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.

Grapevine powdery mildew is one of the most damaging fungal diseases. Therefore, a precise understanding of the grapevine disease resistance system becomes a subject of significant importance. Plant microRNAs(miRNAs) have been implicated to play regulatory roles in plant biotic stress responses. In this study, high-throughput sequencing and miRDeep-P were employed to identify miRNAs in Chinese wild Vitis pseudoreticulata leaves following inoculation with Erysiphe necator. Altogether, 126 previously identified microRNAs and 124 novel candidates of miRNA genes were detected. Among them, 43 conserved miRNAs belong to 20 families and 23 non-conserved but previously-known miRNAs belong to 15 families. Following E. necator inoculation, 119 miRNAs were down-regulated and 131 were up-regulated. Furthermore, the expression changes occurring in 32 miRNAs were significant. The expression patterns of some miRNAs were validated by semi-quantitative RT-PCR and qRT-PCR. A total of 485 target genes were predicted and categorized by Gene Ontology (GO). In addition, 14 vvi-miRNAs were screened with 36 targets which may be involved in powdery mildew resistance in grape. Highly accumulated vvi-NewmiR2118 was detected from accession “Baihe-35-1,” whose targets were mostly NBS-LRR resistance genes. It was down-regulated rapidly and strongly in “Baihe-35-1” leaves after inoculated with E. necator, indicating its involvement in grape powdery mildew resistance. Finally, the study verified interaction between vvi-NewmiR2118 and RPP13 by histochemical staining and GUS fluorescence quantitative assay.

SummaryAs 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.

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 H 2 O 2 accumulation and activated the 1 O 2 signaling pathway in grapevine. RXLR31154 could stabilize PsbP. Together, our data revealed that RXLR31154 reduces H 2 O 2 accumulation and activates the 1 O 2 signaling pathway through stabilizing PsbP, thereby promoting disease.

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.