Abstract
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 Abbreviations
- AA –
- proteins with auxiliary activities
- ATMT –
- Agrobacterium tumefaciens mediated transformation
- CBM –
- proteins with carbohydrate-binding modules
- CE –
- carbohydrate esterases
- CSEPs –
- candidate secreted effector proteins
- CWDEs –
- cell wall-degrading enzymes
- DSI –
- disease severity index
- ETI –
- effector triggered immunity
- GH –
- glycoside hydrolases
- GT –
- glycosyltransferases
- KOG –
- EuKaryotic Orthologous Groups
- NLPs –
- necrosis and ethylene-inducing protein (NEP-1)-like proteins
- NLR –
- nucleotide-binding leucine rich repeat
- Nox –
- NADPH oxidase complex
- PAMPs –
- pathogen-associated molecular patterns
- PHI –
- Pathogen-host interaction database
- PL –
- polysaccharide lyases
- rAUDPC –
- relative area under the disease progress curve
- ROS –
- reactive oxygen species
- SSCPs –
- small secreted cysteine-rich proteins
- SSPs –
- small secreted proteins
- TM –
- transmembrane domain
- XSM –
- xylem simulating medium