A recombinant tobacco mosaic virus-based vector for functional genomic studies in plant pathogenic fungi

Plant viruses have proven to be very versatile tools for biotechnology. They have been used as vectors for protein expression in plants and to trigger gene-silencing mechanisms using recombinant virus vectors carrying fragments of the target gene. The latter process, known as virus-induced gene silencing (VIGS), can be used for functional genomic studies. In this work, we used a plant virus-based vector, Tobacco mosaic virus (TMV), carrying the green fluorescent protein (GFP) gene as a reporter for the infection of taxonomically different phytopathogenic fungi, namely Botrytis cinerea, Fusarium oxysporum f. sp. lycopersici, Monilinia fructicola, and Verticillum dahliae. Confocal microscope observations and molecular analysis at 6, 9, and 13 days post-inoculation (dpi) showed that TMV can replicate and persist in V. dahliae and B. cinerea, whereas it is unable to enter and replicate in F. oxysporum f. sp. lycopersici and M. fructicola. Total RNA preparations extracted at 6, 9 and 13 dpi from infected V. dahliae and B. cinerea mycelia were used to estimate the accumulation of viral RNA and the expression of two key genes involved in the RNA silencing pathway, namely Dicer Like 1 and Argonaute 1, by RT-qPCR. Results indicate that the TMV vector could be a functional VIGS approach to silence constitutive pathogenesis-related genes in V. dahliae and B. cinerea.