Miles R. Armstrong

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world’s population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at

A translocation signal for delivery of oomycete effector proteins into host plant cells

6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at ∼240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for ∼74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

Differential Recognition of Highly Divergent Downy Mildew Avirulence Gene Alleles by RPP1 Resistance Genes from Two Arabidopsis Lines

Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK—representing a change that conserves physicochemical properties of the protein—P. infestans fails to deliver Avr3a or an Avr3a–GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.

An RxLR Effector from Phytophthora infestans Prevents Re-localisation of Two Plant NAC Transcription Factors from the Endoplasmic Reticulum to the Nucleus

Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3aKI, which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3aEM, which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3aKI/Y147del, a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3aKI or Avr3aEM but not the Avr3aKI/Y147del mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.

Towards understanding the virulence functions of RXLR effectors of the oomycete plant pathogen Phytophthora infestans

The potato late blight pathogen Phytophthora infestans secretes an array of effector proteins thought to act in its hosts by disarming defences and promoting pathogen colonisation. However, little is known about the host targets of these effectors and how they are manipulated by the pathogen. This work describes the identification of two putative membrane-associated NAC transcription factors (TF) as the host targets of the RxLR effector PITG_03192 (Pi03192). The effector interacts with NAC Targeted by Phytophthora (NTP) 1 and NTP2 at the endoplasmic reticulum (ER) membrane, where these proteins are localised. Transcripts of NTP1 and NTP2 rapidly accumulate following treatment with culture filtrate (CF) from in vitro grown P. infestans, which acts as a mixture of Phytophthora PAMPs and elicitors, but significantly decrease during P. infestans infection, indicating that pathogen activity may prevent their up-regulation. Silencing of NTP1 or NTP2 in the model host plant Nicotiana benthamiana increases susceptibility to P. infestans, whereas silencing of Pi03192 in P. infestans reduces pathogenicity. Transient expression of Pi03192 in planta restores pathogenicity of the Pi03192-silenced line. Moreover, colonisation by the Pi03192-silenced line is significantly enhanced on N. benthamiana plants in which either NTP1 or NTP2 have been silenced. StNTP1 and StNTP2 proteins are released from the ER membrane following treatment with P. infestans CF and accumulate in the nucleus, after which they are rapidly turned over by the 26S proteasome. In contrast, treatment with the defined PAMP flg22 fails to up-regulate NTP1 and NTP2, or promote re-localisation of their protein products to the nucleus, indicating that these events follow perception of a component of CF that appears to be independent of the FLS2/flg22 pathway. Importantly, Pi03192 prevents CF-triggered re-localisation of StNTP1 and StNTP2 from the ER into the nucleus, revealing a novel effector mode-of-action to promote disease progression.

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

Plant pathogens establish infection by secretion of effector proteins that may be delivered inside host cells to manipulate innate immunity. It is increasingly apparent that the ubiquitin proteasome system (UPS) contributes significantly to the regulation of plant defences and, as such, is a target for pathogen effectors. Bacterial effectors delivered by the type III and IV secretion systems have been shown to interact with components of the host UPS. Some of these effectors possess functional domains that are conserved in UPS enzymes, whilst others contain novel domains with ubiquitination activities. Relatively little is known about effector activities in eukaryotic microbial plant pathogens. Nevertheless, effectors from oomycetes that contain an RXLR motif for translocation to the inside of plant cells have been shown to suppress host defences. Annotation of the genome of one such oomycete, the potato late blight pathogen Phytophthora infestans, and protein-protein interaction assays to discover host proteins targeted by the RXLR effector AVR3a, have revealed that this eukaryotic plant pathogen also has the potential to manipulate host plant UPS functions.

Relocalization of Late Blight Resistance Protein R3a to Endosomal Compartments Is Associated with Effector Recognition and Required for the Immune Response

Oomycete pathogens of plants secrete effectors to perturb host cell physiology. This study reveals that the P. infestans RXLR-type effector protein PexRD2 interferes with host MAPKKKε immunity-related signaling to the benefit of the pathogen. 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.

A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease

This work examines effector recognition and signaling by NB-LRR resistance protein R3a, finding that recognition of AVR3A involves R3a relocalization to vesicular compartments in the endocytic pathway and attenuation of this relocalization suppressed the resultant hypersensitive cell death response. An important objective of plant–pathogen interactions research is to determine where resistance proteins detect pathogen effectors to mount an immune response. Many nucleotide binding–Leucine-rich repeat (NB-LRR) resistance proteins accumulate in the plant nucleus following effector recognition, where they initiate the hypersensitive response (HR). Here, we show that potato (Solanum tuberosum) resistance protein R3a relocates from the cytoplasm to endosomal compartments only when coexpressed with recognized Phytophthora infestans effector form AVR3aKI and not unrecognized form AVR3aEM. Moreover, AVR3aKI, but not AVR3aEM, is also relocalized to endosomes in the presence of R3a. Both R3a and AVR3aKI colocalized in close physical proximity at endosomes in planta. Treatment with brefeldin A (BFA) or wortmannin, inhibitors of the endocytic cycle, attenuated both the relocalization of R3a to endosomes and the R3a-mediated HR. No such effect of these inhibitors was observed on HRs triggered by the gene-for-gene pairs Rx1/PVX-CP and Sto1/IpiO1. An R3a(D501V) autoactive MHD mutant, which triggered HR in the absence of AVR3aKI, failed to localize to endosomes. Moreover, BFA and wortmannin did not alter cell death triggered by this mutant. We conclude that effector recognition and consequent HR signaling by NB-LRR resistance protein R3a require its relocalization to vesicles in the endocytic pathway.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor

Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c–1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease.