Trudy Torto-Alalibo

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

Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome.

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.

A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B.

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.

Cellulose Binding Domains of a Phytophthora Cell Wall Protein Are Novel Pathogen-Associated Molecular Patterns

The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and consequently are thought to have developed alternative mechanisms of pathogenicity. The oomycete Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We describe and functionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant similarity to the Kazal family of serine protease inhibitors. Database searches suggested that Kazal-like proteins are mainly restricted to animals and apicomplexan parasites but appear to be widespread and diverse in the oomycetes. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato in intercellular fluids. The epi1 and P69B genes were coordinately expressed and up-regulated during infection of tomato by P. infestans. Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism between plants and microbial pathogens. In addition, this study points to a common virulence strategy between the oomycete plant pathogen P. infestans and several mammalian parasites, such as the apicomplexan Toxoplasma gondii.

Large-Scale Gene Discovery in the Oomycete Phytophthora infestans Reveals Likely Components of Phytopathogenicity Shared with True Fungi

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.

Common and contrasting themes in host cell-targeted effectors from bacterial, fungal, oomycete and nematode plant symbionts described using the Gene Ontology

To overview the gene content of the important pathogen Phytophthora infestans, large-scale cDNA and genomic sequencing was performed. A set of 75,757 high-quality expressed sequence tags (ESTs) from P. infestans was obtained from 20 cDNA libraries representing a broad range of growth conditions, stress responses, and developmental stages. These included libraries from P. infestans-potato and -tomato interactions, from which 963 pathogen ESTs were identified. To complement the ESTs, onefold coveragethe P. infestans genome was obtained and regions of coding potential identified. A unigene set of 18,256 sequences was derived from the EST and genomic data and characterized for potential functions, stage-specific patterns of expression, and codon bias. Cluster analysis of ESTs revealed major differences between the expressed gene content of mycelial and spore-related stages, and affinities between some growth conditions. Comparisons with databases of fungal pathogenicity genes revealed conserved elements of pa...

Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors

A wide diversity of plant-associated symbionts, including microbes, produce proteins that can enter host cells, or are injected into host cells in order to modify the physiology of the host to promote colonization. These molecules, termed effectors, commonly target the host defense signaling pathways in order to suppress the defense response. Others target the gene expression machinery or trigger specific modifications to host morphology or physiology that promote the nutrition and proliferation of the symbiont. When recognized by the hosts surveillance machinery, which includes cognate resistance (R) gene products, defense responses are engaged to restrict pathogen proliferation. Effectors from diverse symbionts may be delivered into plant cells via varied mechanisms, including whole organism cellular entry (viruses, some bacteria and fungi), type III and IV secretion (in bacteria), physical injection (nematodes and insects) and protein translocation signal sequences (oomycetes and fungi). This mini-review will summarize both similarities and differences in effectors and effector delivery systems found in diverse plant-associated symbionts as well as how these are described with Plant-Associated Microbe Gene Ontology (PAMGO) terms.

Expressed Sequence Tags from Phytophthora sojae Reveal Genes Specific to Development and Infection

BackgroundThe oomycete Saprolegnia parasitica is one of the most economically important fish pathogens. There is a dramatic recrudescence of Saprolegnia infections in aquaculture since the use of the toxic organic dye malachite green was banned in 2002. Little is known about the molecular mechanisms underlying pathogenicity in S. parasitica and other animal pathogenic oomycetes. In this study we used a genomics approach to gain a first insight into the transcriptome of S. parasitica.ResultsWe generated 1510 expressed sequence tags (ESTs) from a mycelial cDNA library of S. parasitica. A total of 1279 consensus sequences corresponding to 525944 base pairs were assembled. About half of the unigenes showed similarities to known protein sequences or motifs. The S. parasitica sequences tended to be relatively divergent from Phytophthora sequences. Based on the sequence alignments of 18 conserved proteins, the average amino acid identity between S. parasitica and three Phytophthora species was 77% compared to 93% within Phytophthora. Several S. parasitica cDNAs, such as those with similarity to fungal type I cellulose binding domain proteins, PAN/Apple module proteins, glycosyl hydrolases, proteases, as well as serine and cysteine protease inhibitors, were predicted to encode secreted proteins that could function in virulence. Some of these cDNAs were more similar to fungal proteins than to other eukaryotic proteins confirming that oomycetes and fungi share some virulence components despite their evolutionary distanceConclusionWe provide a first glimpse into the gene content of S. parasitica, a reemerging oomycete fish pathogen. These resources will greatly accelerate research on this important pathogen. The data is available online through the Oomycete Genomics Database [1].

Agroinfection-based high throughput screening reveals specific recognition of INF elicitins in Solanum

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae.

SUMMARY We adapted and optimized the use of the Agrobacterium tumefaciens binary PVX expression system (PVX agroinfection) to screen Solanum plants for response to pathogen elicitors and applied the assay to identify a total of 11 clones of Solanum huancabambense and Solanum microdontum, out of 31 species tested, that respond to the elicitins INF1, INF2A and INF2B of Phytophthora infestans. Prior to this study, response to INF elicitins was only known in Nicotiana spp. within the Solanaceae. The identified S. huancabambense and S. microdontum clones also exhibited hypersensitivity-like cell death following infiltration with purified recombinant INF1, INF2A and INF2B, thereby validating the screening protocol. Comparison of INF elicitin activity revealed that Nicotiana plants responded to significantly lower concentrations than Solanum, suggesting variable levels of sensitivity to INF elicitins. We exploited natural variation in response to INF elicitins in the identified Solanum accessions to evaluate the relationship between INF recognition and late blight resistance. Interestingly, several INF-responsive Solanum plants were susceptible to P. infestans. Also, an S. microdontum xSolanum tuberosum (potato) population that segregates for INF response was generated but failed to identify a measurable contribution of INF response to resistance. These results suggest that in Solanum, INF elicitins are recognized as general elicitors and do not have a measurable contribution to disease resistance.