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. 2019 Jun 19;180(4):2227–2239. doi: 10.1104/pp.18.00625

Phytophthora infestans RXLR Effectors Target Parallel Steps in an Immune Signal Transduction Pathway1,[OPEN]

Yajuan Ren a,b, Miles Armstrong c,d, Yetong Qi a,b, Hazel McLellan c, Cheng Zhong a, Bowen Du a,b, Paul RJ Birch c,d,2, Zhendong Tian a,b,2,3
PMCID: PMC6670088  PMID: 31217198

Two P. infestans effectors, PexRD2 and Pi22926, target two parallel MAP3K proteins in the same signal transduction pathway to promote P. infestans colonization.

Abstract

The potato (Solanum tuberosum) blight pathogen Phytophthora infestans delivers Arg-X-Leu-Arg (RXLR) effector proteins into host cells to subvert plant immune responses and promote colonization. We show that transient expression and stable transgenic expression of the RXLR effector Pi22926 in Nicotiana benthamiana promotes leaf colonization by P. infestans. Pi22926 suppresses cell death triggered by coexpression of the Cladosporium fulvum avirulence protein Avr4 and the tomato (Solanum lycopersicum) resistance protein Cf4. Pi22926 interacts with a potato mitogen-activated protein kinase kinase kinase, StMAP3Kβ2, in the nucleoplasm. Virus-induced gene silencing (VIGS) of the ortholog NbMAP3Kβ2 in N. benthamiana enhances P. infestans colonization and attenuates Cf4/Avr4-induced cell death, indicating that this host protein is a positive regulator of immunity. Cell death induced by Cf4/Avr4 is dependent on NbMAP3Kε and NbMAP3Kβ2, indicating that these MAP3Ks function in the same signaling pathway. VIGS of NbMAP3Kβ2 does not compromise cell death triggered by overexpression of MAP3Kε. Similarly, VIGS of NbMAP3Kε does not attenuate cell death triggered by MAP3Kβ2, demonstrating that these MAP3K proteins function in parallel. In agreement, Pi22926 or another RXLR effector, PexRD2, only suppresses cell death triggered by expression of StMAP3Kβ2 or StMAP3Kε, respectively. Our data reveal that two P. infestans effectors, PexRD2 and Pi22926, promote P. infestans colonization by targeting MAP3K proteins that act in parallel in the same signal transduction pathway.

Plants have a two-layer surveillance system to respond to pathogens and mount defenses against attack (Jones and Dangl, 2006; Dodds and Rathjen, 2010). The first layer is initiated at the host cell surface by pattern recognition receptors (PRRs) that detect microbe-associated molecular patterns (MAMPs), such as bacterial flagellin, elongation factor EF-Tu, peptidoglycans, and lipopolysaccharide (Couto and Zipfel, 2016). This detection system results in pattern-triggered immunity, accompanied by activation of mitogen-activated protein kinase (MAPK) signaling cascades, production of reactive oxygen species, callose deposition in the cell wall, and the induction of pathogenesis-related (PR) protein expression (Chisholm et al., 2006; Jones and Dangl, 2006). In turn, successful plant pathogens deliver a range of effector proteins that act in the apoplast or within plant cells to attenuate pattern-triggered immunity. Our understanding of how effectors manipulate host targets to interfere with defense pathways and processes has been led by the studies of bacterial type III secreted effectors (Block and Alfano, 2011; Deslandes and Rivas, 2012; Dou and Zhou, 2012). More recently, the targets of effectors from filamentous plant pathogens such as fungi and oomycetes have been revealed (Anderson et al., 2015; Toruño et al., 2016; Whisson et al., 2016). Plants possess resistance proteins (R proteins) that perceive effectors or effector activities, leading to effector-triggered immunity. This causes rapid and localized programmed cell death, reactive oxygen species production, and prolonged MAPK activation (Jones and Dangl, 2006).

The MAPK cascade is a core module for signal transduction in response to extracellular stimuli in plants. MAPK pathways play important roles in the activation of plant immune responses mediated by both PRR and R proteins (Pedley and Martin, 2005; Pitzschke et al., 2009). MAPK pathways generally include three protein kinases: MAP kinase kinase kinase (MAP3K), MAP2K, and MAPK. The MAPK is phosphorylated by a MAP2K, which itself is phosphorylated by a MAP3K. In the Arabidopsis (Arabidopsis thaliana) genome, there are at least 20 putative MAPKs, 10 MAP2Ks, and >80 MAP3Ks (Pitzschke et al., 2009). MAP3Ks can be divided further into three groups, the MAPK/extracellular signal-regulated kinase (ERK) kinase kinase (MEKK)-like subgroup, whose members function as MAP3Ks in plants and mainly participate in linear cascades, and the ZR1-interacting kinase (ZIK)-like and rapidly accelerated fibrosacoma (Raf)-like groups, functional characterization of which largely comes from organisms other than plants (MAPK Group, 2002; Colcombet and Hirt, 2008). Only a limited number of MAP3Ks are associated with regulating plant immunity. For example, Arabidopsis MEKK1 is activated downstream of the PRR FLS2, which detects the bacterial MAMP flg22. Studies have shown that MEKK1 regulates MEKK1-MAPK kinase1 (MKK1)/MKK2-mitogen-activated protein kinase 4 (MPK4), which negatively regulates plant defense responses (Suarez-Rodriguez et al., 2007; Gao et al., 2008). More recently, the MAP3Ks MAPKKK3 and MAPKKK5, which are activated by receptor-like cytoplasmic kinase VII family members, were found to be responsible for activating the MAPKs MPK3 and MPK6, respectively (Bi et al., 2018). Conversely, the MAP3K YODA, which promotes stomatal development, directly inhibits MAPKKK3 and MAPKKK5 activation of MPK3 and MPK6, respectively, demonstrating the antagonism that exists between plant growth and immunity (Sun et al., 2018).

Nicotiana benthamiana MAP3K NPK1 is essential for regulating the resistance responses mediated by the R proteins N, Bs2, and Rx, and may play a role in one or more MAPK cascades (Jin et al., 2002). Tomato (Solanum lycopersicum) MAP3Kα is involved in two distinct MAPK cascades, either MEK2-SIPK (salicylic acid-induced protein kinase) or MEK1-Nicotiana Fus-3-like kinase 6 (NTF6), to regulate plant immunity (del Pozo et al., 2004). Tomato MAP3Kε activates the MEK2-SIPK/WIPK (wound-induced protein kinase) cascade to positively regulate defense (Melech-Bonfil and Sessa, 2010). Finally, the Arabidopsis Raf-like MAP3K enhanced disease resistance1 (EDR1) was found to negatively regulate plant immunity (Frye et al., 2001).

Oomycete pathogens, ranging from obligate biotrophs to necrotrophs, deploy a variety of apoplastic and intracellular effectors (Kamoun et al., 2015). The best studied intracellular effectors are the Arg-X-Leu-Arg (RXLR) class, which contain a signal peptide followed by a conserved RXLR motif. It has been reported that the RXLR peptide motif acts as a host-targeting signal for translocation into host plant cells to suppress plant immunity (Whisson et al., 2007; Dou et al., 2008). Recently, delivery of RXLR effectors from the oomycete potato (Solanum tuberosum) blight pathogen Phytophthora infestans into plant cells has been visualized, revealing that they are secreted via a noncanonical pathway (Wang et al., 2017, 2018).

Since the bacterial type 3 effector HopAI1 was shown to suppress activation of MPK3 and MPK6 in Arabidopsis, a range of phytopathogen effectors have been implicated in targeting MAPK pathways (Bi and Zhou, 2017). RXLR effectors from P. infestans have been shown to suppress MAPK signaling cascades, or to interact with MAP3K components to interfere with immunity (Whisson et al., 2016). Three RXLRs, Pi13628/SFI5/PexRD27, Pi13959/SFI6, and Pi18215/SFI7/avirulence protein 3b (Avr3b), are able to suppress flg22-triggered MAMP signaling at, or upstream of, the MAPK cascade in tomato (Zheng et al., 2014). The effector Pi11383/PexRD2 specifically targets the kinase domain (KD) of MAP3Kε, directly inhibiting its activity to perturb plant defense responses (King et al., 2014). Recently, Murphy et al. (2018) reported that the effector Pi17316 interacts with the host MAP3K, StVIK, which acts as a susceptibility factor to enhance P. infestans colonization.

In this study we show that the P. infestans effector PITG_22926 (Pi22926) targets the potato MAP3K, StMAP3Kβ2, a positive regulator of immunity, to facilitate disease. Transient or stable expression of the RXLR effector Pi22926 in the model host N. benthamiana promotes the growth of P. infestans and specifically suppresses cell death induced by coexpression of the tomato R protein Cf4 with the Cladosporium fulvum avirulence protein Avr4. Pi22926 interacts with the KD of StMAP3Kβ2 in a yeast two-hybrid (Y2H) library screen and in planta. Virus-induced gene silencing (VIGS) of MAP3Kβ2 in N. benthamiana enhanced P. infestans colonization and attenuated Cf4/Avr4-induced cell death, indicating that it is a positive regulator of plant immunity. Overexpression of StMAP3Kβ2 or its KD-induced cell death in N. benthamiana, which is suppressed by Pi22926. Epistasis experiments revealed that StMAP3Kβ2 acts in parallel to StMAP3Kε and upstream of MEK2. Our results reveal a P. infestans effector protein that interacts with host StMAP3Kβ2 to target the same signaling pathway as PexRD2 for immune suppression.

RESULTS AND DISCUSSION

The RXLR Effector Pi22926 Promotes P. infestans Colonization

The RXLR effector gene PITG_22926 (Pi22926) was shown previously to be up-regulated at 2 and 3 d post P. infestans infection of potato leaves in both genotype T30-4 and genotype 13_A2 (Haas et al., 2009; Cooke et al., 2012), and more recently in diverse potato genotypes in China and during tuber infection (Ah-Fong et al., 2017; Yin et al., 2017). Here, we confirmed that Pi22926 is also up-regulated in P. infestans isolate HB09-14-2 at 24, 48, and 72 h postinoculation of a Chinese potato variety ‘E-potato-3’ (Supplemental Fig. S1). The time course suggests that Pi22926 contributes to the biotrophic phase of infection, similar to other PiRXLR effectors (Whisson et al., 2016). Recently, Pi22926 was shown to be secreted from P. infestans haustoria and delivered into host cells to accumulate in the nucleus (Wang et al., 2018), where it enhances P. infestans colonization of N. benthamiana (Wang et al., 2019). We confirmed that the disease lesion diameters on the half leaves transiently expressing GFP-Pi22926 (the signal peptide was deleted) were significantly larger compared to the GFP control 6 d postinoculation (dpi; Supplemental Fig. S2). The GFP-Pi22926 fusion protein was intact when transiently expressed in N. benthamiana leaves (Supplemental Fig. S3A).

To explore this phenomenon further, transgenic N. benthamiana plants were created for stable expression of GFP-Pi22926. GFP-Pi22926 expression was detected in four transgenic lines (Supplemental Fig. S4). All four lines showed growth and morphology similar to the wild type, and two lines were thus taken forward for detailed study. Leaves from transgenic plants were infected with P. infestans and were found to sustain significantly larger P. infestans lesions compared to wild-type control plants (Fig. 1). The GFP-Pi22926 fusion protein was intact in transgenic N. benthamiana leaves (Supplemental Fig. S3C). These results reveal that GFP-Pi22926 activity within host cells is beneficial to P. infestans colonization.

Figure 1.

Figure 1.

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The RXLR effector Pi22926 enhances P. infestans colonization of N. benthamiana (N.b) leaves. A, Representative images taken under UV light at 5 dpi show that transgenic lines overexpressing GFP-Pi22926 enhance pathogen colonization compared to the untransformed N. benthamiana control. Bar = 1 cm. B, Graph shows a significant increase in P. infestans lesions in transgenic lines overexpressing GFP-Pi22926 compared to the wild-type (WT) N. benthamiana control (P < 0.001, ANOVA, three repetitions, n = 120). Lowercase letters above bars denote statistically significant groups. Error bars represent ± se.

Pi22926 Specifically Suppresses Avr4/Cf4- and AvrPto/Pto-Triggered Cell Death

Coexpression of the C. fulvum avirulence protein Avr4 and the tomato R protein Cf4, or the Pseudomonas syringae avirulence protein AvrPto and the corresponding tomato R protein component Pto, triggers cell death in N. benthamiana via activation of a common signaling pathway. The PiRXLR effector PexRD2 was previously shown to specifically suppress Avr4/Cf4- and AvrPto/Pto-triggered cell death (King et al., 2014), and we confirmed these results as a positive control in this study (Fig. 2). In addition, our results reveal that Avr4/Cf4- and AvrPto/Pto-triggered cell death were significantly attenuated by coexpression with Pi22926 compared with the empty vector control (Fig. 2). We also tested whether Pi22926 is able to suppress cell death triggered by the P. infestans avirulence protein (Avr3aKI) and R protein R3a (Avr3a/R3a) pairs (Armstrong et al., 2005), or potato virus X coat protein and R protein (CP/RX) pairs (Moffett et al., 2002), and by the P. infestans MAMP INF1 (Kamoun et al., 1998). No change to the mean percentage of cell death mediated by these elicitors was observed in the presence of Pi22926 (Fig. 2B), indicating that they are independent of the signal transduction cascade(s) manipulated by Pi22926 or PexRD2. These results indicate that Pi22926 and PexRD2 may suppress the same specific signaling pathway to promote disease.

Figure 2.

Figure 2.

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Pi22926 specifically suppresses Avr4/Cf4- and AvrPto/Pto-induced cell death. A, Representative leaf image taken under UV light at 5 d showing Avr4/Cf4 and AvrPto/Pto cell death with empty vector (EV), Pi22926 and PexRD2 positive control. B, Graph showing Pi22926 and PexRD2 expression lead to a significant decrease (P < 0.001, three repetitions, n = 94) in cell death percentage triggered by Avr4/Cf4 and AvrPto/Pto. Lowercase letters above bars denote statistically significant groups by one-way ANOVA, with pairwise comparisons performed with the Holm-Sidak method. Error bars represent ± se. HR, Hypersensitive response.

Pi22926 Specifically Targets Potato MAP3Kβ2

To identify possible host targets of Pi22926, a yeast-2-hybrid (Y2H) library composed of complementary DNA (cDNA) from potato plants infected with P. infestans (Bos et al., 2010) was screened with Pi22926 as a bait. The screen involved ∼1.2 × 106 yeast cotransformants. Two yeast cotransformants growing on selection plates contained identical, partial sequences corresponding to a potato MAP3K (XP_006349414.1). Alignment of full-length amino acid sequences of MAP3Ks from potato, N. benthamiana, and tomato showed that the potato interacting protein shares high identities with both a tomato protein (XP_004230523.1; 94.59% identity) and a N. benthamiana protein (Nbv6.1trP19888; 82.64% identity). The tomato and potato proteins were reciprocal best Blast hits, and thus candidate orthologs, of NbMAP3Kβ2 (Supplemental Fig. S5). The potato interacting protein was hence named StMAP3Kβ2. StMAP3Kβ2 contains a KD at the C terminus (residues 402–653; Supplemental Fig. S5).

To investigate the specificity of the interaction between Pi22926 and StMAP3Kβ2, a pairwise Y2H assay was performed in which the full-length StMAP3Kβ2, its active KD, and an inactive form in which the active site Lys in the ATP binding site (Lys 430) was substituted with an Arg, were used as prey clones against the bait Pi22926. In addition, two other RXLR effectors, PexRD2 (Pi11383) and Pi04089, were used as controls. PexRD2 targets another MAP3K in the cytoplasm, StMAP3Kε (King et al., 2014), and Pi04089 shows a similar nuclear localization to Pi22926 but interacts with the RNA binding protein StKRBP1 (Wang et al., 2015). While all yeast transformants grew on control +His plates, the interactions of Pi22926 with full-length StMAP3Kβ2 or with its active KD were indicated by induction of β-galactosidase activity and growth on media lacking His (−His). The Pi04089 or PexRD2 combinations did not activate either reporter (Fig. 3A; Supplemental Fig. S6). In addition, whereas PexRD2 interacted with StMAP3Kε, no such interaction was observed between Pi22926 and StMAP3Kε (Supplemental Fig. S6). Importantly, the mutant StMAP3Kβ2(KD)Lys-430Arg also failed to interact with Pi22926 (Fig. 3A). This suggests that the intact active KD of StMAP3Kβ2 is necessary and sufficient for the specific interaction with Pi22926.

Figure 3.

Figure 3.

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Pi22926 interacts with the KD of StMAP3Kβ2 in Y2H and immunoprecipitation assays. A, Yeast coexpressing Pi22926 with StMAP3Kβ2 and its KD grow on –His medium and had β-galactosidase (β-gal) activity, wheareas those coexpressed with the inactive mutant KD StMAP3Kβ2(KD)Lys-430Arg or the control Pi04089 did not. B, Coimmunoprecipitation from leaf extracts using GFP-trap (GFP immunoprecipitation) confirmed that cMyc-tagged StMAP3Kβ2(KD) specifically interacted with GFP-Pi22926 and not with Pi04089. Expression of constructs is indicated by +. Protein size markers are indicated in KDa, and protein loading is indicated by Coomassie brilliant blue (CBB) staining.

To confirm that specific interactions also occur in vivo, a coimmunoprecipitation assay was performed by transiently coexpressing cMyc-StMAP3Kβ2(KD) or cMyc-StMAP3Kβ2(KD)Lys-430Arg with GFP-Pi22926, or with the GFP-Pi04089 control, following immunoprecipitation with GFP-TRAP_M beads. Intact GFP- or cMyc-labeled proteins were all stably expressed when corresponding constructs were transiently expressed in N. benthamiana leaves as indicated in the input samples. The cMyc-StMAP3Kβ2(KD) was only pulled down in the presence of Pi22926, but not with the Pi04089 control (Fig. 3B). These results indicate that Pi22926 specifically interacts with StMAP3Kβ2 by its active KD both in yeast and in planta.

Pi22926 Interacts with StMAP3Kβ2 in the Nucleoplasm

To investigate the subcellular localization of Pi22926 and StMAP3Kβ2, GFP was fused to their N or C terminus to form GFP-Pi22926 and StMAP3Kβ2-GFP and viewed following Agrobacterium-mediated expression in N. benthamiana using confocal microscopy. GFP-Pi22926 localized predominantly in the nucleus and nucleolus (Fig. 4A). StMAP3Kβ2-GFP localized in the cytoplasm and showed weak fluorescence in the nucleoplasm, but was not observed in the nucleolus (Fig. 4B). GFP-Pi22926 and StMAP3Kβ2-GFP were stable as fusion proteins in planta (Supplemental Fig. S3, A and B). When red fluorescent protein (RFP)-Pi22926 and StMAP3Kβ2-GFP were coexpressed by Agrobacterium-mediated expression in N. benthamiana, RFP-Pi22926 and StMAP3Kβ2-GFP were colocalized in the nucleus (Fig. 4C), but StMAP3Kβ2-GFP still retained cytoplasmic fluorescence background. We thus investigated the interaction using bimolecular fluorescence complementation. When yellow fluorescent protein (YFP) N-terminus (YN)-Pi22926 and C-terminus (YC)-StMAP3Kβ2 were coexpressed by Agrobacterium-mediated expression in N. benthamiana, reconstituted YFP fluorescence was observed only in the nucleoplasm (Fig. 4D). By contrast, when YC-StMAP3Kβ2 was coexpressed with YN-Pi04089 (Wang et al., 2015), only weak background fluorescence was observed (Fig. 4E). YN-Pi22926, YN-Pi04089, and YC-StMAP3Kβ2 were stable as fusion proteins in planta (Supplemental Fig. S3, D and E). This demonstrates that the interaction between these proteins occurs in the nucleoplasm, despite both showing some level of cytoplasmic localization. Further work is needed to look at substrates of StMAP3Kβ2 and where it phosphorylates them. Interestingly, StMAP3Kε and PexRD2 interacted in the cytoplasm, suggesting that there are alternative substrates for phosphorylation at that location that contribute to cell death (King et al., 2014).

Figure 4.

Figure 4.

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Pi22926 interacts with StMAP3Kβ2 in nucleoplasm. A, Confocal images show that GFP-Pi22926 is localized in the nucleoplasm and nucleolus. B, StMAP3Kβ2-GFP is localized in the cytoplasm and nucleoplasm. For images of StMAP3Kβ2-GFP, the left one is a Z-stack, whereas the right one with higher magnification is a single optical section from the stack. C, Images show transient coexpression of StMAP3Kβ2-GFP with RFP-Pi22926. D, Images show transient coexpression of YC-StMAP3Kβ2 with YN-Pi22926. The inset image is a nucleus at higher magnification. E,The image shows transient coexpression of YC-StMAP3Kβ2 with YN-Pi04089. Scale bars represent 10 µm. OD600 of Agrobacteria suspension is 0.1 for GFP and RFP constructs and 0.03 for split-YFP.

Silencing of NbMAP3Kβ2 Promotes P. infestans Colonization

To investigate the potential role of StMAP3Kβ2 in plant defense responses to P. infestans, VIGS was employed to knock down the expression of NbMAP3Kβ2 (ortholog of StMAP3Kβ2) in the model host plant N. benthamiana. We confirmed that GFP-Pi22926 is able to interact with the KDs of both StMAP3Kβ2 and NbMAP3Kβ2 in yeast and in planta (Supplemental Fig. S7, A and B). Two VIGS vectors containing independent portions of the NbMAP3Kβ2 gene, pBinary Tobacco Rattle Virus (TRV):NbMAP3Kβ2-5′ and TRV:NbMAP3Kβ2-3′, were generated to specifically knock down this gene in N. benthamiana (Supplemental Fig. S8A). Transcript levels of the target gene in plants expressing each of the TRV:NbMAP3Kβ2 constructs was reduced by 70% to 80%, but NbMAP3Kε transcript levels were unaltered (Supplemental Fig. S8B). VIGS plants showed a developmentally normal phenotype when compared with the TRV:GFP control. TRV:NbMAP3Kβ2 and TRV:GFP plants were infected with P. infestans isolate 88069. At 7 dpi, measurements of both P. infestans lesion diameter and sporangia production on TRV:GFP and TRV:NbMAP3Kβ2-5′ and TRV:NbMAP3Kβ2-3′-expressing N. benthamiana plants showed that silencing of NbMAP3Kβ2 significantly enhanced P. infestans colonization compared with the TRV:GFP control (Fig. 5). This indicates that NbMAP3Kβ2 is potentially a positive regulator of plant immunity.

Figure 5.

Figure 5.

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Silencing of NbMAP3Kβ2 enhances P. infestans leaf colonization. A, Images taken at 7 dpi with sporangia indicate more pathogen colonization on TRV:NbMAP3Kβ2 plants compared to the TRV:GFP control. Scale bar = 1 cm. B, Graph showing a significant increase (P < 0.001, ANOVA, three repetitions, n = 120) in P. infestans lesion diameter in plants expressing TRV:NbMAP3Kβ2-3′ and TRV:NbMAP3Kβ2-5′, compared with a TRV-GFP control. C, Graph showing an increase in the average numbers of sporangia mL−1 collected from infected leaves expressing TRV:NbMAP3Kβ2-3′ and TRV:NbMAP3Kβ2-5′, compared with a TRV:GFP control (P < 0.001, ANOVA, three repetitions, n = 120). Lowercase letters above bars denote statistically significant groups. Error bars represent ± se.

Cell Death Induced by Avr4/Cf4 and AvrPto/Pto Is Dependent on MAP3Kβ2

As Pi22926 suppresses Cf4/Avr4 and Pto/AvrPto cell death, we hypothesized that MAP3Kβ2 may act in the signal transduction pathways leading to these cell death responses. To test that, TRV:NbMAP3Kβ2-5′ and TRV:NbMAP3Kβ2-3′ were used to silence NbMAP3Kβ2 in N. benthamiana plants, and the leaves of VIGS plants were infiltrated with the Agrobacterium tumefaciens harboring effector/R protein pairs Avr4/Cf4 and AvrPto/Pto. As expected, similar to the silencing of MAP3Kε (specific silencing efficiency shown in Supplemental Fig. S9), we observed that silencing of NbMAP3Kβ2 significantly reduced the percentage of sites developing cell death upon coexpression of Avr4/Cf4 or AvrPto/Pto, compared with the TRV:GFP empty vector control (Fig. 6). However, silencing of NbMAP3Kβ2 and NbMAP3Kε did not compromise INF1, Avr3a/R3a or CP/RX triggered cell death (Supplemental Fig. S10), indicating that silencing of NbMAP3Kβ2 specifically compromises Avr4/Cf4- or AvrPto/Pto-triggered cell death. Taken together, our results suggest that NbMAP3Kβ2, like MAP3Kε (King et al., 2014), plays an essential role in the signaling pathway activated by Avr4/Cf4 or AvrPto/Pto.

Figure 6.

Figure 6.

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Cell death induced by Avr4/Cf4 and AvrPto/Pto is dependent on MAP3Kε and NbMAP3Kβ2. A, Graph showing a significant suppression of cell death (P < 0.001, ANOVA, three repetitions, n = 72) induced by Avr4/Cf4 in NbMAP3Kβ2-silenced plants, and in positive control NbMAP3Kε-silenced plants, compared to the TRV2:GFP control. B, Graph showing a significant decrease in cell death (P < 0.001, ANOVA, three repetitions, n = 72) triggered by AvrPto/Pto in NbMAP3Kβ2-silenced plants and positive control NbMAP3Kε-silenced plants, compared to the TRV2:GFP control. Error bars represent ± se. Cell death numbers were counted at 6 d. Stars indicate significant difference from the TRV:GFP control. HR, Hypersensitive response.

Pi22926 Suppresses Cell Death Induced by Expression of StMAP3Kβ2 and Its KD

Expression of the full length and KD of some MAP3Ks in N. benthamiana induces cell death (Hashimoto et al., 2012). To determine whether overexpression of StMAP3Kβ2 is able to induce cell death, we used Agrobacterium to transiently express the full-length StMAP3Kβ2, its active KD, or the inactive form (KD)Lys-430Arg in N. benthamiana leaves. We observed that overexpression of StMAP3Kβ2 or its KD alone resulted in pathogen- and elicitor-independent cell death compared to the GFP control. In contrast, cell death was not observed in leaves expressing inactive (KD)Lys-430Arg, suggesting that an intact kinase catalytic domain is essential for StMAP3Kβ2 to trigger cell death (Fig. 7, A and C). As Pi22926 was shown to interact with StMAP3Kβ2 (Fig. 3), this prompted us to test whether Pi22926 has any effect on cell death induced by StMAP3Kβ2. We coexpressed GFP-Pi22926 with StMAP3Kβ2 or its KD in N. benthamiana using Agrobacterium-mediated expression. Seven days postagroinfiltration, we observed that StMAP3Kβ2- and KD-induced cell death was significantly suppressed by coexpression with GFP-Pi22926 compared to the GFP control. This indicates that Pi22926 is a suppressor of cell death triggered by StMAP3Kβ2 (Fig. 7, B and D).

Figure 7.

Figure 7.

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Overexpression of StMAP3Kβ2 or its KD induces cell death that is suppressed by Pi22926. A, Images taken under UV light at 7 dpi showing that transient overexpression of StMAP3Kβ2 and its KD induce cell death in N. benthamiana, whereas no cell death was triggered by the expression of the inactive mutant StMAP3Kβ2 (KD)Lys-430Arg or the empty vector control. B, The cell death triggered by StMAP3Kβ2 and its active KD is suppressed by coexpression with Pi22926, but not the EV control. Images were taken under UV light at 7 dpi. C, Graph showing a significant increase in percentage of cell death compared to the EV control and the inactive mutant KD (P < 0.001, ANOVA, three repetitions, n = 72). D, Graph showing that transient overexpression of Pi22926 can significantly suppress the cell death (P < 0.001, ANOVA, three repetitions, n = 72) induced by StMAP3Kβ2 and its active KD compared to the EV control. Lowercase letters above bars denote statistically significant groups. Error bars represent ± se. HR, Hypersensitive response.

MEK2 and SIPK Act Downstream of StMAP3Kε and StMAP3Kβ2

To test whether StMAP3Kε and StMAP3Kβ2 share the same downstream signaling cascade, VIGS constructs that silence MEK1 and MEK2 (encoding MAP2Ks), or constructs for silencing the wound-induced protein kinase gene WIPK, salicylic acid-induced protein kinase gene SIPK or NTF6 (MAPKs) were generated and used to silence corresponding genes in N. benthamiana plants. The efficiency of silencing was assessed by reverse transcription quantitative PCR (RT-qPCR) analysis that measured the expression of each target gene in silenced plants relative to control TRV:GFP plants (Supplemental Fig. S11). Typical cell death was observed on the leaves of TRV:GFP control plants when expressing intact KDs of StMAP3Kε and StMAP3Kβ2, but mutated KDs (as a negative control) did not induce cell death (Fig. 8A). Intact KDs of StMAP3Kε and StMAP3Kβ2 were also able to induce cell death in both TRV:MEK1 and TRV:NTF6 VIGS plants (Fig. 8A). However, silencing of MEK2, WIPK, and SIPK significantly inhibited cell death triggered by the expression of intact KDs of StMAP3Kε (Fig. 8, A and B). This result is in agreement with the tomato SlMAP3Kε, which mediated a cell death signaling cascade involving the MAP2K MEK2 and the two MAPKs WIPK and SIPK rather than MEK1 or NTF6 (Melech-Bonfil and Sessa, 2010). Interestingly, although the cell death triggered by StMAP3Kβ2 needed MEK2 and SIPK, similar to StMAP3Kε, silencing WIPK did not abolish StMAP3Kβ2-triggered cell death (Fig. 8, A and C). This indicates that StMAP3Kε and StMAP3Kβ2 share the same signal transduction pathway but there is a difference downstream of MEK2.

Figure 8.

Figure 8.

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MEK2 and SIPK act downstream of StMAP3Kβ2. A, N. benthamiana plants were infected with TRV:GFP only or were silenced for the indicated MAP2K (MEK1 or MEK2) and MAPK (SIPK, WIPK, or NTF6) genes. StMAP3Kβ2 KD or the inactive KD mutant was expressed in the leaves to measure cell death. Photos were taken under UV light at 7 d. B and C, Graph showing a significant suppression of cell death (HR) triggered by StMAP3Kε(KD) or StMAP3Kβ2(KD) in TRV2:MEK2 and TRV2:SIPK plants compared to the TRV2:GFP control (P < 0.001, ANOVA, three repetitions, n ≥ 155). Error bars represent ± se.

Pi22926 Suppresses StMAP3Kβ2-Triggered Cell Death But Does Not Suppress StMAP3Kε-Triggered Cell Death

A previous study reported that the P. infestans RXLR effector PexRD2 suppresses cell death triggered by activity of the KD of MAP3Kε (King et al., 2014). We show that Pi22926 suppresses StMAP3Kβ2(KD) triggered cell death, whereas the effectors PexRD2, Pi13959, Pi13628, and Pi18215 failed to do so (Fig. 9, A and B). To test whether Pi22926 suppresses StMAP3Kε-triggered cell death, Pi22926 and StMAP3Kε(KD) were transiently coexpressed in N. benthamiana. We found that Pi22926 cannot suppress StMAP3Kε(KD)-triggered cell death, whereas PexRD2 does (Fig. 9, C and D). These results indicate that StMAP3Kβ2 and StMAP3Kε likely act at the same level in the cell death signaling pathway.

Figure 9.

Figure 9.

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Pi22926 suppresses cell death triggered by StMAP3Kβ2, whereas PexRD2 suppresses StMAP3Kε-induced cell death. A and C, Images showing StMAP3Kβ2(KD) and StMAP3Kε(KD) cell death at 7 dpi, following coexpression with the indicated effectors. B and D, Graphs showing the percentage of inoculation sites developing cell death at 7 d after coexpression of StMAP3Kβ2(KD) or StMAP3Kε(KD) with indicated effectors. A significant decrease in cell death percentage was observed when Pi22926 was coexpressed with StMAP3Kβ2(KD) or when PexRD2 was coexpressed with StMAP3Kε(KD), compared to coexpression with other effectors and the EV control (P < 0.001, ANOVA, 4 reps, n = 73). Lowercase letters above bars denote statistically significant groups. Error bars represent ± se. E, Graph showing no significant decrease in the mean percentage of cell death induced by StMAP3Kε in TRV2:NbMAP3Kβ2-3′ and TRV2:NbMAP3Kβ2-5′ plants compared to the TRV2-GFP control (7 d; P < 0.001, ANOVA, four repetitions, n = 92). F, Graph showing that VIGS of MAP3Kε by TRV2:NbMAP3Kε-3′ and TRV2-NbMAP3Kε-5′ had no significant effect on StMAP3Kβ2-induced cell death compared to the TRV:GFP control (7 d). (P < 0.001, ANOVA, four repetitions, n = 132). Error bars represent ± se. HR, Hypersensitive response.

StMAP3Kβ2 Acts in Parallel with StMAP3Kε in Cf4/AVR4 Induction of Cell Death

Epistasis analysis of the functional relationships among NbMAP3Kβ, NbMAP3Kγ, and NbMAP3Kα suggested that these three MAP3Ks form a linear signaling pathway that proceeds from NbMAP3Kβ to NbMAP3Kγ to NbMAP3Kα, leading to cell death (Hashimoto et al., 2012). We have shown that NbMAP3Kβ2 and MAP3Kε are involved in the same signaling pathway in that they positively regulate Avr4/Cf4 and AvrPto/Pto signal transduction (Fig. 6). The observation that Pi22926 or PexRD2 can only suppress cell death during transient coexpression with NbMAP3Kβ2 or MAP3Kε, respectively, suggests that MAP3Kε and NbMAP3Kβ2 may function at the same level in the signal transduction pathway.

To confirm this, we silenced each gene using VIGS in N. benthamiana. Silencing of NbMAP3Kβ2 did not significantly (P < 0.001, ANOVA) suppress MAP3Kε-triggered cell death (Fig. 9E) compared to the TRV:GFP control. VIGS of NbMAP3Kε did not suppress the cell death induced by transient expression of NbMAP3Kβ2 (Fig. 9F). Taken together, these results confirm that MAP3Kε and MAP3Kβ2 act in parallel in the same signaling pathway in Cf4/AVR4 cell death induction.

Perception of the P. infestans MAMP INF1 triggers a MAPK pathway that is independent of MAP3Kε and MAP3Kβ2, and is thus not suppressed by the effectors PexRD2 (King et al., 2014) or Pi22926. In contrast, as yet unidentified receptor(s) activated by unknown P. infestans elicitor(s) trigger a MAPK cascade that includes StMAP3Kε and StMAP3Kβ2, resulting in activation of MAP2K MEK2 and finally SIPK/WIPK (Fig. 10), ultimately leading to cell death. The importance of the StMAP3Kε/StMAP3Kβ2-MEK2-SIPK/WIPK pathway in immunity to P. infestans is highlighted by the fact that these two RXLR effectors from distinct MCL cluster families (Haas et al., 2009), PexRD2 (RXLRfam6) and Pi22926 (RXLRfam52), act to suppress parallel regulatory steps (Fig. 10).

Figure 10.

Figure 10.

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Model of how PexRD2 and Pi22926 suppress two parallel MAPK signaling pathways triggered by Avr4/Cf4 or AvrPto/Pto. The schematic diagram illustrates P. infestans delivering PexRD2 and Pi22926 into the host cell during infection. The cell death following recognition of the C. fulvum effector Avr4 by Cf4 and the P. syringae effector AvrPto mediated by Pto/Prf are dependent on MAP3Kε or MAP3Kβ2 is suppressed by the presence of PexRD2 or Pi22926, respectively. PexRD2 and Pi22926 specifically interact with MAP3Kε and MAP3Kβ2, respectively. In planta, MAP3Kε and MAP3Kβ2 confer enhanced resistance against P. infestans likely due to recognition of an unidentified pathogen-associated molecular pattern by a PRR or recognition of an effector/avirulence protein (AVR) by an R protein, as proposed by King et al. (2014).

Future work will reveal whether other P. infestans RXLRs target additional members of this MAPK cascade to redundantly suppress this immune pathway, or indeed may target additional MAPK signal transduction pathways associated with immune responses. In the large-scale effector Y2H screens of Mukhtar et al. (2011) and Weßling et al. (2014) MAPK signaling components did not emerge as “hubs” that are targeted by effectors from different pathogens. Nevertheless, type III effectors from bacterial plant pathogens, such as HopAI1 from P. syringae, which targets MPK3 and MPK6, can also directly inactivate MAPK cascade components that positively regulate immunity. Moreover, the P. syringae effector AvrB targets MPK4 to promote its activity as a negative regulator of immunity (Cui et al., 2010), and the P. infestans effector Pi17316 targets the susceptibility factor VASCULAR HIGHWAY1-interacting kinase (VIK), also to exploit its role as a negative immune regulator (Murphy et al., 2018). Thus bacterial and oomycete effectors directly target both positive and negative regulators of immunity within MAPK cascades.

In conclusion, this study emphasizes the power of effectors as probes to dissect and understand the regulation of plant immune signaling pathways. However, the PRR that perceives a P. infestans molecule to initiate the StMAP3Kε/StMAP3Kβ2-MEK2-SIPK/WIPK pathway is unknown, highlighting the need to more deeply invest in identifying cell surface receptors and the pathogen ligands that they detect to activate defense.

MATERIALS AND METHODS

Plant Materials

Nicotiana benthamiana plants were grown in individual plots in the greenhouse with 16 h days at 22°C and 8 h nights at 18°C. Approximately 4- to 5-week-old N. benthamiana plants were used for experiments. A Chinese potato (Solanum tuberosum) variety ‘E-potato-3′’ was used for Pi22926 expression tests. In vitro cultured plantlets were grown in the greenhouse as above. Leaves from 8-week-old plants were used for Phytophthora infestans inoculation.

Plasmid Construction

The RXLR effectors Pi22926 and Pi04089 were cloned without signal peptides from genomic DNA of P. infestans isolate T30-4 in a two-step PCR to add flanking attB recombination sites to the coding sequences. The potato StMAP3Kβ2 coding sequence was amplified from the original Y2H prey library using the same strategy. The Y2H library was the same as that used by McLellan et al. (2013) and Yang et al. (2016).

The PCR products were purified and cloned into pDONR221 (Invitrogen) to generate entry clones via BP reactions. The effector entry clones were transferred into pB7WGF2 (for N-terminal enhanced GFP [eGFP] fusion), pK7WGR2 (for N-terminal RFP fusion), and pDEST32 (for Y2H; Invitrogen). The StMAP3Kβ2 and StMAP3Kβ2 (KD) were recombined with pK7FWG2 (for C-terminal eGFP fusion) and pDEST22 (for Y2H; Invitrogen). For N-terminal cMyc tagging, pH7LIC was generated using the ClonExpress Entry One Step Cloning Kit (Vazyme Biotech). For split-YFP constructs, Pi04089 and Pi22926 were recombined with pCL112 (for N-terminal YN- fusion). StMAP3Kβ2 was recombined with pCL113 (for N-terminal YC- fusion).

Site-directed mutation of Lys-430Arg in the StMAP3Kβ2 kinase catalytic domain was introduced using the Mut Express II Fast Mutagenesis Kit (Vazyme).The entry clone containing the mutated form of StMAP3Kβ2 was recombined into pDEST22 for Y2H and pK7FWG2 for in planta assays. For N-terminal tagging with the cMyc epitope, pH7LIC was generated for coimmunoprecipitation analyses. Primer sequences used for PCR amplification and vector construction are shown in the Supplemental Table S1.

N. benthamiana Transformation

Agrobacterium tumefaciens containing overexpression vector pB7WGF2 was used to transform leaf discs of N. benthamiana. Positive lines were first screened on differential medium (Murashige and Skoog + 2 mg/L 6-benzylaminopurine + 0.2 mg/L naphthylacetic acid + 1.5 mg/L Bialaphos (sodium salt) + 400 mg/L cefalexin + 30 g/L Suc, pH 5.7) and then transferred to root generation medium (Murashige and Skoog + 0.36 mg/L Bialaphos (sodium salt) + 200 mg/L cefalexin + 0.1 mg/L naphthylacetic acid + 30 g/L Suc, pH 5.7). The positive lines were confirmed by semiquantitative RT-PCR. Primers are shown in the Supplemental Table S1).

Agro-Infiltration and P. infestans Infection Assay

A. tumefaciens strain GV3101 harboring plasmid constructs was grown overnight in yeast extract beef medium with appropriate antibiotics at 28°C at 200 rpm. The bacteria were pelleted, resuspended in sterile 10 mm MES, 10 mm MgCl2, and 200 μm acetosyringone, and subsequently adjusted to the appropriate final OD600 before pressure infiltration into N. benthamiana leaves (generally 0.1 for infection assays, 0.3–0.5 for cell death, and 0.5–1.0 for western blot and coimmunoprecipitation assays). For coexpression, agrobacteria cultures containing the appropriate vector constructs were mixed at a 1:1 ratio before infiltration. Each assay consisted of at least eight plants inoculated on three to four leaves.

P. infestans strain 88069 was grown on Rye Suc Agar plates at 18°C in the dark for 14 d. Sporangia were harvested from Rye Suc Agar plates by adding 3 mL H2O to the plates ,and zoospores were collected after 1 h of incubation at 4°C. Droplets (10 μL) of a solution of 100,000 zoospores per mL were applied onto the abaxial side of detached N. benthamiana leaves and incubated for several days on wet paper towels in 100% relative humidity. Agrobacterium tumefaciens transient assays (ATTAs) in combination with P. infestans infection were carried out as described (McLellan et al., 2013). For VIGS, the mean lesion diameter was measured at 7 dpi and compared to the GFP control. Sporangia counts were performed on 10 dpi leaves from VIGS plants that had been washed in 5 mL H2O and vortexed to release sporangia. The number of sporangia recovered from each leaf was counted using a hemocytometer.

Y2H

A Y2H screen with pDEST32-Pi22926 was performed as described (McLellan et al., 2013) using the ProQuest two-hybrid system (Invitrogen). The coding sequences of StMAP3Kβ2, StMAP3Kβ2(KD), and StMAP3Kβ2(KD)Lys-430Arg were recombined into pDEST22 and retested with pDEST32-Pi22926 (with pDEST32-Pi04089 as a negative control) in pairwise interactions. The transformants were selected using SD/-Leu-Trp-His selective medium and the 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside acid assay to detect the reporter gene activation.

Coimmunoprecipitation

Leaves of 5-week-old N. benthamiana plants were respectively agro-infiltrated with GFP-Pi22926 (with GFP-Pi040489 as a negative control), cMyc-tagged StMAP3Kβ2(KD), and cMyc-tagged StMAP3Kβ2(KD)Lys-430Arg. Two days after agro-infiltration, four leaf discs (9 mm in diameter) were harvested and proteins were extracted. GFP-tagged Pi22926/Pi04089 fusions were immunoprecipitated using GFP-Trap-M magnetic beads (MBL Biological Laboratories).The resulting samples were separated by SDS-PAGE and western blotted. Immunoprecipitated GFP fusions and coimmunoprecipitated c-Myc fusions were detected using appropriate antisera (Sungene Biotech).

Confocal Microscopy

A. tumefaciens (OD600 = 0.03–0.1)-containing target protein fusions were infiltrated into leaves of 4-week-old N. benthamiana plants. N. benthamiana leaf cells expressing fluorescent protein fusions were imaged no later than 2 d after agroinfiltration using a CLSM (Leica TCS-SPE) confocal microscope. GFP was excited with 488 nm from an argon laser and its emissions were detected between 500 and 530 nm. Monomeric RFP was excited with 561 nm and its emissions were detected between 600 and 630 nm. Split-YFP was excited using 514 nm from an argon laser with emissions detected from 530 to 575 nm. Images were collected from leaf cells expressing low levels of the fluorescence to minimize artifacts of ectopic protein expression.

VIGS

Plasmids pTRV1 and pTRV2 were used for VIGS (Liu et al., 2002; Ekengren et al., 2003). Constructs pTRV2:NbSIPK, pTRV2:NbWIPK, pTRV2:NbMEK1, pTRV2:NbMEK2, and pTRV2:NbNTF6 were generated using the same gene fragments based on the construct information previously published (Asai et al., 2008; Melech-Bonfil and Sessa, 2010). pTRV2:NbMAPKKKε-5′ and pTRV2:NbMAPKKKε-3′, used in this study, have been described (Melech-Bonfil and Sessa, 2010). For pTRV2:NbMAP3kβ2, 300 bp PCR fragments were cloned from N. benthamiana cDNA and inserted into TRV vectors (Liu et al., 2002) between BamH I and EcoR I sites in the antisense orientation. A TRV construct expressing GFP, described previously, was used as a control (McLellan et al., 2013). A. tumefaciens strains harboring pTRV2 vectors combined with that harboring the pTRV1 vector were mixed at a 1:1 ratio and adjusted to OD600 = 0.5. The cocultures were then infiltrated into two primary leaves of a plant at the four-leaf stage. Plants were used for assays or to check gene silencing levels by RT-qPCR 2 to 3 weeks later. The primers and constructs used in this study are shown in the Supplemental Table S1.

Gene Expression Assay

Three leaf discs (9 mm in diameter) were collected from N. benthamiana VIGS plants to extract total RNA using the PLANTpure Plant RNA Kit (Aidlab Biotechnologies). The first-strand cDNA was synthesized from 2 μg of RNA using the TRUEscript 1st Strand cDNA Synthesis Kit with gDNA Eraser (Aidlab Biotechnologies). RT-qPCR reactions were performed using Power SYBR Green (Bio-Rad). The N. benthamiana gene EF1α was used as a reference control. Primer pairs were designed outside the region of the cDNA targeted for silencing. The primers are shown in Supplemental Table S1. Gene expression levels were calculated by a comparative ΔΔCt method as described in Bio-Rad instructions.

Statistical Analyses

All data and statistical analysis were carried out using one-way ANOVA and pairwise or multiple comparisons in Graphpad Prism 6.0 software (GraphPad Prism Software Inc.). All values and error bars presented are means ± sd or se of three or more experimental replicates.

Accession Numbers

Sequence data from this article can be found in the GenBank and Web site under the following accession numbers. EEY57148 (P. infestans PITG_22926), EEY62542 (P. infestans PexRD2), XP_006360216.1 (potato StMAP3Kβ2), KJ504180 (potato StMAP3Kε), XP_010323778.1 (tomato SlMAPKKKβ1), XP_004230523.1 (tomato SlMAPKKKβ2), BAM36967.1 (N. benthamiana NbMAPKKKβ), Nbv6.1trP19888 (N. benthamiana NbMAP3Kβ2; http://benthgenome.qut.edu.au/), ADK36643 (N. benthamiana NbMAP3Kε), and BAM36969 (N. benthamiana NbMAP3Kε).

Supplemental Materials

The following supplemental materials are available.

Footnotes

1

This work was supported by the National Natural Science Foundation of China (grant nos. 31761143007 and 31471550 to Z.T.), the Fundamental Research Funds for the Central Universities of China (grant no. 2662017PY069 to Z.T.), the Biotechnology and Biological Sciences Research Council (BBSRC; grants BB/G015244/1, BB/K018183/1, and BB/L026880/1 to P.R.J.B., H.M., and M.A.), and The Scottish Government Rural and Environment Science and Analytical Services Division (RESAS; P.R.J.B.).

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