Characterization of a novel broad-spectrum antifungal protein from virus-infected Helminthosporium (Cochliobolus) victoriae.

A broad-spectrum anti-fungal protein of approximately 10 kDa, designated victoriocin, was purified from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium victoriae (teleomorph: Cochliobolus victoriae) by a multistep procedure involving ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences, obtained by automated Edman degradation sequencing of RP-HPLC-purified victoriocin-derived peptides, were used to design primers for degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification from H. victoriae DNA and cDNA templates. An open reading frame coding for a victoriocin precursor of 183 amino acids with calculated molecular mass of approximately 20 kDa was amplified by PCR from H. victoriae genomic DNA but not from the control fungus Penicillium chrysogenum. Southern hybridization analysis confirmed the presence of the victoriocin gene in all H. victoriae strains tested. Sequence analysis indicated that victoriocin has a sequence motif similar to that found in scorpion short toxin/charybdotoxin and a consensus sequence similar to that found in defensins. Victoriocin, like some other antifungal proteins, including the totivirus-encoded killer proteins, is predicted to be expressed in vivo as a preprotoxin precursor consisting of a hydrophobic N-terminal secretion signal followed by a pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N terminus of the mature victoriocin. A putative cell wall protein of approximately 30 kDa (P30) co-purified with victoriocin from cultural filtrates. The potential role of P30 in the antifungal activity of H. victoriae culture filtrates is discussed.

Overexpression of the victoriocin gene in Helminthosporium (Cochliobolus) victoriae enhances the antifungal activity of culture filtrates.

We have previously reported the isolation and characterization of the broad-spectrum antifungal protein, victoriocin, from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium (teleomorph: Cochliobolus) victoriae. We predicted that the 10-kDa mature victoriocin is derived in vivo from a preprotoxin precursor that is processed by a signal peptidase and kexin-like endopeptidase. We also presented evidence that the victoriocin precursor is encoded by a host gene, designated the victoriocin (vin) gene. In the present study, an H. victoriae genomic DNA library was constructed in the cosmid vector pMLF-2, and a cosmid clone carrying the vin gene and flanking sequences was isolated and used to generate constructs for transformation of virus-free and virus-infected H. victoriae isolates with the vin gene. Culture filtrates of the virus-free vin transformants exhibited high levels of antifungal activity compared with that revealed by the nontransformed virus-free wild-type strain, which exhibited little or no antifungal activity. Moreover, transformation of the wild-type virus-infected H. victoriae strain with the vin gene resulted in still higher production of victoriocin and higher antifungal activity in the culture filtrates of the vin transformants compared with the virus-infected wild-type strain. As previously predicted, the presence in the vin transformants of the preprovictoriocin and its post-translationally generated products, the provictoriocin and the mature victoriocin, was clearly demonstrated. Processing of the victoriocin preprotoxin requires eukaryotic host factors because no processing occurred in an in vitro translation system or in bacteria. It is of interest that some of the virus-free isolates transformed with the vin gene exhibited some features of the virus-induced disease phenotype, including moderate stunting and sectoring. Present data suggests that victoriocin may play an indirect role in disease development. Taken together, these results indicate that victoriocin is the primary protein responsible for the antifungal activity in culture filtrates of virus-infected H. victoriae isolates and that virus infection upregulates the expression of victoriocin. Overproduction of victoriocin may give the slower-growing virus-infected fungal strains some competitive advantage by inhibiting the growth of other fungi.