented Bax-induced plant programmed cell death. Among the cell death-suppressing members, PvCRN17, PvCRN20, and PvCRN23 were found to promote the susceptibility of N. benthamiana to Phytophthora capsici, which is a semi-biotrophic oomycete. Moreover, the nucleus-targeting member, PvCRN19, promoted the susceptibility of N. benthamiana to P. capsici. Therefore, these PvCRN proteins were estimated to be virulent effectors involved in the pathogenicity of P. viticola YL. Collectively, this study provides comprehensive insight into the CRN effector repertoire of P. viticola YL, which will help further elucidate the molecular mechanisms of the pathogenesis of grapevine downy mildew.

A Plasmopara viticola RXLR effector targets a chloroplast protein PsbP to inhibit ROS production in grapevine.

Pathogens secrete a large number of effectors that manipulate host processes to create an environment conducive to pathogen colonization. However, the underlying mechanisms by which Plasmopara viticola effectors manipulate host plant cells remain largely unclear. In this study, we reported that RXLR31154, a P. viticola RXLR effector, was highly expressed during the early stages of P. viticola infection. In our study, stable expression of RXLR31154 in grapevine (Vitis vinifera) and Nicotiana benthamiana promoted leaf colonization by P. viticola and Phytophthora capsici, respectively. By yeast two-hybrid screening, the 23-kDa oxygen-evolving enhancer 2 (VpOEE2 or VpPsbP), encoded by the PsbP gene, in Vitis piasezkii accession Liuba-8 was identified as a host target of RXLR31154. Overexpression of VpPsbP enhanced susceptibility to P. viticola in grapevine and P. capsici in N. benthamiana, and silencing of NbPsbPs, the homologs of PsbP in N. benthamiana, reduced P. capcisi colonization, indicating that PsbP is a susceptibility factor. RXLR31154 and VpPsbP protein were co-localized in the chloroplast. Moreover, VpPsbP reduced H2 O2 accumulation and activated the 1 O2 signaling pathway in grapevine. RXLR31154 could stabilize PsbP. Together, our data revealed that RXLR31154 reduces H2 O2 accumulation and activates the 1 O2 signaling pathway through stabilizing PsbP, thereby promoting disease.

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.).

Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from the cultivar "Thompson Seedless" using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance. Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred. Infection assays using leaf discs showed that, compared to wild-type plants, the VvPR4b knockout lines had increased susceptibility to P. viticola. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of P. viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

Insight Into Function and Subcellular Localization of Plasmopara viticola Putative RxLR Effectors.

Grapevine downy mildew, caused by oomycete fungus Plasmopara viticola, is one of the most devastating diseases of grapes across the major production regions of the world. Although many putative effector molecules have been identified from this pathogen, the functions of the majority of these are still unknown. In this study, we analyzed the potential function of 26 P. viticola effectors from the highly virulent strain YL. Using transient expression in leaf cells of the tobacco Nicotiana benthamiana, we found that the majority of the effectors could suppress cell death triggered by BAX and INF1, while seven could induce cell death. The subcellular localization of effectors in N. benthamiana was consistent with their localization in cells of Vitis vinifera. Those effectors that localized to the nucleus (17/26) showed a variety of subnuclear localization. Ten of the effectors localized predominantly to the nucleolus, whereas the remaining seven localized to nucleoplasm. Interestingly, five of the effectors were strongly related in sequence and showed identical subcellular localization, but had different functions in N. benthamiana leaves and expression patterns in grapevine in response to P. viticola. This study highlights the potential functional diversity of P. viticola effectors.

New insights into the heat responses of grape leaves via combined phosphoproteomic and acetylproteomic analyses.

Heat stress is a serious and widespread threat to the quality and yield of many crop species, including grape (Vitis vinifera L.), which is cultivated worldwide. Here, we conducted phosphoproteomic and acetylproteomic analyses of leaves of grape plants cultivated under four distinct temperature regimes. The phosphorylation or acetylation of a total of 1011 phosphoproteins with 1828 phosphosites and 96 acetyl proteins with 148 acetyl sites changed when plants were grown at 35 °C, 40 °C, and 45 °C in comparison with the proteome profiles of plants grown at 25 °C. The greatest number of changes was observed at the relatively high temperatures. Functional classification and enrichment analysis indicated that phosphorylation, rather than acetylation, of serine/arginine-rich splicing factors was involved in the response to high temperatures. This finding is congruent with previous observations by which alternative splicing events occurred more frequently in grapevine under high temperature. Changes in acetylation patterns were more common than changes in phosphorylation patterns in photosynthesis-related proteins at high temperatures, while heat-shock proteins were associated more with modifications involving phosphorylation than with those involving acetylation. Nineteen proteins were identified with changes associated with both phosphorylation and acetylation, which is consistent with crosstalk between these posttranslational modification types.

The Nuclear-Localized RxLR Effector PvAvh74 From Plasmopara viticola Induces Cell Death and Immunity Responses in Nicotiana benthamiana.

Downy mildew is one of the most serious diseases of grapevine (Vitis spp). The causal agent of grapevine downy mildew, Plasmopara viticola, is an obligate biotrophic oomycete. Although oomycete pathogens such as P. viticola are known to secrete RxLR effectors to manipulate host immunity, there have been few studies of the associated mechanisms by which these may act. Here, we show that a candidate P. viticola RxLR effector, PvAvh74, induces cell death in Nicotiana benthamiana leaves. Using agroinfiltration, we found that nuclear localization, two putative N-glycosylation sites, and 427 amino acids of the PvAvh74 carboxyl terminus were necessary for cell-death-inducing activity. Using virus-induced gene silencing (VIGS), we found that PvAvh74-induced cell death in N. benthamiana requires EDS1, NDR1, SGT1, RAR1, and HSP90, but not BAK1. The MAPK cascade components MEK2, WIPK, and SIPK were also involved in PvAvh74-induced cell death in N. benthamiana. Transient expression of PvAvh74 could suppress Phytophthora capsici colonization of N. benthamiana, which suggests that PvAvh74 elicits plant immune responses. Suppression of P. capsici colonization also was dependent on nuclear localization of PvAvh74. Additionally, PvAvh74-triggered cell death could be suppressed by another effector, PvAvh8, from the same isolate. This work provides a framework to further investigate the interactions of PvAvh74 and other RxLR effectors with host immunity.

Genome-wide analysis of glyoxalase-like gene families in grape (Vitis vinifera L.) and their expression profiling in response to downy mildew infection.

BACKGROUND: The glyoxalase system usually comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII). This system converts cytotoxic methylglyoxal (MG) into non-toxic D-lactate in the presence of reduced glutathione (GSH) in two enzymatic steps. Recently, a novel type of glyoxalase III (GLYIII) activity has observed in Escherichia coli that can detoxify MG into D-lactate directly, in one step, without a cofactor. Investigation of the glyoxalase enzymes of a number of plant species shows the importance of their roles in response both to abiotic and to biotic stresses. Until now, glyoxalase gene families have been identified in the genomes of four plants, Arabidopsis, Oryza sativa, Glycine max and Medicago truncatula but no similar study has been done with the grapevine Vitis vinifera L. RESULTS: In this study, four GLYI-like, two GLYII-like and three GLYIII-like genes are identified from the genome database of grape. All these genes were analysed in detail, including their chromosomal locations, phylogenetic relationships, exon-intron distributions, protein domain organisations and the presence of conserved binding sites. Using quantitative real-time PCR analysis (qRT-PCR), the expression profiles of these genes were analysed in different tissues of grape, and also when under infection stress from downy mildew (Plasmopara viticola). The study reveals that most VvGLY-like genes had higher expressions in stem, leaf, tendril and ovule but lower expressions in the flower. In addition, most of the VvGLY-like gene members were P. viticola responsive with high expressions 6-12 h and 96-120 h after inoculation. However, VvGLYI-like1 was highly expressed 48 h after inoculation, similar to VvPR1 and VvNPR1 which are involved in the defence response. CONCLUSIONS: This study identified the GLYI-like, GLYII-like and GLYIII-like full gene families of the grapevine. Based on a phylogenetic analysis and the presence of conserved binding sites, we speculate that these glyoxalase-like genes in grape encode active glyoxalases. Moreover, our study provides a basis for discussing the roles of VvGLYI-like, VvGLYII-like and VvGLYIII-like genes in grape's response to downy mildew infection. Our results shed light on the selection of candidate genes for downy mildew tolerance in grape and lay the foundation for further functional investigations of these glyoxalase genes.

Transcriptomic analysis of Chinese wild Vitis pseudoreticulata in response to Plasmopara viticola.

Downy mildew, resulted from Plasmopara viticola, is one of most severe fungal diseases of grapevine. Since Vitis vinifera is susceptible to downy mildew, much effort has been focused on improving the resistance of V. vinifera. The Chinese wild V. pseudoreticulata accession Baihe-35-1 (BH) shows resistance to P. viticola; however, the molecular mechanism underlying its resistance to P. viticola is largely unknown. In order to better understand the cellular processes, the transcriptomic changes were investigated at 0, 12, 24, 48, 96, and 120 h post infection (hpi). Transcriptome analysis identified a total of 175 differentially expressed genes. Most of them were found to be associated with oxidative stress, cell wall modification, and protein modification. Moreover, the BH resistance to P. viticola was involved in metabolism process, including terpene synthesis and hormone synthesis. In addition, we verified 12 genes to ensure the accuracy of transcriptome data using quantitative real-time PCR (qRT-PCR). This study broadly characterizes a molecular mechanism in which oxidative stress and cell wall biosynthesis and modification play important roles in the response of BH to P. viticola and provides a basis for further analysis of key genes involved in the resistance to P. viticola.

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation.

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures. However, the accumulation of metabolites during cold acclimation (CA) in V. amurensis remains largely unknown. In this study, plantlets of V. amurensis and V. vinifera cv. Muscat of Hamburg were treated at 4 °C for 24 and 72 h, and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry. Most of the identified metabolites, including carbohydrates, amino acids, and organic acids, accumulated in the two types of grape after CA. Galactinol, raffinose, fructose, mannose, glycine, and ascorbate were continuously induced by cold in V. amurensis, but not in Muscat of Hamburg. Twelve metabolites, including isoleucine, valine, proline, 2-oxoglutarate, and putrescine, increased in V. amurensis during CA. More galactinol, ascorbate, 2-oxoglutarate, and putrescine, accumulated in V. amurensis, but not in Muscat of Hamburg, during CA, which may be responsible for the excellent cold tolerance in V. amurensis. The expression levels of the genes encoding ß-amylase (BAMY), galactinol synthase (GolS), and raffinose synthase (RafS) were evaluated by quantitative reverse transcription-PCR. The expression BAMY (VIT_02s0012 g00170) and RafS (VIT_05s0077 g00840) were primarily responsible for the accumulation of maltose and raffinose, respectively. The accumulation of galactinol was attributed to different members of GolS in the two grapes. In conclusion, these results show the inherent differences in metabolites between V. amurensis and V. vinifera under CA.

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

KEY MESSAGE: Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10 µM, 100 µM and 1 mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1 mM PUT and 100 µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

Pathogen development and host responses to Plasmopara viticola in resistant and susceptible grapevines: an ultrastructural study.

The downy mildew disease in grapevines is caused by Plasmopara viticola. This disease poses a serious threat wherever viticulture is practiced. Wild Vitis species showing resistance to P. viticola offer a promising pathway to develop new grapevine cultivars resistant to P. viticola which will allow reduced use of environmentally unfriendly fungicides. Here, transmission and scanning microscopy was used to compare the resistance responses to downy mildew of three resistant genotypes of V. davidii var. cyanocarpa, V. piasesezkii and V. pseudoreticulata and the suceptible V. vinifera cultivar 'Pinot Noir'. Following inoculation with sporangia of P. viticola isolate 'YL' on V. vinifera cv. 'Pinot Noir', the infection was characterized by a rapid spread of intercellular hyphae, a high frequency of haustorium formation within the host's mesophyll cells, the production of sporangia and by the absence of host-cell necrosis. In contrast zoospores were collapsed in the resistant V. pseudoreticulata 'Baihe-35-1', or secretions appeared arround stomata at the beginning of the infection period in V. davidii var. cyanocarpa 'Langao-5' and V. piasezkii 'Liuba-8'. The main characteristics of the resistance responses were the rapid depositions of callose and the appearance of empty hyphae and the plasmolysis of penetrated tissue. Moreover, collapsed haustoria were observed in V. davidii var. cyanocarpa 'Langao-5' at 5 days post inoculation (dpi) and in V. piasezkii 'Liuba-8' at 7 dpi. Lastly, necrosis extended beyond the zone of restricted colonization in all three resistant genotypes. Sporangia were absent in V. piasezkii 'Liuba-8' and greatly decreased in V. davidii var. cyanocarpa 'Langao-5' and in V. pseudoreticulata 'Baihe-35-1' compared with in V. vinifera cv. 'Pinot Noir'. Overall, these results provide insights into the cellular biological basis of the incompatible interactions between the pathogen and the host. They indicate a number of several resistant Chinese wild species that could be used in developing new cultivars having good levels of downy mildew resistance.