Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges

Effector proteins are mostly secretory proteins that stimulate plant infection by manipulating the host response. Identifying fungal effector proteins and understanding their function is of great importance in efforts to curb losses to plant diseases. Recent advances in high-throughput sequencing technologies have facilitated the availability of several fungal genomes and 1000s of transcriptomes. As a result, the growing amount of genomic information has provided great opportunities to identify putative effector proteins in different fungal species. There is little consensus over the annotation and functionality of effector proteins, and mostly small secretory proteins are considered as effector proteins, a concept that tends to overestimate the number of proteins involved in a plant–pathogen interaction. With the characterization of Avr genes, criteria for computational prediction of effector proteins are becoming more efficient. There are 100s of tools available for the identification of conserved motifs, signature sequences and structural features in the proteins. Many pipelines and online servers, which combine several tools, are made available to perform genome-wide identification of effector proteins. In this review, available tools and pipelines, their strength and limitations for effective identification of fungal effector proteins are discussed. We also present an exhaustive list of classically secreted proteins along with their key conserved motifs found in 12 common plant pathogens (11 fungi and one oomycete) through an analytical pipeline.

[1]  R. Terauchi,et al.  Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor , 2015, eLife.

[2]  K. Tamura,et al.  Metabolic engineering of plant alkaloid biosynthesis. Proc Natl Acad Sci U S A , 2001 .

[3]  S. Reissmann,et al.  Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem. , 2015, European journal of cell biology.

[4]  Zsolt Karányi,et al.  FSRD: fungal stress response database , 2013, Database J. Biol. Databases Curation.

[5]  Christina A. Cuomo,et al.  Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici , 2011 .

[6]  R. Siezen,et al.  LAB-Secretome: a genome-scale comparative analysis of the predicted extracellular and surface-associated proteins of Lactic Acid Bacteria , 2010, BMC Genomics.

[7]  Kay Hofmann,et al.  Tmbase-A database of membrane spanning protein segments , 1993 .

[8]  B. Maček,et al.  Metabolic priming by a secreted fungal effector , 2011, Nature.

[9]  J. Stuart Insect effectors and gene-for-gene interactions with host plants. , 2015, Current opinion in insect science.

[10]  R. Deshmukh,et al.  FUNGICIDAL INTERFERENCE DURING INFECTION RELATED DEVELOPMENTAL STAGES IN MAGNAPORTHE GRISEA , 2012 .

[11]  Jaeyoung Choi,et al.  Fungal Secretome Database: Integrated platform for annotation of fungal secretomes , 2010, BMC Genomics.

[12]  S. Kamoun,et al.  RXLR effectors of plant pathogenic oomycetes. , 2007, Current opinion in microbiology.

[13]  Antonio Di Pietro,et al.  The Top 10 fungal pathogens in molecular plant pathology. , 2012, Molecular plant pathology.

[14]  Marcin J. Skwark,et al.  Sequence analysis SPOCTOPUS: a combined predictor of signal peptides and membrane protein topology , 2008 .

[15]  Jeppe Emmersen,et al.  Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif , 2010, BMC Genomics.

[16]  Shiv D. Kale,et al.  Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry. , 2013, Molecular plant-microbe interactions : MPMI.

[17]  Yang Wang,et al.  Bioinformatics Analysis Reveals Abundant Short Alpha-Helices as a Common Structural Feature of Oomycete RxLR Effector Proteins , 2015, PloS one.

[18]  Erik L. L. Sonnhammer,et al.  Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server , 2007, Nucleic Acids Res..

[19]  Kuo-Chen Chou,et al.  Signal-CF: a subsite-coupled and window-fusing approach for predicting signal peptides. , 2007, Biochemical and biophysical research communications.

[20]  Christina A. Cuomo,et al.  Obligate biotrophy features unraveled by the genomic analysis of rust fungi , 2011, Proceedings of the National Academy of Sciences.

[21]  Erik L. L. Sonnhammer,et al.  MetaTM - a consensus method for transmembrane protein topology prediction , 2009, BMC Bioinformatics.

[22]  E. Danchin,et al.  Horizontal gene transfer in nematodes: a catalyst for plant parasitism? , 2011, Molecular plant-microbe interactions : MPMI.

[23]  T. Kigawa,et al.  Phosphatidylinositol monophosphate-binding interface in the oomycete RXLR effector AVR3a is required for its stability in host cells to modulate plant immunity , 2011, Proceedings of the National Academy of Sciences.

[24]  B. Valent,et al.  Filamentous plant pathogen effectors in action , 2013, Nature Reviews Microbiology.

[25]  H. Sonah,et al.  Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.) , 2014 .

[26]  M. Syvanen,et al.  Horizontal Gene Transfer , 2015, Evolution, Medicine, and Public Health.

[27]  François Belzile,et al.  The Transition from a Phytopathogenic Smut Ancestor to an Anamorphic Biocontrol Agent Deciphered by Comparative Whole-Genome Analysis[W][OPEN] , 2013, Plant Cell.

[28]  T. Rouxel,et al.  Major Gene and Polygenic Resistance to Leptosphaeria maculans in Oilseed Rape (Brassica napus) , 2005, European Journal of Plant Pathology.

[29]  Gisbert Schneider,et al.  Prediction of Type III Secretion Signals in Genomes of Gram-Negative Bacteria , 2009, PloS one.

[30]  J. Holton,et al.  Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces , 2011, Proceedings of the National Academy of Sciences.

[31]  P. D. de Wit,et al.  Fungal effector proteins. , 2009, Annual review of phytopathology.

[32]  J. A. Oguiza,et al.  SECRETOOL: integrated secretome analysis tool for fungi , 2013, Amino Acids.

[33]  Qing Zhang,et al.  High-accuracy prediction of bacterial type III secreted effectors based on position-specific amino acid composition profiles , 2011, Bioinform..

[34]  S. Reissmann,et al.  Fungal effectors and plant susceptibility. , 2015, Annual review of plant biology.

[35]  Ram Samudrala,et al.  Accurate Prediction of Secreted Substrates and Identification of a Conserved Putative Secretion Signal for Type III Secretion Systems , 2009, PLoS pathogens.

[36]  R. Deshmukh,et al.  Intron gain, a dominant evolutionary process supporting high levels of gene expression in rice , 2015, Journal of Plant Biochemistry and Biotechnology.

[37]  Genetic architecture of cyst nematode resistance revealed by genome-wide association study in soybean , 2015, BMC Genomics.

[38]  István Simon,et al.  The HMMTOP transmembrane topology prediction server , 2001, Bioinform..

[39]  H. Nguyen,et al.  Genomic-assisted phylogenetic analysis and marker development for next generation soybean cyst nematode resistance breeding. , 2016, Plant science : an international journal of experimental plant biology.

[40]  Zhixiong Xie,et al.  Horizontal Gene Transfer , 2003, Methods in Molecular Biology.

[41]  Leighton Pritchard,et al.  A translocation signal for delivery of oomycete effector proteins into host plant cells , 2007, Nature.

[42]  S J Hamodrakas,et al.  A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm. , 1999, Protein engineering.

[43]  A. Mohanty,et al.  Pep1, a Secreted Effector Protein of Ustilago maydis, Is Required for Successful Invasion of Plant Cells , 2009, PLoS pathogens.

[44]  Sarah Calvo,et al.  Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis , 2006, Nature.

[45]  S. Reissmann,et al.  Genome editing in Ustilago maydis using the CRISPR-Cas system. , 2016, Fungal genetics and biology : FG & B.

[46]  I. Baldwin,et al.  New insights into plant responses to the attack from insect herbivores. , 2010, Annual review of genetics.

[47]  S. Singh,et al.  Molecular mapping of the downy mildew resistance gene Ppa3 in cauliflower (Brassica oleracea var. botrytis L.) , 2012 .

[48]  B. Thomma,et al.  Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae. , 2005, Molecular plant pathology.

[49]  K. Shirasu,et al.  Sequence Divergent RXLR Effectors Share a Structural Fold Conserved across Plant Pathogenic Oomycete Species , 2012, PLoS pathogens.

[50]  John Meinken,et al.  FunSecKB2: a fungal protein subcellular location knowledgebase , 2014 .

[51]  Burkhard Rost,et al.  The PredictProtein server , 2003, Nucleic Acids Res..

[52]  G. von Heijne,et al.  Prediction of membrane-protein topology from first principles , 2008, Proceedings of the National Academy of Sciences.

[53]  External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells. , 2010, Cell.

[54]  Rashmi Pant,et al.  The Pathogen-Host Interactions database (PHI-base): additions and future developments , 2014, Nucleic Acids Res..

[55]  Zixin Deng,et al.  SecReT4: a web-based bacterial type IV secretion system resource , 2012, Nucleic Acids Res..

[56]  F. Govers,et al.  RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members , 2008, Proceedings of the National Academy of Sciences.

[57]  Liam J McGuffin,et al.  Structure and evolution of barley powdery mildew effector candidates , 2012, BMC Genomics.

[58]  Christopher J. Rawlings,et al.  PHI-base update: additions to the pathogen–host interaction database , 2007, Nucleic Acids Res..

[59]  James K. Hane,et al.  A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi , 2013, BMC Genomics.

[60]  Jonathan D. G. Jones,et al.  The plant immune system , 2006, Nature.

[61]  Fan Zhang,et al.  T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System , 2010, BMC Bioinformatics.

[62]  Jingyu Yang,et al.  SOMRuler: A Novel Interpretable Transmembrane Helices Predictor , 2011, IEEE Transactions on NanoBioscience.

[63]  S. Kamoun A catalogue of the effector secretome of plant pathogenic oomycetes. , 2006, Annual review of phytopathology.

[64]  J. Holland,et al.  New insight into a complex plant–fungal pathogen interaction , 2015, Nature Genetics.

[65]  M. Banfield,et al.  On the front line: structural insights into plant–pathogen interactions , 2013, Nature Reviews Microbiology.

[66]  B. Matthews,et al.  Manipulation of two α-endo-β-1,4-glucanase genes, AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots. , 2014, Molecular plant pathology.

[67]  Xiang Jia Min,et al.  FunSecKB: the Fungal Secretome KnowledgeBase , 2011, Database J. Biol. Databases Curation.

[68]  Shiv D. Kale,et al.  Entry of oomycete and fungal effectors into plant and animal host cells , 2011, Cellular microbiology.

[69]  A. Vergunst,et al.  Exploitation of Eukaryotic Ubiquitin Signaling Pathways by Effectors Translocated by Bacterial Type III and Type IV Secretion Systems , 2007, PLoS pathogens.

[70]  Tal Pupko,et al.  Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine-learning approach. , 2016, Molecular plant pathology.

[71]  A. Ware,et al.  Breakdown of resistance to the fungal disease, blackleg, is averted in commercial canola (Brassica napus) crops in Australia , 2014 .

[72]  Rainer Winnenburg,et al.  The pathogen-host interactions database (PHI-base) provides insights into generic and novel themes of pathogenicity. , 2006, Molecular plant-microbe interactions : MPMI.

[73]  Rangel C. Souza,et al.  AtlasT4SS: A curated database for type IV secretion systems , 2012, BMC Microbiology.

[74]  S. Malhi,et al.  Blackleg disease of canola mitigated by resistant cultivars and four-year crop rotations in western Canada , 2013 .

[75]  S. Kamoun,et al.  How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells? , 2014, PLoS biology.

[76]  G. Jander,et al.  Myzus persicae (green peach aphid) salivary components induce defence responses in Arabidopsis thaliana. , 2009, Plant, cell & environment.

[77]  R. V. D. van der Hoorn,et al.  Enzyme-inhibitor interactions at the plant-pathogen interface. , 2008, Current opinion in plant biology.

[78]  Paul Horton,et al.  Nucleic Acids Research Advance Access published May 21, 2007 WoLF PSORT: protein localization predictor , 2007 .

[79]  B. Usadel,et al.  Reprogramming a maize plant: transcriptional and metabolic changes induced by the fungal biotroph Ustilago maydis. , 2008, The Plant journal : for cell and molecular biology.

[80]  H. Sonah,et al.  Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice , 2009, Molecular Breeding.

[81]  B. McDonald,et al.  The Cysteine Rich Necrotrophic Effector SnTox1 Produced by Stagonospora nodorum Triggers Susceptibility of Wheat Lines Harboring Snn1 , 2012, PLoS pathogens.

[82]  J. Nigou,et al.  Pathogen-Associated Molecular Patterns (PAMPs) , 2016 .

[83]  J. Batley,et al.  Molecular mapping of qualitative and quantitative loci for resistance to Leptosphaeria maculans causing blackleg disease in canola (Brassica napus L.) , 2012, Theoretical and Applied Genetics.

[84]  Jeff A. Bilmes,et al.  Transmembrane Topology and Signal Peptide Prediction Using Dynamic Bayesian Networks , 2008, PLoS Comput. Biol..

[85]  S. Brunak,et al.  SignalP 4.0: discriminating signal peptides from transmembrane regions , 2011, Nature Methods.

[86]  A. Elling,et al.  Nematode effector proteins: an emerging paradigm of parasitism. , 2013, The New phytologist.

[87]  David A. Jones,et al.  The genome sequence and effector complement of the flax rust pathogen Melampsora lini , 2014, Front. Plant Sci..

[88]  T. Kanneganti,et al.  Synergistic interactions of the plant cell death pathways induced by Phytophthora infestans Nepl-like protein PiNPP1.1 and INF1 elicitin. , 2006, Molecular plant-microbe interactions : MPMI.

[89]  Shiv D. Kale,et al.  External Lipid PI3P Mediates Entry of Eukaryotic Pathogen Effectors into Plant and Animal Host Cells , 2010, Cell.

[90]  J. McCutcheon,et al.  Horizontal Gene Transfer from Diverse Bacteria to an Insect Genome Enables a Tripartite Nested Mealybug Symbiosis , 2013, Cell.

[91]  E. Stukenbrock,et al.  Evidence for Extensive Recent Intron Transposition in Closely Related Fungi , 2011, Current Biology.

[92]  Masami Ikeda,et al.  ConPred II: a consensus prediction method for obtaining transmembrane topology models with high reliability , 2004, Nucleic Acids Res..

[93]  William Stafford Noble,et al.  FIMO: scanning for occurrences of a given motif , 2011, Bioinform..

[94]  M. Banfield,et al.  Structures of Phytophthora RXLR Effector Proteins , 2011, The Journal of Biological Chemistry.

[95]  S. Raffaele,et al.  Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi , 2012, PloS one.

[96]  Thomas Kroj,et al.  Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi , 2015, PLoS pathogens.

[97]  J. Berger,et al.  Structural Elucidation and Functional Characterization of the Hyaloperonospora arabidopsidis Effector Protein ATR13 , 2011, PLoS pathogens.

[98]  M. Thines,et al.  The fungal core effector Pep1 is conserved across smuts of dicots and monocots. , 2015, The New phytologist.

[99]  Dieter Jahn,et al.  PrediSi: prediction of signal peptides and their cleavage positions , 2004, Nucleic Acids Res..

[100]  Tao Lu,et al.  DFVF: database of fungal virulence factors , 2012, Database J. Biol. Databases Curation.

[101]  Burkhard Rost,et al.  The PredictProtein server , 2003, Nucleic Acids Res..

[102]  Ziding Zhang,et al.  Using Weakly Conserved Motifs Hidden in Secretion Signals to Identify Type-III Effectors from Bacterial Pathogen Genomes , 2013, PloS one.

[103]  K. Chou,et al.  Signal-3L: A 3-layer approach for predicting signal peptides. , 2007, Biochemical and biophysical research communications.

[104]  U. Güldener,et al.  Endophytic Life Strategies Decoded by Genome and Transcriptome Analyses of the Mutualistic Root Symbiont Piriformospora indica , 2011, PLoS pathogens.

[105]  Priyanka Pandey,et al.  Mycosec - A database for signal peptide bearing genes of mycobacterium , 2011 .

[106]  Li Cheng,et al.  DBSecSys: a database of Burkholderia mallei secretion systems , 2014, BMC Bioinformatics.

[107]  C. Hollier,et al.  Crop losses due to diseases and their implications for global food production losses and food security , 2012, Food Security.