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

Abstract

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 <i>Plasmopara viticola</i>, 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 <i>CRN-like</i> genes were cloned from the <i>P. viticola</i> isolate YL genome, hereafter referred to as <i>PvCRN</i> genes, and characterized <i>in silico</i> and <i>in planta. PvCRN</i> genes in 'YL' share high sequence identities with their ortholog genes in the other three previously sequenced <i>P. viticola</i> isolates. Sequence divergence among the genes in the <i>PvCRN</i> family indicates that different <i>PvCRN</i> genes have different roles. Phylogenetic analysis of the PvCRN and the CRN proteins encoded by genes in the <i>P. halstedii</i> genome suggests that various functions might have been acquired by the <i>CRN</i> superfamily through independent evolution of <i>Plasmopara</i> 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 <i>Vitis riparia</i>. This was in accordance with the result that most PvCRN proteins, except PvCRN11, failed to induce necrosis in <i>Nicotiana benthamiana</i>. 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 <i>N. benthamiana</i> to <i>Phytophthora capsici</i>, which is a semi-biotrophic oomycete. Moreover, the nucleus-targeting member, PvCRN19, promoted the susceptibility of <i>N. benthamiana</i> to <i>P. capsici</i>. Therefore, these PvCRN proteins were estimated to be virulent effectors involved in the pathogenicity of <i>P. viticola</i> YL. Collectively, this study provides comprehensive insight into the CRN effector repertoire of <i>P. viticola</i> YL, which will help further elucidate the molecular mechanisms of the pathogenesis of grapevine downy mildew.