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

Quentin Gascuel,Luis Buendia,Yann Pecrix,Nicolas Blanchet,Stéphane Muños,Felicity Vear,Laurence Godiard

doi:10.3389/fpls.2016.01887

Abstract

Plasmopara halstedii is an obligate biotrophic oomycete causing downy mildew disease on sunflower, Helianthus annuus, 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. Pl resistance genes conferring resistance to specific P. halstedii 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 P. halstedii pathotypes recorded in France, pathotype 710 is frequently found, and therefore continuously controlled in sunflower by different Pl genes. High-throughput sequencing of cDNA from P. halstedii led us to identify potential effectors with the characteristic RXLR or CRN motifs described in other oomycetes. Expression of six P. halstedii 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 P. halstedii 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 Pl loci-mediated resistances.

Highlights

Sunflower (Helianthus annuus L.) is the fourth most important oil crop in world trade after oil palm, soybean and rapeseed, with an annual production of 12.6 million tons of oil
P. halstedii belongs to the Peronosporales, the most devastating group of plant pathogenic oomycetes which includes the hemibiotroph genus Phytophthora causing late blight diseases and a large group of obligate biotrophs causing downy mildews, such as P. viticola on grapevine, Bremia lactucae on lettuce, and Hyaloperonospora arabidopsidis on Arabidopsis thaliana (Michelmore and Wong, 2008; Thines and Kamoun, 2010; Gessler et al, 2011)
The five putative RXLR proteins showed a predicted signal peptide of 17–23 amino acids (SignalP4.1) and putative translocation patterns described in RXLR effectors (Table 1)

Summary

Introduction

Sunflower (Helianthus annuus L.) is the fourth most important oil crop in world trade after oil palm, soybean and rapeseed, with an annual production of 12.6 million tons of oil. Downy mildew caused by Plasmopara halstedii is one of the major diseases affecting sunflower yield (Gascuel et al, 2015). This pathogen has been reported in most of the sunflower seed producing countries. Thirty six pathotypes of P. halstedii, often known as races, have so far been identified worldwide They are defined by an international nomenclature system, based on differential virulence profiles on a set of sunflower inbred lines containing different resistance loci called Pl (Tourvieille de Labrouhe et al, 2012; Gascuel et al, 2015)

Methods

Results

Conclusion