A novel alcohol oxidase/RNA-binding protein with affinity for mycovirus double-stranded RNA from the filamentous fungus Helminthosporium (Cochliobolus) victoriae - Molecular and functional characterization

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the fila mentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies wi th mycoviral double-stranded RNAs, Sequence analysis revealed that Hv-p68 b elongs to the large family of FAD-dependent glucose methanol choline oxidor eductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcoho l oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gen e was found to contain four introns, Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was no t induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared w ith virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-bind ing beta-alpha-beta fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the fila mentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophi c yeasts examined. This study represents the first reported case that a mem ber of the FAD-dependent glucose methanol choline oxidoreductase family, Hv -p68, may function as an RNA-binding protein.