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.

Keywords: Agrobacterium-mediated transient expression; Plasmopara halstedii; downy mildew; hypersensitive response; oomycete effectors; resistant sunflower; subcellular localization.

FIGURE 1

Q-RT PCR gene expression patterns of six putative effector genes and a control gene, PhRIBS3A, in P. halstedii spores and in infected sunflower seedlings at 3, 7, and 11 days post inoculation (dpi). Relative expression values of P. halstedii effector genes compared to housekeeping gene PhRIBS3A were calculated by the ΔCT method. PhRIBS3A expression ratio was normalized by the mean of plant constitutive genes by the ΔCT method. Standard error was calculated for each point for three biological repetitions and three technical repetitions.

FIGURE 2

Subcellular localizations of PhRXLR and PhCRN fusion proteins in sunflower leaf tissues. Sunflower (A–K) and Nicotiana benthamiana (L) leaf tissues showing expression of the YFP-RXLR and GFP-CRN fusion protein genes driven by the 35S promoter after Agrobacterium-transient expression. Results presented (A–K) are from susceptible NIL161S sunflower line. Scale bar = 10 μm. (A) Subcellular localization of the YFP protein in the nucleus and cytoplasm. Nuclear localization of PhRXLR02 (B) and PhRXLR03 (C) fused to YFP showing intense nucleolar fluorescence. Representative images of in planta plasma membrane-like localization of PhRXLR08 (D) and PhRXLR14 (E) fusion proteins. (F,G) PhRXLR31-YFP localizes at the nucleus, showing a dynamic subnuclear localization varying from nucleolar localization to speckles-like structures. (H) Subcellular localization of the GFP protein in the nucleus and cytoplasm. (I) PhCRN37-GFP showed a nucleoplasmic localization. Detail of the plasma-membrane like localization of PhRXLR08 (J) and PhRXLR14 (K). (L) DAPI staining of nuclei expressing PhRXLR03 in transiently transformed N. benthamiana epidermal cells.

FIGURE 3

Plasmopara halstedii CRN effector, PhCRN37, does not exhibit a necrosis phenotype in N. benthamiana. (A) Alignment of CRN2 necrotic domain (CRN2_NecD, 173–407 amino acids) of Phytophthora infestans CRN2 protein with the corresponding domain of PhCRN37 (PhCRN37_D, 126–369 amino acids). (B) Unlike CRN2 effector from Phytophthora infestans, PhCRN37 does not induce cell death after infiltration in N. benthamiana leaf. Picture was taken at 4 days post infiltration.

FIGURE 4

Scoring scale of sunflower leaves used in Agrobacterium-transient experiments.

FIGURE 5

Recognition of P. halstedii RXLR and CRN effectors in resistant sunflower genotypes by HR-like responses. Phenotypes observed in sunflower leaves of six different genotypes (susceptible genotypes: NIL161S, CAY, and GB, and resistant genotypes to pathotype 710 of P. halstedii: NIL161R, CAYRM and GBRM; see text for explanations) after Agrobacterium-transient expression of the different YFP-PhRXLR, or the GFP-PhCRN37 fusions driven by the 35S promoter. Pictures were taken 11 days post infiltration.

FIGURE 6

Mean scores estimated on sunflower leaf sectors 11 days post infiltration with the P. halstedii YFP-RXLR and GFP-CRN effector fusions. Twenty four leaf sectors were scored in two representative independent experiments. Statistical analysis was performed with Wilcoxon test. Asterisks indicate significant differences with the control constructs in the same sunflower genotype (YFP for PhRXLRs and GFP for PhCRN37), calculated for the two biological replicates. ^∗∗∗P-value < 0.001.