An exploration of the function of specific components of the predicted secretome of Fusarium graminearum during wheat infection

Abstract

Fusarium graminearum is a major fungal pathogen of wheat and other small grain cereal crops globally, causing Fusarium ear blight (FEB) disease. Like many other plant pathogens, F. graminearum is predicted to produce in planta secreted effector proteins that modulate plant metabolism to suppress or re-programme plant defences. Understanding the molecular functions of Fg effectors will help to elucidate the processes underlying wheat spike colonisation and fungal pathogenicity. With the aim of identifying Fg effector proteins that can suppress host plant defences, I selected using next generation sequencing and bioinformatic analysis, a set of small secreted proteins (SSP) to express in planta using the Barley stripe mosaic virus over-expression system (BSMV-VOX). I then tested whether expression of any of these SSPs enhanced Fg fungal infection of susceptible wheat spikes. Amongst the set of Fg SSP tested, FgSSP8, which encodes a ribonuclease protein, induced strong symptoms of necrosis in N. benthamiana leaves when infiltrated via the BSMV:FgSSP8. Three other genes tested (FgSSP7, FgSSP6 and FgSSP5) enhance FEB disease formation in the majority of the experiments when overexpressed in wheat ears prior to infecting with F. graminearum. FgSSP6 and FgSSP7 belong to the cerato-platanin protein (CPP) family. In several other plant pathogenic fungi, CPPs have been implicated in a number of virulence and plant protection mechanisms, including induction of host plant cell death, binding specific polymers and/or expansin-like activity. FgSSP5 encodes a protein that possesses the pfam domain RALF (Rapid alkalinization factor; PF05498.6). RALF domain-containing proteins are predominately found in plants and play a role in plant development regulating tissue expansion and/or negatively regulating pollen tube elongation. BLAST analyses identified RALF domain containing proteins in a restricted range of different pathogen species. Based on the VOX results and biochemical tests, our hypothesis is that pre-elevated cerato-platanins (FgSSP6 and FgSSP7) levels in the apoplast/surrounding the hyphae could initially shield the hyphae from detection by the plant, but late induce an intense defence response culminating in cell death to benefit the necrotrophic phase of Fg by increasing nutrient availability. FgSSP5 may be a specific virulence factor that manipulates a key plant process, by alkalinising the plant environment during infection, and using the same plant receptor repertoire used to recognise plant proteins. Once the mechanisms are further understood, these genes/proteins could potentially be novel intervention targets either for conventional chemistries and/or for methods such as host-induced gene silencing to achieve FEB disease and/or mycotoxin control. The characterisation of single and double gene deletion F. graminearum mutants is in progress.