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

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.

Random mutagenesis screen shows that Phytophthora capsici CRN83_152‐mediated cell death is not required for its virulence function(s)

“…However, PsCRN115 has been shown to be a potent suppressor of PsCRN63-mediated cell death whilst having no effect on PsCRN63 virulence boost, suggesting that PsCRN63-mediated cell death and virulence boost consist of two independent mechanisms (Zhang et al, 2015a). Further insights into the biological meaning of CRN-mediated cell death were obtained in a recent work analysing the cell death-inducing capacities of a CRN effector (PhCRN37) from Plasmopara halstedii in sunflower nearisogenic lines (NILs) (Gascuel et al, 2016). PhCRN37 was shown to induce cell death only in resistant sunflower NILs carrying the Pl5 resistance locus and not in the corresponding sunflower susceptible parent.…”

“…PhCRN37 was shown to induce cell death only in resistant sunflower NILs carrying the Pl5 resistance locus and not in the corresponding sunflower susceptible parent. These observations suggest that PhCRN37mediated cell death is associated with recognition events putatively mediated by one of the numerous resistance gene analogues encoded in the Pl5 locus (Gascuel et al, 2016). From these studies, it is apparent that the role(s) of cell death in the biology of CRN and pathogen virulence strategies has yet to be clarified.…”

Identification of Downy Mildew Resistance Loci in Sunflower Germplasm

“…To date, thirty six pathotypes of P. halstedii have been recognized (Gascuel et al, 2016). These pathotypes are often defined by an international nomenclature system, based on differential virulence profiles on a set of sunflower inbred lines containing different resistance gene called Pl (Tourvieille de Labrouhe et al, 2012;Gascuel et al, 2015 (Mouzeyar et al, 1995;Roeckel-Drevet et al, 1996;Vear et al, 1997;MolineroRuiz et al, 2003;Yu et al, 2003;Mulpuri et al, 2009;de Romano et al, 2010;Bachlava et al, 2011;Qi et al, 2015), but none has been cloned so far (Gascuel et al, 2015;Qi et al, 2015Qi et al, , 2016.…”

“…Sunflower produces a healthy oil rich in unsaturated fatty acids as well as high vitamin E content (Gascuel et al, 2016). Downy mildew is one of the major fungal diseases in most sunflower producing areas of the world (Gascuel et al, 2015).…”

Nonhost wild lettuce as a donor for resistance to downy mildew in cultivated lettuce

“…Application of (candidate) effectors to host plant leaf tissue through transient expression triggered HR responses that were associated with novel and already known R genes (Vleeshouwers et al, 2008;Rietman et al, 2012;Gascuel et al, 2016;Lenman et al, 2016). Recognition of effectors in nonhost species is often assumed to contribute to NHR (Wroblewski et al, 2009;Schulze-Lefert & Panstruga, 2011;Lee et al, 2014;Adlung et al, 2016).…”