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: 3.23). 01/2012; 7(1):e29847. DOI: 10.1371/journal.pone.0029847
Source: PubMed


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,
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    • "In contrast, no RCPs could be identified among the 1852 secreted proteins predicted from the proteome of P. graminis f. sp. tritici (Saunders et al., 2012). As such, RCP effectors are expected to play an important role in promoting the colonization of some, but not all, plant-associated organisms. "
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    ABSTRACT: Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.
    Frontiers in Plant Science 10/2015; 6:872. DOI:10.3389/fpls.2015.00872 · 3.95 Impact Factor
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    • "n the secretome of fungal pathogens ( Saunders et al . , 2012 ; Guyon et al . , 2014 ) . Typical effector properties include the presence of a N - terminal secretion signal , small protein size , high Cys content , the absence of characterized protein domains , high rate of non - synonymous over synonymous substitutions ( Hacquard et al . , 2012 ; Saunders et al . , 2012 ; Persoons et al . , 2014 ; Sperschneider et al . , 2014 ) . However , validated virulence factors do not all comply with these properties , such as Verticillium dahlia isochorismatase VdIsc1 harboring an isochorismatase domain but no conventional secretion signal ( Liu et al . , 2014 ) or Melampsora lini AvrM that lacks any Cys ( Catan"
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    ABSTRACT: Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases.
    Frontiers in Plant Science 09/2015; 6. DOI:10.3389/fpls.2015.00776 · 3.95 Impact Factor
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    • "Scale bars: 10 µm. such as RXLR and LXLFLAK (Haas et al. 2009; Raffaele et al. 2010; Whisson et al. 2007), the identification of translocated effectors in fungi is more problematic (Cantu et al. 2013; Hacquard et al. 2012; Petre and Kamoun 2014; Saunders et al. 2012). In the rust fungi, haustorial-expression has been used to predict host-translocated effectors (Catanzariti et al. 2006; Kemen et al. 2005). "
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    ABSTRACT: Rust fungi are devastating crop pathogens that deliver effector proteins into infected tissues to modulate plant functions and promote parasitic growth. The genome of the poplar leaf rust fungus Melampsora larici-populina revealed a large catalogue of secreted proteins, some of which have been considered candidate effectors. Unravelling how these proteins function in host cells is key to understanding pathogenicity mechanisms and developing resistant plants. In this study, we used an effectoromics pipeline to select, clone, and express 20 candidate effectors in Nicotiana benthamiana leaf cells to determine their subcellular localisation and identify the plant proteins they interact with. Confocal microscopy revealed that six candidate effectors target the nucleus, nucleoli, chloroplasts, mitochondria and discrete cellular bodies. We also used coimmunoprecipitation and mass spectrometry to identify 606 N. benthamiana proteins that associate with the candidate effectors. Five candidate effectors specifically associated with a small set of plant proteins that may represent biologically relevant interactors. We confirmed the interaction between the candidate effector MLP124017 and the TOPLESS-Related Protein 4 from poplar by in planta coimmunoprecipitation. Altogether, our data enable us to validate effector proteins from M. larici-populina and reveal that these proteins may target multiple compartments and processes in plant cells. It also shows that N. benthamiana can be a powerful heterologous system to study effectors of obligate biotrophic pathogens.
    Molecular Plant-Microbe Interactions 02/2015; 28(6). DOI:10.1094/MPMI-01-15-0003-R · 3.94 Impact Factor
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