FcRav2, a gene with a ROGDI domain involved in Fusarium head blight and crown rot on durum wheat caused by Fusarium culmorum

Abstract

Fusarium culmorum is a soil-borne fungal pathogen which causes foot and root rot and Fusarium head blight on small-grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in the contamination of kernels with type B trichothecene mycotoxins. Our knowledge of the pathogenicity factors of this fungus is still limited. A transposon tagging approach based on the mimp1/impala double-component system has allowed us to select a mutant altered in multiple metabolic and morphological processes, trichothecene production and virulence. The flanking regions of mimp1 were used to seek homologies in the F. culmorum genome, and revealed that mimp1 had reinserted within the last exon of a gene encoding a hypothetical protein of 318 amino acids which contains a ROGDI-like leucine zipper domain, supposedly playing a protein-protein interaction or regulatory role. By functional complementation and bioinformatic analysis, we characterized the gene as the yeast Rav2 homologue, confirming the high level of divergence in multicellular fungi. Deletion of FcRav2 or its orthologous gene in F. graminearum highlighted its ability to influence a number of functions, including virulence, trichothecene type B biosynthesis, resistance to azoles and resistance to osmotic and oxidative stress. Our results indicate that the FcRav2 protein (and possibly the RAVE complex as a whole) may become a suitable target for new antifungal drug development or the plant-mediated resistance response in filamentous fungi of agricultural interest.

Keywords: Fusarium graminearum; Fusarium head blight; fungal pathogens; fungicide; molecular target; transposon tagging; virulence genes.

Figure 1

Southern blot analysis of Fusarium culmorum (A) and F. graminearum (B) transformants. Top: EcoRV‐digested DNAs were blotted and hybridized with a gene upstream probe obtained with primers 1F/2R (expected size for wild‐type and ectopic transformant, 3800 bp; expected size for deletion mutants, 4068 bp). Bottom: BglIII‐digested DNAs were blotted and hybridized with an internal FcRav2 gene upstream probe obtained with primers NF/NR (expected size for wild‐type and ectopic transformant, 2409 bp).

Figure 2

Comparison of Fusarium culmorum ΔFcRav2 mutant phenotype and in vitro effect of the vacuolar type H(+)‐ATPase inhibitor bafilomycin A1. (A) Fusarium culmorum wild‐type strain UK99 colony border after 48 h of growth on Czapek dox agar. (B) UK99 grown on Czapek dox agar containing 11 μmol of bafilomycin A1. (C) Fusarium culmorum mutant ΔFcRav2 B51 grown on Czapek dox agar. Microscopic detail (40×) shows hyperbranching and thickening of the hyphal tips in the ΔFcRav2 mutant and in the wild‐type exposed to bafilomycin A1.

Figure 3

Phenetic heat map of carbon utilization patterns of Fusarium culmorum deletion mutant ΔFcRav2 B24 relative to its wild‐type strain UK99 and to ectopic transformant strain FcB6. Average OD₇₅₀ (optical density at 750 nm) readings recorded during the 60–72‐h interval on the 30 most differentiating carbon sources are listed in order of decreasing proportional growth difference between ΔFcRav2 B24 and UK99.

Figure 4

Expression patterns of different Affymetrix array experiments carried out on Fusarium graminearum in different environmental conditions obtained from the plexdb.org platform. The results are the average of three repetitions, with bars indicating standard error. CMC, carboxy‐methyl cellulose liquid medium; DON, deoxynivalenol.