Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway

Center for Biosystems Research, University of Maryland Biotechnology Institute, Shady Grove Campus, Rockville, MD 20850

^* To whom correspondence should be addressed. Email: nuss{at}umbi.umd.edu.

	Abstract

Disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus, Cryphonectria parasitica, was recently shown to increase susceptibility to mycovirus infection [Segers G, Zhang X, Deng F, Sun Q Nuss D (2007) Proc. Natl. Acad. Sci. USA 104:12902-12906]. We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild type and dicer gene dcl-1 mutant C. parasitica strains, but not in hypovirus infected dcl-2 and double dcl-1/dcl-2 mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands with a ratio of 3:2 and in a non-random distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12 to 20 fold) in dcl-2 expression while expression of dcl-1 is only modestly increased (2 fold). Expression of dcl-2 is further increased (35 fold) following infection by a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.