Plasmopara viticola effector PvRXLR53 suppresses innate immunity in Nicotiana benthamiana

ABSTRACT

Plasmopara viticola, the casual oomycete of grapevine downy mildew, could cause yield loss and compromise berry quantity. Previously, we have identified several PvRXLR effectors that could suppress plant immunity to promote infection and disease development. In this study, the role of effector, PvRXLR53, in plant–microbe interaction was investigated. PvRXLR53 has several orthologs in other oomycetes and contains a functional signal peptide. Expression level of PvRXLR53 was already detected upon inoculation, further induced in the early stage after P. viticola inoculation and decreased to low level in the late infection stage in grapevine (Vitis vinifera ‘Cabernet Sauvignon’). PvRXLR53 is localized in both nucleus and cytoplasm. When transiently expressed in Nicotiana benthamiana, PvRXLR53 suppressed oomycete elicitor INF1-triggered programmed cell death and defense gene expression, and Phytophthora capsici-induced reactive oxygen species production (ROS) and eventually resistance to P. capsici. In summary, these findings suggest that P. viticola secretes PvRXLR53 to suppress host immunity from the very early stage of infection.

KEYWORDS:

• Effector
• Nicotiana benthamiana
• Plasmopara viticola
• PvRXLR53

Introduction

Through long-term co-evolutionary struggle, plants have evolved complicated and highly effective innate immune defense system to resist attack by microbial pathogens.^1,2 Recent molecular studies of the plant–pathogen interaction reveal that plants have evolved two layers of immune responses to resist pathogens. PAMP-triggered immunity (PTI) acts as the first one, which effectively prevents a broad range of potential pathogens by detection of conserved pathogen-associated molecular patterns (PAMPs).^1,3 However, a variety of effectors are secreted by plant pathogens to manipulate plant immunity thereby interfering with PTI and facilitating infection.^4,5 In response, plants acquire resistance proteins (R proteins) to monitor effectors specifically, resulting in effector-triggered immunity (ETI).² ETI is a kind of faster and stronger disease resistance response. Due to the natural selection, pathogens are compelled to avoid ETI either by escaping from the recognition via shedding or diversifying, or by acquiring additional effectors. Programmed cell death (PCD), callose depositions, production of reactive oxygen species (ROS), and a series of defense-related genes expression are common plant immune responses.⁶ Numerous studies have shown that pathogen effectors tend to suppress these defense responses to promote pathogenicity.

The obligately biotrophic Plasmopara viticola is the agent of grapevine downy mildew, one of the most destructive disease of the global grape and wine industry.^7–9 It is known that P. viticola secrets a series of effectors containing the RXLR motif (for Arg, any amino acid, Leu and Arg) to suppress host immunity.⁷ At least 188 candidate P. viticola RXLR effectors have been reported in previous studies, and some of which have been functionally characterized.^10–15 For example, the virulence function of PvRXLR131 is mediated by interacting with plant BRI1 kinase inhibitor 1 to promote infection.¹⁶ During P. viticola infection, PvRXLR159 is induced dramatically and secreted into plant to suppress host resistance.¹⁷ Here, we unravel the role of PvRXLR53 in pathogen–plant interaction to understand the infection strategies of the plant pathogens.

Materials and methods

Bioinformatics analysis

Multiple amino acid sequences were aligned using BioEdit3.3.19.0 software with ClustalW. The signal peptide of PvRXLR53 was predicted by SingalP5.0 Server (http://www.cbs.dtu.dk/services/SignalP/).¹⁸

Plant materials and culture conditions

The grapevine (Vitis vinifera ‘Cabernet Sauvignon’) and Nicotiana benthamiana were both grown in a greenhouse at 25°C under 16/8 h light/dark.

Gene cloning and vector construction

The full-length of predicted PvRXLR53 genes was amplified using 2 × PrimeSTAR Max DNA Polymerase (Takara Bio Inc., Beijing, China). The open reading frame of PvRXLR53 lacking the signal peptide sequence was ligated into the vector both pGR106 (Potato virus X-based binary expression vector, PVX) and pHB-GFP. The signal peptide of PvRXLR53 was ligated into vector pSUC2 (pSUC2T7M13jORI).¹⁹ Green fluorescent protein, GFP. The primers are listed in Table S1.

Yeast signal sequence trap assay

To validate the function of the predicted signal peptide of PvRXLR53, the signal sequence trap assay was performed using the yeast strain YTK12 and the recombinant plasmid pSUC2-PvRXLR53 according to previous study.^20–22

Agrobacterium tumefacien-mediated transient expression in N. benthamiana

Agrobacterium tumefaciens GV3101 carrying the indicated plasmids were used for transient expression in N. benthamiana according to our previous methods.^23,24 For subcellular localization, the transferred A. tumefaciens cultures containing PvRXLR53-GFP/GFP were infiltrated in N. benthamiana leaves, respectively. The accumulation of fluorescence signals was monitored 2 days post-infiltration using a confocal microscope (Leica TCS SP5II).²⁵

Pathogen infection assay

Each grapevine leaf disc (1 cm in diameter) was inoculated with 20 μL of 1 × 10⁴ sporangia/mL P. viticola suspension on the abaxial surface. Excess water was removed at 36 hours post inoculation (hpi).²⁶ The samples were taken at 0, 6, 12, 36, 72 and 120 hpi for qRT-PCR analysis.²⁷

For P. capsici infection assay, agar discs (5 mm in diameter) harboring mycelium were inoculated on the abaxial surface of excised N. benthamiana leaves according to the methods previously reported.²⁸ The lesion areas were measured at 48 hpi by Image J software.

DAB staining

Reactive oxygen species production was analyzed by 3, 3ʹ-diaminobenzidine (DAB) staining. The excised leaves were placed in 1 mg/mL DAB (dissolved in 50 mM Tris-HCl, pH 5.0) and vacuum infiltrated three times for 5 min until the leaves were entirely soaked. And then the soaked leaves were maintained for 24 h in the dark. The leaf tissues were decolorized in boiling 95% ethanol for 10 min and subjected to imaging.²⁹

Electrolyte leakage assay

Electrolyte leakage assay was performed to analyze the cell death of N. benthamiana.³⁰ For each sample, five leaf discs (8 mm in diameter) were immersed into 5 mL sterile water for 3 h. The conductivity was detected using a conductivity meter (DDS-307, Rex Shanghai, China). Electrolyte leakage (%) = 100 × A/B. The ‘value A’ and ‘value B’ represent the conductivity before and after boiling. All experiments were repeated three times.

RNA extraction and real-time quantitative RT-PCR

The RNA extraction kit (OMEGA, Norcross, GA, USA), iScript cDNA Synthesis kit (BIO-RAD, Hercules, CA, USA) and iTaq Universal SYBR Green Supermix (BIO-RAD, Hercules, CA, USA) were respectively used for RNA extraction, cDNA synthesis and quantitative RT-PCR reactions in our study.³¹ The primers used are listed in Table S1.

Protein extraction and western blotting

Total proteins were extracted according to previous studies.³² The proteins of plant tissues were extracted in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.2% (v/v) Triton X-100, 1 × protease inhibitor cocktail. The extract was boiled with 5 × SDS loading buffer at 100°C for 5 min and detected by western blotting.

Statistical analysis

The SPSS 20 (SPSS Inc., Chicago, IL) statistical software was used to determine the significant differences. All reaction data of qRT-PCR were analyzed using BIO-RAD CFX software.

Results

Identification and sequence analysis of effector PvRXLR53

About 100 genes encoding candidate RXLR effectors were identified in the genome of P. viticola isolate “JL-7-2”¹¹ One of them, PvRXLR53, encompassed an N-terminal signal peptide with 20 amino acids and a conserved RXLR motif.⁷ Generally, PvRXLRs effector shows only limited similarity to RXLR effectors identified from other oomycete species.¹¹ In the current study, however, PvRXLR53 was found being comparatively conserved across oomycetes as demonstrated that it had more than 47% sequence similarity with Plasmopara halstedii, Phytophthora infestans, Phytophthora parasitica, Phytophthora fragariae, and Phytophthora sojae (Figure 1). The amino acid sequence alignments of the clustered RXLRs revealed that these proteins had an especially higher sequence similarity in the C-terminal domain, a functional domain contributing to the effector activity.

Figure 1. Amino acid sequence alignment of PvRXLR53 and the other similar RXLR effector proteins from different oomycete species

The conserved RXLR motifs are indicated in red box. The alignment was performed using BioEdit software. The shading threshold was set at 50%. Identical and similar amino acid residues are shaded black and gray, respectively.

PvRXLR53 is expressed upon infection and further induced

To investigate the expression pattern of the effector PvRXLR53 during P. viticola attacking the grapevine, qRT-PCR was performed to analyze its expression levels at different time points post inoculation. Expression of PvRXLR53 was already detected upon inoculation, peaked at 6 hpi and then decreased to low level (Figure 2a). This indicates that the effector PvRXLR53 might contribute to the virulence during the infection of P. viticola to grapevine.

Figure 2. Functional characterization of effector PvRXLR53 during the pathogen infection

A. The expression pattern of the effector PvRXLR53 during the infection of P. viticola. Transcriptional levels of PvRXLR53 were monitored by qRT-PCR at different time points post-inoculation using the internal reference gene PvActin (P. viticola Actin). The relative transcription at time 120 hpi was standardized as 1. Error bars (SEs) were from three independent biological replicates. hpi, hours post inoculation. B. Functional validation of the PvRXLR53 signal peptide. The PvRXLR53 signal peptide fragments fused in the pSUC2 vector enabled invertase secretion and the transferred YTK12 yeast cells to grow on YPRAA media. C. Subcellular localization analysis of PvRXLR53. The images of fluorescent accumulation were captured at 48 hpi by confocal microscopy in N. benthamiana. Scale bar, 50 µm.

PvRXLR53 contains a functional signal peptide

The N-terminal 20 amino acids of PvRXLR53 were predicted by singalP5.0 to be a signal peptide with a high probability of 0.9494. The signal sequence trap (SST) assay was performed to validate the secretory activity of the signal peptide in PvRXLR53. The untransformed YTK12 strain and the YTK12 carrying pSUC2-Mg87 vector were used as negative controls, while the YTK12 carrying pSUC2-Avr1b vector was as a positive one. In accord with observation, YTK12 yeast cells carrying pSUC2-PvRXLR53 were able to utilize raffinose as the sole carbon source to grow on YPRAA medium consistent with the positive control, while the negative control yeast strain and the YTK12 strain without transformation could not (Figure 2b). This result confirmed that the effector PvRXLR53 contained a functional signal peptide.

PvRXLR53 is localized in both nucleus and cytoplasm

To determine the localization of PvRXLR53 in plants, PvRXLR53 with green fluorescent protein (GFP) fused to its C terminus was transiently expressed in N. benthamiana leaves. GFP was served as control. The accumulation of fluorescence was visualized at 48 h post-infiltration by confocal microscopy. The results indicate that PvRXLR53 is localized in both nucleus and cytoplasm (Figure 2c).

PvRXLR53 suppresses INF1-triggered cell death and the expression levels of the defense-related genes in N. benthamiana

Oomycete PAMP INF1 is known to trigger a series of plant immune responses.^33,34 We investigated the effects of PvRXLR53 on INF1-triggered immunity. The PvRXLR53 and GFP were transiently expressed in N. benthamiana 24 h followed by expression of INF1, respectively. As shown in Figure 3a, instead of GFP, PvRXLR53 suppressed the INF1-induced PCD in N. benthamiana. The suppression by PvRXLR53 was further confirmed by ion leakage measurement (Figure 3b).

Figure 3. Suppression by PvRXLR53 of INF1-induced immune responses

PvRXLR53 and GFP control were agroinfiltrated into each side of N. benthamiana leaves for 24 h followed by infiltration with INF1. A. Suppression of INF1-triggered programmed cell death by PvRXLR53. Necrosis caused by programmed cell death was captured and analyzed at 4 days after infiltration with INF1 in N. benthamiana. The circles indicated the infiltrated areas of INF1. B. Quantification of cell death was measured by electrolyte leakage. The means and standard deviation are from three independent experiments. An asterisk represents statistically significant differences (*P < .01, Student’s t-test). C. Suppression of INF1-triggered defense-related genes in N. benthamiana. The relative expression levels of NbPR1b, NbPR2b and NbLOX after infiltration with INF1 were assayed by qRT-PCR at different time points with the NbEF1α as a reference gene for normalization. The relative transcription at 0 h was standardized as 1. Mean and standard errors (SEs) were calculated from three biological replicates. Asterisks indicate significant differences based on Dunnett’s test (*P < .01).

In addition, the effect of PvRXLR53 on INF1-induced transcription of defense-related genes, PR1b, PR2b and LOX,^35,36 was also investigated in N. benthamiana by qRT-PCR. As shown in Figure 3c, INF1-triggered NbPR1b, NbPR2b and NbLOX were all significantly suppressed by the expression of PvRXLR53. Taken together, these results clearly indicate that PvRXLR53 could suppress plant basal immune responses in N. benthamiana.

PvRXLR53 enhances plant susceptibility to Phytophthora capsici in N. benthamiana

In order to explore the virulence contribution of PvRXLR53, we investigated the effect of PvRXLR53 during a pathogenic oomycete P. capsici infecting N. benthamiana. When PvRXLR53 and GFP were transiently expressed in N. benthamiana leaves via agroinfiltration followed by inoculation with equal area of P. capsici mycelium, effector PvRXLR53 clearly suppressed the ROS production as indicated by DAB staining (Figure 4a). Meanwhile, the effector also significantly enlarged the lesion size induced by P. capsici (Figure 4b, c) at 2 dpi. To validate the suppression of ROS production and P. capsici infectivity was indeed due to the expression of the effector, PvRXLR53 protein was confirmed by western blotting (Figure 4d). These results indicate that PvRXLR53 enhances N. benthamiana susceptibility to P. capsici.

Figure 4. Inhibition of ROS-mediated resistance and enhanced susceptibility to P. capsici by PvRXLR53 in N. benthamiana.

PvRXLR53-GFP and GFP were transiently expressed in each side of one piece of leave via agroinfiltration followed by inoculation with equal area of P. capsici mycelium after 24 h in N. benthamiana. A. DAB staining was performed at 2 dpi. The circles in solid line and in dotted line indicated the infiltrated sites and P. capsici infection area, respectively. B. Photographs of typical symptoms were taken at 2 dpi. The circles indicated the infiltrated sites. C. Quantification of lesion size diameters in B were measured. Results are mean and standard deviation from three independent biological replicates. Each time at least five leaves were used for inoculation. An asterisk represents significant differences (*P < .01, Student’s t-test) in lesion size between the effector and GFP infiltration. D. Expression validation by western blotting analysis. Scale bars, 2 cm.

Conclusions

In this study, we characterized the function PvRXLR53 with the following results: 1) PvRXLR53 may have orthologs in P. halstedii, P. infestans, P. parasitica, P. fragariae, and P. sojae; 2) it was constitutively expressed in P. viticola and its expression only dominated in the early P. viticola infection stage on grapevine; 3) it has a functional signal peptide and is localized in nucleus and cytoplasm; 4) transient expression of PvRXLR53 suppressed INF1-triggered programmed cell death and the expression of the defense-related genes, and P. capsici-induced ROS production making N. benthamiana more susceptible to P. capsici infection. These findings lead us to hypothesize that P. viticola secretes PvRXLR53 to suppress host immunity from the very early stage of infection. Future research is needed to identify the host targets of PvRXLR53 to further understand its working modes in plants.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by National Key R & D Program of China (2018 YFD 1000301 to J.L., 2019 YFD1002500 to S.S), Shanghai Science and Technology Commission (18391900400 to J.L.), the Start-up Fund from Shanghai Jiao Tong University (WF107115001 to J.L. and WF220515002 to W.Y.), China Agriculture Research System (CARS-29-yc-2 to J.L.), National Natural Science Foundation of China (31901984 to W.Y.) and Shanghai Natural Science Foundation (19ZR1428000 to S.S.).

Supplementary Material

Supplemental data for this article can be accessed on the publisher’s website