Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi

University of California Riverside, United States of America
PLoS ONE (Impact Factor: 3.53). 01/2012; 7(1):e29847. DOI: 10.1371/journal.pone.0029847
Source: PubMed

ABSTRACT Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

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Available from: Sylvain Raffaele, Aug 21, 2015
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    • "These small-secreted proteins have distinct primary and tertiary protein structures (Figs S4 and S5) indicating their functional independence. Like many other effectors associated with fungal virulence (Saunders et al., 2012), these four small secreted proteins share at least one of the following properties: a signal polypeptide, small protein size and cysteine rich, implying their involvement in Piloderma–host symbiosis. All the small secreted proteins encode a small polypeptide of less than 215 amino acids and were predicted as non-cytoplasmic proteins with no transmembrane domains (data not shown). "
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    Environmental Microbiology 09/2014; 16(12). DOI:10.1111/1462-2920.12619 · 6.24 Impact Factor
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    • "These candidate effectors were predicted to form 164 clusters, varying in membership from 2 to 44, and form a striking 10% of all of the expanded families in the stem rust genome. Another bioinformatic pipeline uses Markov and hierarchical clustering to identify protein families of wheat stem rust and poplar leaf rust, and it prioritizes effector candidates using a ranking system based on eight criteria associated with effector properties (Saunders et al., 2012). "
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    ABSTRACT: Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.
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    • "Length is a criteria used to identify small secreted proteins (SSPs) from within putative effector protein sets, as SSPs are effector-like proteins with lengths less than 300 amino acids. Sequence comparisons alone do not provide a reliable means to identify putative effectors since some known effectors are lineage-specific while others are conserved across taxa (Rep, 2005; Saunders et al., 2012; Giraldo and Valent, 2013). Candidate effectors, a further distinction, are putative effectors that have additional support for roles in pathogenicity "
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