RXLR and CRN Effectors from the Sunflower Downy Mildew Pathogen Plasmopara halstedii Induce Hypersensitive-Like Responses in Resistant Sunflower Lines

Abstract

<i>Plasmopara halstedii</i> is an obligate biotrophic oomycete causing downy mildew disease on sunflower, <i>Helianthus annuus</i>, an economically important oil crop. Severe symptoms of the disease (e.g., plant dwarfism, leaf bleaching, sporulation and production of infertile flower) strongly impair seed yield. <i>Pl</i> resistance genes conferring resistance to specific <i>P. halstedii</i> pathotypes were located on sunflower genetic map but yet not cloned. They are present in cultivated lines to protect them against downy mildew disease. Among the 16 different <i>P. halstedii</i> pathotypes recorded in France, pathotype 710 is frequently found, and therefore continuously controlled in sunflower by different <i>Pl</i> genes. High-throughput sequencing of cDNA from <i>P. halstedii</i> led us to identify potential effectors with the characteristic RXLR or CRN motifs described in other oomycetes. Expression of six <i>P. halstedii</i> putative effectors, five RXLR and one CRN, was analyzed by qRT-PCR in pathogen spores and in the pathogen infecting sunflower leaves and selected for functional analyses. We developed a new method for transient expression in sunflower plant leaves and showed for the first time subcellular localization of <i>P. halstedii</i> effectors fused to a fluorescent protein in sunflower leaf cells. Overexpression of the CRN and of 3 RXLR effectors induced hypersensitive-like cell death reactions in some sunflower near-isogenic lines resistant to pathotype 710 and not in susceptible corresponding lines, suggesting they could be involved in <i>Pl</i> loci-mediated resistances.