http://phytophthoradb.org/Blair_et_al_2008.pdf

A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences.

Interactions between plants and microbial pathogens involve complex signal exchanges at the plant surface and intercellular space interface (Baker et al. 1997; Parniske 2000; Hahn and Mendgen 2001). For example, plant pathogens have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants through a diverse array of extracellular effector molecules that can either promote infection or trigger defense responses (Knogge 1996; Lauge and De Wit 1998; Collmer et al. 2000; Kjemtrup et al. 2000; Staskawicz et al. 2001). Typically, such molecules are secreted into the intercellular interface between the pathogen and the plant or delivered inside the host cell to reach their cellular target. Thus, discovery programs that target genes encoding extracellular proteins can be expected to increase the probability of identifying genes involved in virulence. This approach has been taken successfully in the study of bacterial pathogens and symbionts. For example, an early study showed that Sinorhizobium meliloti mutants deficient in extracellular proteins were five times more likely to be affected in symbiosis than random mutants (Long et al. 1988). More recently, the characterization of effector proteins secreted through the type III secretion system of animal- and plant-associated bacteria has emerged as a key strategy for understanding mechanisms of virulence (Collmer et al. 2000; Kjemtrup et al. 2000; Staskawicz et al. 2001). In eukaryotic plant pathogens, genomic studies that focus systematically on extracellular proteins remain limited to nematodes, in which secretions from the esophageal gland cells are thought to play critical roles in infection (Wang et al. 2001). However, several classes of oomycete and fungal effector molecules, such as elicitor proteins that induce plant defense responses and a programmed cell death response termed the “hypersensitive response” (HR), are known to require secretion (Lauge and De Wit 1998; Jia et al. 2000). Therefore, secretion is an essential mechanism for delivery of virulence factors by eukaryotic plant pathogens to their appropriate site in infected plant tissue.

In eukaryotic cells, most secreted and membrane proteins are exported through the general secretory pathway (also known as type II secretion system) via short, N-terminal amino-acid sequences known as signal peptides (von Heijne 1985; Rapoport 1992). Typically, signal peptides contain one or two charged amino acids followed by a hydrophobic core, and the signal peptidase cleavage site is defined by a pair of small uncharged amino acids (von Heijne 1985). Although most of these features can be identified in known extracellular proteins, the particular amino acid sequences are highly degenerate, and cannot be identified using DNA hybridization or PCR-based techniques (Klein et al. 1996). However, with the advent of genomics, large sets of sequence data became available, creating the opportunity to develop and test predictive software to identify extracellular proteins. For example, SignalPv 2.0, a program that was developed using machine learning methods, assigns signal peptide prediction scores and putative cleavage sites to unknown amino acid sequences with a high level of accuracy (Nielsen et al. 1997; Nielsen and Krogh 1998; Menne et al. 2000).

The Irish famine pathogen, Phytophthora infestans, is a eukaryotic oomycete microorganism that causes late blight, a worldwide devastating disease of potato and tomato (Fry and Goodwin 1997a,b). Although it is a pathogen of great economic importance, little is known about the molecular mechanisms involved in the pathogenicity and host specificity of P. infestans, and only a handful of genes have been implicated in interaction with host plants (Kamoun 2000, 2001). Structural genomics of Phytophthora is underway. Pilot cDNA sequencing projects were performed for P. infestans and another species, Phytophthora sojae (Kamoun et al. 1999b; Qutob et al. 2000), resulting in a database of expressed sequence tags (ESTs; Waugh et al. 2000). With the accumulation of sequence data for Phytophthora, the challenge is shifting to data mining and functional analyses. One important goal is to be able to associate a biological function with sequences with no significant similarity to known genes. With this objective in mind, we set up to identify systematically P. infestans cDNAs encoding extracellular proteins from EST databases. Here, we describe PexFinder, an algorithm for the automated identification of putative extracellular proteins from ESTs. We applied PexFinder to a P. infestans EST data set and selected 63 candidate Pex (Phytophthora extracellular proteins) cDNAs for functional expression in plants using a viral vector. This functional genomics strategy resulted in the discovery of a novel family of necrosis-inducing genes that are predicted to encode extracellular proteins with no similarity to sequences in public databases.

/pdf/est-mining-and-functional-expression-assays-identify-4lbtaq8f4o.pdf

EST Mining and Functional Expression Assays Identify Extracellular Effector Proteins From the Plant Pathogen Phytophthora

Phytophthora infestans, the agent of potato and tomato late blight disease, produces a 10-kD extracellular protein, INF1 elicitin. INF1 induces a hypersensitive response in a restricted number of plants, particularly those of the genus Nicotiana. In virulence assays with different P. infestans isolates, five Nicotiana species displayed resistance responses. In all of the interactions, after inoculation with P. infestans zoospores, penetration of an epidermal cell was observed, followed by localized necrosis typical of a hypersensitive response. To determine whether INF1 functions as an avirulence factor in these interactions, we adopted a gene-silencing strategy to inhibit INF1 production. Several transformants deficient in inf1 mRNA and INF1 protein were obtained. These strains remained pathogenic on host plants. However, in contrast to the wild-type and control transformant strains, INF1-deficient strains induced disease lesions when inoculated on N. benthamiana. These results demonstrate that the elicitin INF1 functions as an avirulence factor in the interaction between N. benthamiana and P. infestans.

/pdf/resistance-of-nicotiana-benthamiana-to-phytophthora-1dvko5ya3n.pdf

Resistance of Nicotiana benthamiana to Phytophthora infestans Is Mediated by the Recognition of the Elicitor Protein INF1

Parasitic and pathogenic lifestyles have evolved repeatedly in eukaryotes ([93][1]). Several parasitic eukaryotes represent deep phylogenetic lineages, suggesting that they feature unique molecular processes for infecting their hosts. One such group is formed by the oomycetes. Traditionally, due to

Molecular genetics of pathogenic oomycetes.

The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD3645-1, suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34–amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.

/pdf/in-planta-expression-screens-of-phytophthora-infestans-rxlr-5ggwbefalq.pdf

In Planta Expression Screens of Phytophthora infestans RXLR Effectors Reveal Diverse Phenotypes, Including Activation of the Solanum bulbocastanum Disease Resistance Protein Rpi-blb2

The variation in two aspects of pathogenicity, aggressiveness and virulence (physiologic race), among single zoospore cultures from three wild isolates has been examined. Two components of aggressiveness were measured, rate of growth on tubers and generation time on detached leaflets. Virulence was assessed from the pattern of compatible and incompatible reactions to members of the R gene differential series. Extensive variation in aggressiveness was detected in samples of zoospore cultures from all three isolates. The level of aggressiveness ranged from a high, equivalent to that of the parental wild isolates, to a complete lack of pathogenicity; as much as 45% was of the latter type. No instance of a change in virulence was found among 104 cultures tested.The significance of the observed pathogenic variation is discussed. It is concluded that zoospore variation is not important as a source of new physiologic races or strains adapted to particular, horizontally resistant varieties. Comparison of populati...

C. E. Caten

Papers

Spontaneous variability of single isolates of Phytophthora infestans. II. Pathogenic variation