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

Keywords: CRN effectors; Plasmopara viticola; Vitis; cell death; virulence.

FIGURE 1

Phylogenetic relationships among the PvCRN genes of P. viticola isolate YL. The phylogenetic tree was originally generated in MEGA7.0 using the maximum likelihood method with 1,000 bootstrap replicates. The optimal substitution model was WAG + G. Different subgroups are highlighted with different colors, and bootstrap values greater than 50 are indicated on the nodes.

FIGURE 2

Phylogenetic relationships among the CRN proteins from P. viticola YL and P. halstedii. The phylogenetic tree was generated in MEGA7.0 using the maximum likelihood method with 1,000 bootstrap replicates. The optimal substitution model was WAG + G. Bootstrap values greater than 50 are indicated in green on the nodes.

FIGURE 3

Functional validation of the predicted signal peptides of the PvCRN proteins. (A) Yeast growth assay on CMD-W media (left) and YPRAA media (right). (B) Yeast invertase secretion assay for the predicted signal peptides of the indicated PvCRN proteins.

FIGURE 4

PvCRN proteins are distributed among different plant cell compartments. PvCRN proteins labeled with GFP were transiently expressed in N. benthamiana leaves to determine their subcellular localization in the plant cell by confocal microscopy at 48–72 h post agroinfiltration. p, plasma membrane; c, cytoplasm; n, nucleus; m, membrane. (A) A summary of statistics of subcellular localizations of PvCRN proteins. (B) PvCRN15, PvCRN16, PvCRN30, and PvCRN35 were localized in the membrane system of the host cell, and PM-RK was used as a membrane localization marker. (C) PvCRN19, PvCRN27, and PvCRN29 were localized in the nucleus of the plant cell. PvCRN6 was one of the PvCRN proteins that were localized in the plasma membrane, cytoplasm, and nucleus of the plant cell. (D) PvCRN17 was distributed among both the plasma membrane and nucleus of the plant cell. The scale bar indicates 20 μm.

FIGURE 5

PvCRN11 induced cell death in N. benthamiana leaves but failed to induce cell death in V. riparia and V. vinifera cv. Thompson seedless leaves. (A) PvCRN11 induced spotted necrosis on N. benthamiana leaves. Constructs PVX-GFP and PVX-INF1 were transiently expressed along with PVX1-PvCRN11 in N. benthamiana leaves as the negative and positive controls for plant cell death induction, respectively. The expression of each construct was verified by western blot, except for INF1 since the plant cell death phenomena induced by PVX-INF1 were typical, even though there was no accessible corresponding antibody (B). Photographs were taken 5–8 days after agroinfiltration. (C) PvCRN11 did not induce HR-like cell death on V. riparia (left) or V. vinifera cv. Thompson seedless (right) leaves. PvRXLR77-GFP and GFP were expressed in grapevine leaves simultaneously with PvCRN11-GFP, and their corresponding resulting phenomena served as the positive and negative controls, respectively. The expression of each recombinant protein was confirmed through observing GFP fluorescence in the infiltrated leaves 72–120 h post agroinfiltration (D). Photographs of grapevine leaves were taken 7–10 days after agroinfiltration. The experiment was repeated at least three times with at least six leaves tested in each independent experiment.

FIGURE 6

Transient expression of PvCRN17, PvCRN20 and PvCRN23 in N. benthamiana leaves promoted P. capsici colonization. (A–C) indicate the results for PvCRN17, PvCRN20, and PvCRN23, respectively. Left panel: representative inoculated N. benthamiana leaves stained with trypan blue. The lesions are outlined with red circles. Right panel: mean lesion lengths of N. benthamiana leaf regions in which each PvCRN or the control GFP were expressed. The heights of rectangles represent the mean lesion lengths. Error bars represent SD of 9–12 samples. Asterisks indicate significant differences from the control groups (paired t-test; **p < 0.01). Similar results were obtained from at least three independent experiments.

FIGURE 7

PvCRN10 and PvCRN26 enhanced the resistance of N. benthamiana to P. capsici when transiently expressed in N. benthamiana. (A) Results for PvCRN10, (B) Results for PvCRN26. Left panel: representative inoculated N. benthamiana leaves stained with trypan blue. The lesions are outlined with red circles. Right panel: mean lesion lengths of N. benthamiana leaf regions that were expressing the control GFP plus either PvCRN10 or PvCRN26. The heights of rectangles represent the mean lesion lengths. Error bars represent SD of 9–12 samples. Asterisks indicate significant differences from the control groups (paired t-test; *p < 0.05). Similar results were obtained in three independent experiments.

FIGURE 8

Plant nuclear localized proteins PvCRN19, PvCRN27, and PvCRN29 have different effects on the colonization of N. benthamiana by P. capsici. PvCRN19 made N. benthamiana leaves more susceptible to P. capsici, whereas PvCRN27 and PvCRN29 showed the opposite effect. (A–C) indicate the results for PvCRN19, PvCRN27, and PvCRN29, respectively. Left panel: representative inoculated N. benthamiana leaves stained with trypan blue. The lesions are outlined with red circles. Right panel: mean lesion lengths of N. benthamiana leaf regions in which each PvCRN or the control GFP were expressed. The heights of rectangles represent the mean lesion lengths. Error bars represent SD of 9–12 samples. Asterisks indicate significant differences from the control groups (paired t-test; *p < 0.05; **p < 0.01). Similar results were obtained in three independent experiments.

FIGURE 9

The PvCRN11gene enhanced the resistance of N. benthamiana to P. capsici when transiently expressed in N. benthamiana. Left panel: representative inoculated N. benthamiana leaves stained with trypan blue. The lesions are outlined with red circles. Right panel: mean lesion lengths of N. benthamiana leaf regions in which PvCRN11 or the control GFP were expressed. The heights of rectangles represent the mean lesion lengths. Error bars represent SD of nine samples. Asterisks indicate significant differences from the control groups (paired t-test; **p < 0.01). Similar results were obtained in three independent experiments.

FIGURE 10

Detection of the transcription level of PvCRN genes during infection of P. viticola YL on V. vinifera Pinot Noir by RT-PCR. (A) Transcriptional analysis of the PvCRN family in P. viticola YL sporangia 96 h post inoculation (96 hpi) on Pinot Noir leaves. The corresponding DNA band of each detectable PvCRN gene was marked with a pentagram. (B) Transcriptional analysis of PvCRN genes at 72 hpi (upper) and 120 hpi (lower). (C) Detection of the total RNA and cDNA prepared from Pinot Noir leaves inoculated with P. viticola YL. Experiments were repeated three times with similar results.