PT  - JOURNAL ARTICLE
AU  - Kristina Marton
AU  - Marko Flajšman
AU  - Sebastjan Radišek
AU  - Katarina Košmelj
AU  - Jernej Jakše
AU  - Branka Javornik
AU  - Sabina Berne
TI  - Comprehensive analysis of <em>Verticillium nonalfalfae in silico</em> secretome uncovers putative effector proteins expressed during hop invasion
AID  - 10.1101/237255
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 237255
4099  - http://biorxiv.org/content/early/2017/12/21/237255.short
4100  - http://biorxiv.org/content/early/2017/12/21/237255.full
AB  - Background The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop.Results The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed.Conclusions Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.List of AbbreviationsAA –proteins with auxiliary activitiesATMT –Agrobacterium tumefaciens mediated transformationCBM –proteins with carbohydrate-binding modulesCE –carbohydrate esterasesCSEPs –candidate secreted effector proteinsCWDEs –cell wall-degrading enzymesDSI –disease severity indexETI –effector triggered immunityGH –glycoside hydrolasesGT –glycosyltransferasesKOG –EuKaryotic Orthologous GroupsNLPs –necrosis and ethylene-inducing protein (NEP-1)-like proteinsNLR –nucleotide-binding leucine rich repeatNox –NADPH oxidase complexPAMPs –pathogen-associated molecular patternsPHI –Pathogen-host interaction databasePL –polysaccharide lyasesrAUDPC –relative area under the disease progress curveROS –reactive oxygen speciesSSCPs –small secreted cysteine-rich proteinsSSPs –small secreted proteinsTM –transmembrane domainXSM –xylem simulating medium