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A Phytophthora capsici virulence effector associates with NPR1 and suppresses plant immune responses
Phytopathology Researchvolume 1, Article number: 6 (2019)
Salicylic acid (SA) plays a crucial regulatory role in plant immunity. NPR1 (non-expressor of pathogenesis related-1) is a SA receptor and plays a pivotal role in SA signaling. However, pathogen effectors which target NPR1 to promote infection have rarely been reported. Here, we identified a Phytophthora capsici effector RxLR48 that associates with NPR1, facilitates P. capsici infection and is required for pathogen virulence. Furthermore, we demonstrated that RxLR48 promotes NPR1’s nuclear localization and inhibits its proteasome-mediated degradation, suggesting that RxLR48 suppresses SA signaling by targeting the central regulator NPR1. In addition, we showed that RxLR48 also suppresses pattern-triggered immunity (PTI). Together, our research indicates that P. capsici suppresses plant immunity by targeting SA and PTI pathways.
The plant pathogenic oomycetes, which are evolutionarily close to algae in the kingdom Stramenopila, represent one of the greatest threats to agriculture and natural ecosystems in the worldwide (Kamoun 2003). Within oomycetes, Phytophthora that literally means plant destroyer, contains the most devastating pathogens causing notorious plant diseases (Tyler et al. 2006). For example, Phytophthora infestans, the causal agent of potato late blight, caused the Great Irish Famine in nineteenth century (Kroon et al. 2012). P. sojae causes soybean root and stem rot, leading to serious yield losses every year (Tyler 2007). P. ramorum, which affects trees and shrubs, is the most destructive disease of oaks worldwide (Kamoun et al. 2015). Thus, there are urgent needs to understand the infection process and mechanism of Phytophthora pathogens. P. capsici causes many devastating diseases on a broad range of plant species, including the model plant Arabidopsis thaliana, Nicotiana benthamiana and hundreds of vegetable crops such as pepper and cucumber (Lamour et al. 2012a; Wang et al. 2013). In recent years, the exploitation of P. capsici as a model pathogen is widely accepted and the molecular interaction between P. capsici and its hosts attracts increasing attentions (Wang et al. 2013).
Plant pathogens deliver molecules termed effectors to manipulate host immunity during infection, usually by targeting vital immune components or physiological processes. Phytophthora pathogens secrete hundreds of effectors, a prominent category among which is RxLR effectors. The RxLR effectors contain a conserved RxLR (Arg-any amino acid-Leu-Arg) motif in the N-terminal for translocation into plant cells (Tyler et al. 2006; Whisson et al. 2007; Dou et al. 2008a). Therefore, numerous researches have been implemented to uncover the biological function of effectors by identifying their host targets. For instance, P. infestans effector AVR3a can bind to and stabilize potato E3 ubiquitin ligase CMPG1 to prevent INF1-mediated cell death (Bos et al. 2010). Another P. infestans effector PexRD2 interacts with the kinase domain of potato MAPKKKε to perturb immunity-related signaling pathways (King et al. 2014). In addition, P. sojae effector PSR1 directly targets host PINP1, which is required for accumulation of distinct classes of endogenous small RNAs, to promote infection (Qiao et al. 2015). Like other Phytophthora species, P. capsici produces about four hundred cytoplasmic RxLR effectors (Lamour et al. 2012b). However, few studies have identified the host targets of these P. capsici RxLR effectors and the molecular mechanisms involved are still largely unknown.
To cope with pathogen infection, plants develop two layers of immune system to recognize pathogen signatures and activate defense responses at a molecular level (Jones and Dangl 2006). The first layer is dependent on host pattern recognition receptors (PRRs) through the recognition of conserved microbial features, leading to pattern-triggered immunity (PTI) that provides basal resistance to a wide range of pathogens (Jones and Dangl 2006). Plants also evolve intracellular receptors to specifically recognize corresponding avirulence effectors and activate effector-triggered immunity (ETI), resulting in robust disease resistance to certain pathogens (Cui et al. 2015).
SAR (systemic acquired resistance) is usually induced in distal tissues upon the onset of ETI at the primary infection sites, protects plants from secondary infection by activating long-lasting (up to several months) and broad-spectrum resistance (Kachroo and Robin 2013). Salicylic acid (SA) is a key plant hormone that is required for both local and systemic acquired resistance (Dong 2004). Plants that are defective in SA synthesis/accumulation always exhibit enhanced susceptibility to pathogens (Wildermuth et al. 2001). And the SA receptor NPR1 (non-expressor of pathogenesis related-1), which functions as a transcriptional co-activator, acts as the central signaling regulator during SAR (Mukhtar et al. 2009).
NPR1 was first cloned using a map-based approach in Arabidopsis and was found to encode a novel protein containing ankyrin repeats (Cao et al. 1997). Subsequent studies demonstrated that nuclear localization of NPR1 is essential for its activity in inducing PR genes and NPR1-mediated DNA binding of a basic leucine zipper transcription factor TGA2 is critical for activation of defense genes (Despres et al. 2000; Kinkema et al. 2000). Normally, NPR1 is present in the cytoplasm as an oligomer through intermolecular disulfide bonds. However, SAR results in reduction of NPR1 to a monomeric form, which accumulates in the nucleus to activate gene expression (Mou et al. 2003). S-nitrosylation of NPR1 by S-nitrosoglutathione (GSNO) facilitates its oligomerization, while the SAR-induced NPR1 monomerization is catalyzed by thioredoxins (TRXs) (Tada et al. 2008). In addition, proteasome-mediated turnover of phosphorylated NPR1 through a Cullin3-based ubiquitin ligase in the nucleus is required for full induction of target genes and establishment of systemic immunity (Spoel et al. 2009). The systemic immunity also requires SnRK2.8-mediated phosphorylation of NPR1, which is necessary for the nuclear import of NPR1 (Lee et al. 2015). Recent study showed that sumoylation of NPR1 activates gene expression by switching its association with the WRKY transcription repressors to TGA transcription activators (Saleh et al. 2015). Considering NPR1 is a master regulator of SA-mediated responses, we proposed that Phytophthora pathogens would had evolved certain effectors to target host NPR1 for virulence function.
In this study, we screened P. capsici effectors by using NPR1 as a bait in yeast two-hybrid (Y2H) system and obtained an interacting effector RxLR48. We confirmed the interaction between RxLR48 and NPR1 by Y2H and co-immunoprecipitation (co-IP) assays. We found that transient expression of RxLR48 facilitates P. capsici infection, and silencing of RxLR48 impairs the pathogenicity of P. capsici. Furthermore, we demonstrated that co-expression of NPR1 with RxLR48 results in enhanced localization of NPR1 in the nucleus and elevated protein accumulation of NPR1 by inhibiting its proteasome-mediated degradation. Finally, we found that RxLR48 could suppress PTI-related immune responses and reduce the expression levels of PTI marker gene FRK1. Together, our results revealed that the P. capsici effector RxLR48 could target plant NPR1 and PTI signaling pathway for virulence and suggested potential mechanisms underlying this interaction to manipulate plant immunity.
RxLR48 interacts with NPR1
To identify P. capsici effectors targeting plant NPR1, we exploited yeast two-hybrid approach to screen P. capsici RxLR effectors by using Arabidopsis NPR1 as a bait. Fourty-two RxLR effectors from P. capsici (Lamour et al. 2012b) were separately cloned into Y2H prey vector pGADT7 (Additional file 1: Table S1), and used for Y2H screening. Finally, we identified RxLR48 associating with plant NPR1.
To confirm this association, we cloned RxLR48 into the bait vector pGBKT7, and NPR1 into the prey vector pGADT7 for reciprocal Y2H assay. The result clearly showed that RxLR48 associates with NPR1 in yeast (Fig. 1a). To further confirm the association between RxLR48 and NPR1, we performed co-IP assay in Arabidopsis protoplasts. RxLR48-FLAG and NPR1-HA were transiently expressed in Arabidopsis protoplasts, and then the protoplasts were subsequently subjected to co-IP assay. A specific signal for NPR1-HA was clearly observed in the RxLR48-FLAG immunoprecipitate (Fig. 1b), indicating that RxLR48 associates with NPR1 in planta.
We also found that RxLR48 homologs were present in other tested Phytophthora species (Additional file 2: Figure S1). This result indicates that RxLR48 is a conserved effector in Phytophthora pathogens.
RxLR48 enhances P. capsici infection in N. benthamiana
To investigate RxLR48’s virulence function, GFP-RxLR48 was transiently expressed in N. benthamiana and GFP alone was used as a negative control. The infiltrated leaves were then inoculated with P. capsici. Expression of RxLR48 significantly promoted infection of P. capsici (Fig. 2a, b). The photograph of disease symptoms and statistical analysis of lesion diameters together showed that ectopic expression of RxLR48 led to development of bigger lesions, compared with the GFP expression control (Fig. 2a, b). The GFP-RxLR48 fluorescence was observed in the infiltrated leaves using a confocal microscope to ensure protein expression (Fig. 2c). Together, these results demonstrated that RxLR48 is able to effectively enhance P. capsici colonization in planta.
RxLR48 contributes to pathogen virulence
To explore whether RxLR48 contributes to pathogen virulence, we silenced RxLR48 in P. capsici. To check silencing efficiency, total RNA was extracted from independent transformants and the transcription levels of RxLR48 were measured by quantitative real-time PCR (qRT-PCR). We obtained two RxLR48- silenced transformants, named T12 and T129, whose transcription levels were significantly decreased to 0.5% and 3.8% of the wild-type strain LT263, respectively (Fig. 3a). An additional transformant, T108, was selected as a control in which RxLR48 expression remained unaffected (Fig. 3a). Next, we evaluated the virulence of the RxLR48-silenced transformants on N. benthamiana leaves. The detached N. benthamiana leaves were drop-inoculated with zoospore suspensions of T12 or T129 on the right side while T108 zoospores on the left side (Fig. 3b). T12 and T129 developed smaller lesion diameters compared to T108 36 h post inoculation (hpi) (Fig. 3b). Meanwhile, statistical analysis showed that the lesion diameters caused by T12 and T129 were reduced to 31% and 77% relative to that caused by T108, respectively (Fig. 3c). Thus, these results indicate that RxLR48 is required for pathogen infection.
RxLR48 promotes nuclear accumulation of NPR1
It was reported that NPR1 was primarily localized in the cytoplasm and nucleus under normal circumstances, while accumulated in the nucleus in response to SA signal (Kinkema et al. 2000). To investigate whether RxLR48 could alter the subcellular localization of NPR1, GFP-NPR1 and mCherry-RxLR48 were transiently expressed in N. benthamiana. GFP-NPR1 distributed in the cytoplasm and nucleus when expressed alone in planta (Fig. 4a), and mCherry-RxLR48 exhibited similar subcellular localization as NPR1 (Fig. 4b). When mCherry was co-expressed, GFP-NPR1 showed similar localization pattern as GFP-NPR1 alone (Fig. 4c). Interestingly, the fluorescence intensity of NPR1 was significantly enhanced in the nucleus when mCherry-RxLR48 was co-expressed (Fig. 4d). Hence, our results suggest that RxLR48 could alter the subcellular localization of NPR1 by promoting its nuclear accumulation.
RxLR48 inhibits protein degradation of NPR1
Previous studies reported that proteasome-mediated turnover of NPR1 plays an important role in modulating transcription of its target genes (Spoel et al. 2009). In order to test whether RxLR48 could affect the stability of NPR1 during interaction, RxLR48-FLAG and NPR1-HA were transiently co-expressed in Arabidopsis protoplasts, and protein accumulation of NPR1-HA was detected by immunoblot. Results showed that the abundance of NPR1 was significantly higher when co-expressed with RxLR48 compared to NPR1 alone (Fig. 5a). It was shown that SA treatment induces NPR1 monomer formation in the nucleus, resulting in an actual increase in total NPR1 protein (Spoel et al. 2009). We treated NPR1-expressing protoplasts with SA, and immunoblot results proved that the abundance of NPR1 was greatly elevated regardless of the presence of RxLR48 (Fig. 5a). Together, these results demonstrated that co-expression of RxLR48 increases the accumulation of NPR1 protein.
Next, we analyzed NPR1 protein accumulation in the presence of MG132, an inhibitor of 26S proteasome (Zhang et al. 2015). When expressing NPR1 alone, the conspicuously elevated protein accumulation of NPR1 was observed after MG132 treatment (Fig. 5b), consistent with previous findings that NPR1 was degraded through the 26S proteasome (Spoel et al. 2009). Moreover, immunoblot analysis revealed that the abundance of NPR1 remained the same upon MG132 treatment regardless of the presence of RxLR48, whereas the negative control of dimethyl sulfoxide (DMSO) treatment showed similar result to Fig. 5a. Taken together, these results indicate that RxLR48 is able to promote NPR1 protein accumulation by suppressing its proteasome-mediated degradation.
RxLR48 suppresses ROS production and callose deposition in plants
In addition to interfering with SA signaling by affecting NPR1 subcellular localization and protein accumulation, we want to clarify if RxLR48 also suppresses PTI responses. Reactive oxygen species (ROS) production is a typical response in PTI signaling pathway (Dodds and Rathjen 2010). To test if RxLR48 is able to suppress ROS production, we conducted inoculation assay using the RxLR48-silenced transformant. The ROS production was visualized by DAB staining. Compared with the T108 control, inoculation of the silencing transformant T12 resulted in obviously elevated H2O2 accumulation in N. benthamiana leaves at 9 hpi (Fig. 6a). Callose deposition is another typical response in PTI signaling and is regulated by ROS (Dodds and Rathjen 2010). The leaves challenged with the silencing transformant T12 showed significantly enhanced callose deposition compared with T108 control (Fig. 6b). Taken together, these results suggest that RxLR48 suppresses PTI-related immune responses.
RxLR48 down-regulates the expression of PTI-responsive gene FRK1
To further clarify the virulence function of RxLR48, we subsequently detected the expression of defense-related genes in RxLR48-expressing Arabidopsis protoplasts. First, RxLR48 and EV (empty vector) were transiently expressed in protoplasts, then total RNA was isolated from protoplasts for qRT-PCR analysis. Four defense-related genes were selected for qRT-PCR analysis (Fig. 7a). NPR3, a paralog of NPR1, acts as a receptor for the immune signal SA (Fu et al. 2012); PR1 (pathogenesis-related 1), is a marker gene in SA signaling pathway (Cao et al. 1997); FRK1 has been widely used as a PTI marker gene (Li et al. 2005). The expression of NPR1 and NPR3 showed no significant differences in the RxLR48-expressing protoplasts compared to that in the EV control (Fig. 7a). We constantly observed reduced PR1 expression in the RxLR48-expressing protoplasts compared with the EV control, although it is not statistically significant when we set P < 0.01. Interestingly, transient expression of RxLR48 resulted in the reduction of FRK1 transcripts to 37% relative to that in the EV negative control (Fig. 7a). This result further confirmed that RxLR48 is able to suppress PTI signaling.
MAPK activation is considered as an early biochemical event of PTI signaling pathways (Zhang et al. 2010; Segonzac and Zipfel 2011; Feng et al. 2012). To test if RxLR48 also affects MAPK activation in host cells, protoplasts transiently expressing EV, RXLR48 and HopAI1 were treated with a 22-amino acid peptide conserved in the N-terminus of bacterial flagellin (flg22) at the indicated time points. The expression of RxLR48 was unable to prevent phosphorylation of MAPKs upon flg22 treatment, compared with that in the EV control (Fig. 7b). In contrast, Pseudomonas syringae effector HopAI1, the positive control, completely blocked MAPKs activation as reported (Zhang et al. 2007) (Fig. 7b). These results suggest that RxLR48 mainly suppresses ROS production but not MAPK cascades.
NPR1 plays an important role in SAR and plant immunity. However, how pathogen effectors target NPR1 to diminish SA-dependent signaling pathway remained a mystery until recently. An impressive recent research reported that the P. syringae type III effector AvrPtoB, a U-box type E3 ubiquitin ligase, mediates the degradation of NPR1 via host 26S proteasome, resulting in disrupting SA signaling, pattern-triggered immunity and systemic acquired resistance (Chen et al. 2017). In addition, a conserved rust protein from Puccinia striiformis interacts with wheat NPR1 in the nucleus, and competes with NPR1’s interaction partner TGA2.2 in barley (Wang et al. 2016). In this study, we adopted the Y2H system to screen the candidate effectors of P. capsici by using NPR1 as a bait. We identified an NPR1-interacting effector RxLR48 and confirmed the interaction by Y2H and co-IP assays. Considering subcellular localization and protein stability of NPR1 are important for its function, we successively demonstrated that RxLR48 affects these aspects to interfere with SA signaling pathway.
Previous studies reported that some pathogen effectors can suppress plant immunity, termed as effector triggered susceptibility (ETS), thereby enhancing pathogenicity by using diversified strategies in susceptible hosts (Gohre and Robatzek 2008). One of the familiar strategies is to alter subcellular localization of their host targets. For example, the P. infestans RxLR effector PITG_03192 interacts with two putative NAC transcription factors StNTP1 and StNTP2 at the endoplasmic reticulum (ER) membrane, and prevents culture filtrate-triggered re-localization of StNTP1 and StNTP2 from entering nucleus (McLellan et al. 2013). Furthermore, a potential effector SsSSVP1 from Sclerotinia sclerotiorum interacts with QCR8, and disturbs its subcellular localization by hijacking QCR8 to the cytoplasm before targeting to mitochondria (Lyu et al. 2016). In this study, we found that co-expression of NPR1 with RxLR48 resulted in enhanced localization of NPR1 in the nucleus, which would break the dynamic balance of the distribution ratio of NPR1. Considering NPR1 is present in the cytoplasm as an oligomer in non-active state, while monomerized and translocted into the nucleus to activate the expression of PR genes in the presence of pathogen or SA treatment (Mou et al. 2003). We hypothesized that RxLR48 interfered with distribution of NPR1, leading to aberrant SA-mediated defense response, which needs to be further elucidated in the future.
Accumulating evidence from prior studies supports that post-translational regulation of target proteins, including interfering with the stability of target proteins, is a commonly used strategy utilized by a large variety of pathogens. For example, a type III secretion system effector HopX1 from P. syringae pv. tabaci interacts with and promotes the degradation of JAZ (JASMONATE ZIM DOMAIN) proteins, a key family of jasmonate (JA)-repressors, to enhance the activation of JA-mediated signaling and susceptibility in Arabidopsis (Gimenez-Ibanez et al. 2014). Another effector Tin2 secreted by Ustilago maydis promotes virulence by refueling anthocyanin biosynthesis in maize. Tin2 stabilizes a maize protein kinase ZmTTK1, leading to rewiring metabolites into the anthocyanin pathway to lower their availability for other defense responses (Tanaka et al. 2014). In our study, ectopic expression of RxLR48 resulted in elevated protein accumulation of NPR1 by inhibiting 26S proteasome-mediated degradation. It was reported that proteasome-mediated turnover of NPR1 in the nuclear is required for full induction of target genes and establishment of systemic immunity (Spoel et al. 2009). Thus, our results suggested that RxLR48 blocks proteasome-mediated degradation of NPR1, which is in accord with an excess of nuclear NPR1.
To further elucidate the role of RxLR48 during infection, we also checked the transcription levels of the NPR1-related genes and FRK1. Although RxLR48 promotes total and nuclear accumulation of NPR1, relative expression of PR1 was not significantly affected. Instead, the transcription level of FRK1 was subverted by transient expression of RxLR48. Considering our findings that RxLR48 contributes to the suppression of PTI responses including H2O2 production and callose deposition, we assumed that RxLR48 is involved in obstructing PTI signaling pathway. A number of pathogen effectors suppress flg22-triggered immunity by altering PTI signaling pathway at different stages. For example, the P. infestans RxLR effector AVR3a associates with the Dynamin-Related Protein 2 (DRP2) to suppress the endocytosis of the activated FLS2 receptor (Chaparro-Garcia et al. 2015). Xanthomonas campestris pv campestris type III effector AvrAC specifically uridylylates BIK1 (BOTRYTIS-INDUCED KINASE1) and RIPK (RPM1-INDUCED PROTEIN KINASE) to reduce phosphorylation of these two receptor-like cytoplasmic kinases (Feng et al. 2012). In our study, flg22-induced MAPK activation remained unaffected when RxLR48 was transiently expressed in Arabidopsis protoplasts, suggesting that RxLR48 acts independent or downstream of the MAPK cascades to suppress PTI signaling. This is reminiscent of another Phytophthora effector PsCRN63 (Li et al. 2016). Therefore, additional experiments are required to clarify which stages RxLR48 interferes with PTI.
In summary, we identified a virulence effector RxLR48 from the hemibiotrophic oomycete pathogen P. capsici. RxLR48 not only interferes with plant immunity by associating with and altering NPR1’s protein localization and accumulation, but also suppresses PTI responses by inhibiting PTI-responsive genes. This study provides a new insight into the mechanisms of oomycete pathogen virulence.
For Y2H assay, RxLR48 (lacking the signal peptide-encoding region) was PCR-amplified from P. capsici strain LT263 genomic DNA and NPR1 coding region was amplified by using A. thaliana complementary DNA as templates. Then the corresponding PCR products were inserted into pGBKT7 or pGADT7, respectively. To generate constructs for protoplast transfection, coding sequences of indicated genes were amplified and inserted into the pUC19-35S-FLAG/HA-RBS vector (Li et al. 2005). For transient expression in N. benthamiana, RxLR48 was PCR-amplified and cloned into pBinGFP2 or pBinPLUS::mCherry vector under the control of the 35S promoter (Song et al. 2015). And NPR1 was amplified and cloned into pBinGFP2 as well. For transformation of P. capsici, RxLR48 was amplified and cloned into pHam34 which was maintained in our laboratory. All primers used for PCR amplification are listed in Additional file 1: Table S2.
Yeast two-hybrid assay
The yeast (Saccharomyces cerevisiae) two-hybrid assay was performed according to the Yeast Protocols Handbook (Clontech) using the Y2H Gold yeast reporter strain (Clontech). Yeast cells were co-transformed with the indicated plasmid combinations, and the transformed yeast cells were selected using synthetic dropout (SD/−Leu/−Trp) medium, then further transferred to SD/−Leu/−Trp/-His/−Ade selective medium for growth analysis.
Plant materials and growth
The Columbia ecotype of A. thaliana was used for protoplast isolation. The plants were grown at 23 °C with a 10-h-light/14-h-dark photoperiod for 4 to 5 weeks. For transient expression, N. benthamiana plants were grown and maintained in a greenhouse for 4–6 weeks at 25 °C or 20 °C under a 16 h/8 h day/night photoperiod.
Arabidopsis protoplast transfection and protein extraction
Protoplast isolation and transfection were implemented as described (Li et al. 2005), except that the transfected protoplasts were incubated in W5 medium (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES pH 5.7) instead of 0.4 M mannitol. Total protein was isolated with an extraction buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM EDTA, 0.3% Triton-X 100, 1 mM DTT, complete protease inhibitors (Roche).
Arabidopsis protoplasts were transfected with indicated plasmid combinations and incubated overnight, followed by protein extraction. The α-FLAG IP was carried out as previously described (Li et al. 2016). Total protein and immune precipitates were separated by SDS–PAGE (SDS–polyacrylamide gel electrophoresis), and detected by immunoblots with α-FLAG antibody (Sigma-Aldrich) together with α-HA antibody (Tiangen), respectively.
Transient expression in N. benthamiana
The A. tumefaciens strain GV3101 in our lab was used for this experiment (Liu et al. 2011). For infiltration, recombinant strains were cultured overnight at 28 °C in Luria-Bertani medium with the antibiotics kanamycin and rifampicin. The cells were harvested by centrifugation at 5600 x g and washed three times in 10 mM MgCl2, and then resuspended in Agro-infiltration buffer (10 mM MgCl2, 10 mM MES, pH 5.6, and 150 μM acetosyringone) to an appropriate optical density (OD) at 600 nm (0.2). For co-expression, suspensions carrying each construct were thoroughly mixed before infiltration.
P. capsici culture conditions and inoculation assay
The P. capsici strain LT263 used in the study were routinely cultured at 25 °C in the dark on 10% (v/v) V8 juice medium. For inoculation on N. benthamiana, mycelial plugs were inoculated on one-half of the leaves at 36 h after infiltration. Inoculated leaves were photographed under UV light and lesion diameters were measured at 36 hpi. This assay was repeated at least three times.
Zoospores were obtained by incubating mycelial plugs in 10% (v/v) V8 broth at 25 °C for 3 days, then washed three times with sterilized water at room temperature, numerous sporangia were developed after incubation overnight in sterilized water. To initiate zoospore release, the cultures were transferred into fresh cold water (4 °C) for 0.5 h followed by incubation at 25 °C for 1 h. The concentrations of zoospores were estimated with a hemacytometer. Inoculation assay was performed using droplets of zoospore suspension (10 μL of a 50,000 zoospores/mL solution) and incubated in a growth room at 25 °C in darkness. Inoculated leaves were photographed under UV light and lesion diameters were measured at the indicated time points.
Confocal laser scanning microscopy
N. benthamiana leaf tissues expressing the corresponding constructs were mounted in water under a coverslip at 48 h after infiltration. Images were captured and processed using the Zeiss LSM 710 confocal laser scanning microscope. The GFP fluorescence were excited at 488 nm, and the mCherry fluorescence were excited at 561 nm.
P. capsici transformation and characterization
For transformation of P. capsici, we used the polyethylene glycol (PEG)-mediated protoplast transformation procedure as described (Dou et al. 2008b). Transformants appeared within 4–10 days in pea broth medium containing 30 μg/mL G418 (Sigma) at 25 °C under the dark conditions were multiplied further on 10% V8 solid medium containing 30 μg/mL G418 for selection of putative transformants. To check silencing efficiency, the transcript level of RxLR48 gene of different transformants was measured by qRT-PCR.
RNA isolation and qRT-PCR
For detection of NPR1, NPR3 and PR1 gene expression, protoplasts were harvested directly after incubation; For FRK1 detection, protoplasts were treated with 1 μM flg22 for 8 h before harvest. Total RNA was extracted from P. capsici mycelia or Arabidopsis protoplasts by using RNA-simple Total RNA Kit (Tiangen) according to the manufacturer’s instructions. cDNA was synthesized by using the SuperScriptIII First-Strand Kit (Invitrogen). Real-Time PCR was performed on ABI Prism 7500 Fast Real-Time PCR system by using SYBR Premix Ex Taq Kit (TaKaRa) following manufacturer’s instructions. The gene-specific primers used for qRT-PCR are listed in Additional file 1: Table S2.
DAB staining and callose deposition assay
For determination of H2O2 accumulation, N. benthamiana leaves were stained with 1 mg/mL DAB solution for 8 h in the dark at 10 hpi, and then destained with ethanol before observation by light microscopy. For measurement of callose deposition, N. benthamiana leaves were stained with aniline blue, and visualized with a fluorescence microscope as described (Song et al. 2015). The number of callose deposition was counted using Image J software and significant differences were identified by Student’s t test.
MAPKs activity assay
Protoplasts were transfected with empty vector, RxLR48-FLAG and HopAI1-FLAG, and treated with 1 μM flg22 for 0, 5, 10 min before protein extraction. The protein concentration was determined using a Bio-Rad Bradford protein assay kit, and equal amounts of total protein were electrophoresed on 10% SDS-PAGE. An α-pERK antibody (no. 4370S, Cell Signaling) was used to determine the phosphorylation state of MPK3, MPK4 and MPK6.
Dynamin-Related Protein 2
Effector triggered immunity
Effector triggered susceptibility
- FRK1 :
FLG22-INDUCED RECEPTOR-LIKE KINASE 1
JASMONATE ZIM DOMAIN
Mitogen-activated protein kinases
Non-expressor of pathogenesis related-1
- PR :
Pattern recognition receptors
Quantitative real-time PCR
RPM1-INDUCED PROTEIN KINASE
Reactive oxygen species
Arg-any amino acid-Leu-Arg
Systemic acquired resistance
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We thank Yi-Hsuan Chiang (University of California, Davis) for editing the article.
This work was supported by grants from the National Natural Science Foundation of China (31672008, 31625023 and 31801715) and Special Fund for Agro-scientific Research in the Public Interest (201503112). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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All data generated or analyzed during this study are included in this published article [and its supplementary information files].
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The authors declare that they have no competing interests.
Table S1. Screening of NPR1-interacted effectors in Phytophthora capsici. Table S2. Primers used in this study. (XLSX 28 kb)
Figure S1. Phylogenetic relationships of RxLR48 and homologs in four Phytophthora species. The phylogenetic tree was constructed using MEGA 5 with the neighbor-joining method and 1000 bootstrap replicates. Ps, Pr, Pi, and Pc, correspond to P. sojae, P. ramorum, P. infestans, and P. capsici, respectively. (TIF 568 kb)