A candidate RxLR effector from Plasmopara viticola can elicit immune responses in Nicotiana benthamiana

Abstract

Background: Diverse plant pathogens deliver effectors into plant cells to alter host processes. Oomycete pathogen encodes a large number of putative RxLR effectors which are likely to play a role in manipulating plant defense responses. The secretome of Plasmopara viticola (downy mildew of grapevine) contains at least 162 candidate RxLR effectors discovered in our recent studies, but their roles in infection and pathogenicity remain to be determined. Here, we characterize in depth one of the putative RxLR effectors, PvRxLR16, which has been reported to induce cell death in Nicotiana benthamiana in our previous study.

Results: The nuclear localization, W/Y/L motifs, and a putative N-glycosylation site in C-terminal of PvRxLR16 were essential for cell death-inducing activity. Suppressor of G-two allele of Skp1 (SGT1), heat shock protein 90 (HSP90) and required for Mla12 resistance (RAR1), but not somatic embryogenesis receptor-like kinase (SERK3), were required for the cell death response triggered by PvRxLR16 in N. benthamiana. Some mitogen-activated protein kinases and transcription factors were also involved in the perception of PvRxLR16 by N. benthamiana. PvRxLR16 could also significantly enhance plant resistance to Phytophthora capsici and the nuclear localization was required for this ability. However, some other PvRxLR effectors could suppress defense responses and disease resistance induced by PvRxLR16, suggesting that it may not trigger host cell death or immune responses during physiological infection under natural conditions.

Conclusion: These data demonstrate that PvRxLR16 may be recognized by endogenous proteins in nucleus to trigger immune responses in N. benthamiana, which in turn can be suppressed by other PvRxLR effectors.

Keywords: Nicotiana benthamiana cell death; Plasmopara viticola; RxLR effector; grapevine; immune responses.

Fig. 1

PvRxLR16 induces cell death in N. benthamiana. a Leaves of N. benthamiana were infiltrated with A. tumefaciens carrying PVX-Flag::PvRxLR16 and the indicated controls. The photography was taken 5 d post infiltration. The experiment was repeated three times with similar results. b Quantification of cell death by measuring electrolyte leakage. Error bars represent standard errors from three biological replicates (^**, P < 0.01, Dunnett’s test)

Fig. 2

PvRxLR16 effector domains function in plant nucleus. a Green fluorescence-tagged PvRxLR16 were transiently expressed in Arabidopsis protoplasts via PEG-mediated transformation approach. Scale bar =25 μm. b GFP- PvRxLR16 fusion protein was expressed in onion epidermal cells. Scale bar =50 μm. c Nuclear localization is required for PvRxLR16-triggering cell death. N. benthamiana leaves were agroinfiltrated with the indicated constructs 2 d and 5 d before assessment of GFP confocal imaging and cell death observation, respectively. NES and nes represent the nuclear export signal and nonfunctional NES. Scale bar =20 μm. d Quantification of cell death by measuring electrolyte leakage. Error bars represent standard errors from three biological replicates (^** for P < 0.01 and ^* for P < 0.05, Dunnett’s test)

Fig. 3

Deletion analysis of PvRxLR16. a Left column, schematic diagrams of deletion mutants for PvRxLR16. Right column, deletion mutants of PvRxLR16 were expressed by agroinfiltration in N. benthamiana to investigate induction of cell death. The representative pictures were taken at 5 dpi. b Immunoblot analysis of proteins from N. benthamiana leaves transiently expressing PvRxLR16 and its deletion mutants from a PVX-3 × Flag vector

Fig. 4

Functional characterization of three putative N-glycosylation sites for PvRxLR16. a Expression of PvRxLR16 and its mutants in N. benthamiana by agroinfiltration. Typical symptoms were photographed at 5 dpi. b Immunoblot analysis of proteins from N. benthamiana leaves transiently expressing PvRxLR16 and its site-specific mutations from a PVX-3 × Flag vector. c Quantification of cell death by measuring electrolyte leakage. Error bars represent standard errors from three biological replicates (^**, P < 0.01, Dunnett’s test)

Fig. 5

SGT1, Hsp90, and RAR1 were required for PvRxLR16-induced cell death in N. benthamiana. a PvRxLR16 was transiently expressed in N. benthamiana leaves silenced for pTV00 (control), SGT1, Hsp90, RAR1, and SERK3. GFP and INF1 are control proteins. Typical symptoms were photographed after 5 d after agroinfiltration. b Quantification of cell death by measuring electrolyte leakage. Averages and standard errors were calculated from three independent experimental treatments (^** for P < 0.01 and ^* for P < 0.05, Dunnett’s test). c Transcript levels of indicated genes in silenced N. benthamiana measured by quantitative RT-PCR. Error bars represent standard errors from three biological replicates (^**, P < 0.01, Dunnett’s test)

Fig. 6

MAPK cascades were required for PvRxLR16-induced cell death in N. benthamiana. a PvRxLR16 was transiently expressed in N. benthamiana leaves silenced for indicated MAPK cascades genes. GFP and INF1 are control proteins. Typical symptoms were photographed after 5 d after agroinfiltration. b Quantification of cell death by measuring electrolyte leakage. Averages and standard errors were calculated from three independent experimental treatments (^**, P < 0.01, Dunnett’s test). c Transcript levels of indicated genes in silenced N. benthamiana measured by quantitative RT-PCR. Error bars represent standard errors from three biological replicates (^**, P < 0.01, Dunnett’s test)

Fig. 7

Introduction of disease resistance and H₂O₂ accumulation by PvRxLR16 in N. benthamiana. a Lesions of the N. benthamiana leaves expressing the indicated genes inoculated with P. capsici at 36 hpi. b Lesion diameters of N. benthamiana leaves (**P < 0.01, Dunnett’s test). c DAB staining of the N. benthamiana leaves at 3 dpi expressing the indicated genes. d The relative levels of DAB staining. Asterisks indicate significant differences (**P < 0.01, Dunnett’s test). These experiments were replicated three times with six leaves per biological replicate

Fig. 8

Upregulation of defense- related genes mediated by PvRxLR16 in N. benthamiana. Transcript level of the PR1b, PR2b, LOX and ERF1 genes induced by PvRxLR16 at different time points. Means and standard errors from three independent replicates are shown

Fig. 9

Suppression of PvRxLR16-induced disease resistance and immune responses by PvRXLR effectors in N. benthamiana. a Lesions of the N. benthamiana leaves expressing the indicated genes inoculated with P. capsici at 36 hpi. The leaves were infiltrated with PvRxLR16 constructs 12 h after expressing each PvRxLR or GFP using agroinfiltration. Then the infiltrated leaves were inoculated with P. capsici 48 h after expression of PvRxLR16. The representative pictures were taken at 36 hpi post infection of P. capsici. b DAB staining of the N. benthamiana leaves at 3 dpi expressing PvRxLR16. PvRxLR effectors (PvRxLR1, 10 and 30) and GFP were transiently expressed in N. benthamiana leaves by agroinfiltration 12 h before infiltration of PvRxLR16. c Lesion diameters of N. benthamiana leaves (**P < 0.01, Dunnett’s test). d The relative levels of DAB staining. Significant differences based on Dunnett’s test are indicated by the asterisks (**P < 0.01). These experiments were replicated three times with six leaves per biological replicate. e Relative expression levels of PR1b, PR2b, LOX, and ERF1 genes induced by PvRxLR16 were suppressed by other PvRxLRs (PvRxLR1, 10 and 30) in N. benthamiana. PvRxLR effectors (PvRxLR1, 10 and 30) and GFP were transiently expressed in N. benthamiana leaves by agroinfiltration 12 h before infiltration of PvRxLR16. The relative transcript levels of defense-related genes were detected at 3 dpi expressing PvRxLR16 (+). EF1a was used as an endogenous control. Means and standard errors from three independent replicates are shown. Significant differences based on Dunnett’s test are indicated by the asterisks (**P < 0.01, *P < 0.05)