Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein–protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector–host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

One-sentence summary In planta protein–protein interaction screen of Phytophthora infestans RXLR effectors by proteomics approach reveals host vesicle trafficking as a major effector-targeted process.     

Host Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the Solanum demissum Immune Receptor R2 to Mediate Disease Resistance

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.     

Paranoid potato: Phytophthora-resistant genotype shows constitutively activated defense

Phytophthora is the most devastating pathogen of dicot plants. There is a need for resistance sources with different modes of action to counteract the fast evolution of this pathogen. In order to better understand mechanisms of defense against P. infestans, we analyzed several clones of potato. Two of the genotypes tested, Sarpo Mira and SW93-1015, exhibited strong resistance against P. infestans in field trials, whole plant assays and detached leaf assays. The resistant genotypes developed different sizes of hypersensitive response (HR)-related lesions. HR lesions in SW93-1015 were restricted to very small areas, whereas those in Sarpo Mira were similar to those in Solanum demissum, the main source of classical resistance genes. SW93-1015 can be characterized as a cpr (constitutive expressor of PR genes) genotype without spontaneous microscopic or macroscopic HR lesions. This is indicated by constitutive hydrogen peroxide (H₂O₂) production and PR1 (pathogenesis-related protein 1) secretion. SW93-1015 is one of the first plants identified as having classical protein-based induced defense expressed constitutively without any obvious metabolic costs or spontaneous cell death lesions.     

Phytophthora infestans RXLR Effector PexRD2 Interacts with Host MAPKKKε to Suppress Plant Immune Signaling

Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity–related signaling.     

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2–mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.     

The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans

The interaction between Phytophthora infestans (Mont.) de Bary and Solanum was examined cytologically using a diverse set of wild Solanum species and potato (S. tuberosum L.) cultivars with various levels of resistance to late blight. In wild Solanum species, in potato cultivars carrying known resistance (R) genes and in nonhosts the major defense reaction appeared to be the hypersensitive response (HR). In fully resistant Solanum species and nonhosts, the HR was fast and occurred within 22 h. This resulted in the death of one to three cells. In partially resistant clones, the HR was induced between 16 and 46 h, and resulted in HR lesions consisting of five or more dead cells, from which hyphae were occasionally able to escape to establish a biotrophic interaction. These results demonstrate the quantitative nature of the resistance to P. infestans. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones and correlated with resistance levels. Other responses associated with the defense reaction were deposition of callose and extracellular globules containing phenolic compounds. These globules were deposited near cells showing the HR, and may function in cell wall strengthening. Received: 22 April 1999 / Accepted: 4 November 1999     

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.     

Fine mapping of the Ph-3 gene conferring resistance to late blight (Phytophthora infestans) in tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is a devastating disease for tomato and potato crops. In the past decades, many late blight resistance (R) genes have been characterized in potato. In contrast, less work has been conducted on tomato. The Ph-3 gene from Solanum pimpinellifolium was introgressed into cultivated tomatoes and conferred broad-spectrum resistance to P. infestans. It was previously assigned to the long arm of chromosome 9. In this study, a high-resolution genetic map covering the Ph-3 locus was constructed using an F₂ population of a cross between Solanum lycopersicum CLN2037B (containing Ph-3) and S. lycopersicum LA4084. Ph-3 was mapped in a 0.5 cM interval between two markers, Indel_3 and P55. Eight putative genes were found in the corresponding 74 kb region of the tomato Heinz1706 reference genome. Four of these genes are resistance gene analogs (RGAs) with a typical nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 domain. Each RGA showed high homology to the late blight R gene Rpi-vnt1.1 from Solanum venturii. Transient gene silencing indicated that a member of this RGA family is required for Ph-3-mediated resistance to late blight in tomato. Furthermore, this RGA family was also found in the potato genome, but the number of the RGAs was higher than in tomato.     

An elicitor-and pathogen-induced cdna from potato encodes a stress-responsive cyclophilin

Differential mRNA display was used to identify pathogen-responsive, stress-related genes in potato cell suspensions treated with salicylic acid and a cell wall-derived elicitor from Phytophthora infestans. Among the positive clones identified, one was found to be expressed at a significantly higher level in elicited cells than in control cells. DNA sequencing of this amplicon revealed high homology and identified it as a potato cyclophilin cDNA. The maximum amount of the cyclophilin mRNA was found 9 to 12 h after elicitation. Cyclophilin (CyP) mRNA synthesis was also up-regulated from 12 to 24 h in potato leaves locally infected with zoospores from Phytophthora infestans. However, untreated leaves responding systemically to the pathogen showed only a weak, delayed response at 24 h post infection. The observed accumulation of potato CyP mRNA in response to salicylic acid, P. infestans elicitor and P. infestans infection, suggest that CyPs play an important role in plant stress responses.     

Functional analysis of potato genes involved in quantitative resistance to Phytophthora infestans

The most significant threat to potato production worldwide is the late blight disease, which is caused by the oomycete pathogen Phytophthora infestans. Based on previous cDNA microarrays and cDNA-amplified fragment length polymorphism analysis, 63 candidate genes that are expected to contribute to developing a durable resistance to late blight were selected for further functional analysis. We performed virus-induced gene silencing (VIGS) to these candidate genes on both Nicotiana benthamiana and potato, subsequently inoculated detached leaves and assessed the resistance level. Ten genes decreased the resistance to P. infestans after VIGS treatment. Among those, a lipoxygenase (LOX; EC 1.13.11.12) and a suberization-associated anionic peroxidase affected the resistance in both N. benthamiana and potato. Our results identify genes that may play a role in quantitative resistance mechanisms to late blight.     

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.     

Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

Background

Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta.

Methodology/Principal Findings

We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family.

Conclusions/Significance

We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.     

A nonspecific lipid transfer protein,StLTP10, mediates resistance to Phytophthora infestans in potato

Nonspecific lipidtransfer proteins (nsLTPs), which are small, cysteine-rich proteins, belong to the pathogenesis-related protein family, and several of them act as positive regulators during plant disease resistance. However, the underlying molecular mechanisms of these proteins in plant immune responses are unclear. In this study, a typical nsLTP gene, StLTP10, was identified and functionally analysed in potato. StLTP10 expression was significantly induced by Phytophthora infestans, which causes late blight in potato, and defence-related phytohormones, including abscisic acid (ABA), salicylic acid, and jasmonic acid. Characterization of StLTP10-overexpressing and knockdown lines indicated that StLTP10 positively regulates plant resistance to P. infestans. This resistance was coupled with enhanced expression of reactive oxygen species scavenging- and defence-related genes. Furthermore, we identified that StLTP10 physically interacts with ABA receptor PYL4 and affects its subcellular localization. These two proteins work together to regulate stomatal closure during pathogen infection. Interestingly, we also found that wound-induced protein kinase interacts with StLTP10 and positively regulates its protein abundance. Taken together, our results provide insight into the role of StLTP10 in resistance to P. infestans and suggest candidates to enhance broad-spectrum resistance to pathogens in potato.     

AB-QTL analysis reveals new alleles associated to proline accumulation and leaf wilting under drought stress conditions in barley (Hordeum vulgareL.)

Background

Phytophthora infestans (Mont.) de Bary, the causal organism of late blight, is economically the most important pathogen of potato and resistance against it has been one of the primary goals of potato breeding. Some potentially durable, broad-spectrum resistance genes against this disease have been described recently. However, to obtain durable resistance in potato cultivars more genes are needed to be identified to realize strategies such as gene pyramiding or use of genotype mixtures based on diverse genes.

Results

A major resistance gene, Rpi-rzc1, against P. infestans originating from Solanum ruiz-ceballosii was mapped to potato chromosome X using Diversity Array Technology (DArT) and sequence-specific PCR markers. The gene provided high level of resistance in both detached leaflet and tuber slice tests. It was linked, at a distance of 3.4 cM, to violet flower colour most likely controlled by the previously described F locus. The marker-trait association with the closest marker, violet flower colour, explained 87.1% and 85.7% of variance, respectively, for mean detached leaflet and tuber slice resistance. A genetic linkage map that consisted of 1,603 DArT markers and 48 reference sequence-specific PCR markers of known chromosomal localization with a total map length of 1204.8 cM was constructed.

Conclusions

The Rpi-rzc1 gene described here can be used for breeding potatoes resistant to P. infestans and the breeding process can be expedited using the molecular markers and the phenotypic marker, violet flower colour, identified in this study. Knowledge of the chromosomal localization of Rpi-rzc1 can be useful for design of gene pyramids. The genetic linkage map constructed in this study contained 1,149 newly mapped DArT markers and will be a valuable resource for future mapping projects using this technology in the Solanum genus.     

A potato pathogenesis-related protein gene, StPRp27, contributes to race-nonspecific resistance against Phytophthora infestans

Late blight caused by Phytophthora infestans is the most important disease of potato. Many efforts have been made to understand molecular mechanism of the durable resistance to address the challenge raised by rapid evolution of the pathogen. A pathogenesis related protein (PR) gene StPRp27 was previously isolated from the potato leaves challenged by P. infestans. The sequence analysis and expression pattern reveal that StPRp27 may be associated with resistance to P. infestans. In present research, transient expression of StPRp27 in Nicotiana benthamiana enhanced resistance to P. infestans isolates 99189 and PY23 indicating its potential contribution to the disease resistance. These findings were also confirmed by over-expression of StPRp27 in potato cv. E-potato 3, which significantly slowed down the development of the disease after inoculation with a mixture of P. infestans races. Further, silencing of StPRp27 homologous genes in N. benthamiana harboring dominant Phytophthora resistance gene Rpi-blb1 or Rpi-blb2 showed no effects on the resistance triggered by these R genes. Our results suggest that StPRp27 contributes to a race-nonspecific resistance against P. infestans by inhibiting the disease development and has a potential use in selection and breeding for durable resistance to late blight.