Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

Abstract

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.transfection | plant pathogen adaptation | host species jump

Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens

“…Multiple studies in the 1970s suggested that tobacco mosaic virus (TMV) could replicate in and debilitate oomycetes in the genus Pythium (90)(91)(92), which provided early support to the infection of fungus-like organisms by tobamoviruses. More recently, Mascia et al (93) infected Colletotrichum acutatum with a TMV-based vector for virus-induced gene silencing using Agrobacterium-mediated transformation. In addition, tobamo-like viruses were observed naturally infecting cultures of Agaricus bisporus (Basidiomycota) and Plicaria spp.…”

Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species

“…At ten days after TRV treatment, plants were challenged with either the V. dahliae race 1 strain JR2 , or an Ave1 deletion mutant (V. dahliae JR2△Ave1;, and inspected for Verticillium wilt symptoms (stunting and wilting) up to 14 days post inoculation (dpi). As expected, no significant disease symptoms were observed on TRV::GUStreated plants that were inoculated with the wild-type race 1 V. dahliae strain (Song et al, 2016 It was recently demonstrated that Tobacco mosaic virus (TMV) may infect fungi in addition to plants, remaining for up to six subcultures, and also persisted in plants infected by the virus-infected fungus (Mascia et al, 2014 Figure S1A). Furthermore, the fungal isolates were used to infect Ve1 tomato plants and tomato plants that lack…”

Something new to explore

“…Recently, however, it was shown that TMV could indeed infect and replicate in fungi (Fig. 1), specifically three species of Colletotrichum ( C. acutatum , C. clavatum , and C. theobromicola ) 21 . Infection by TMV did not alter the growth rate, morphology or pathogenicity of C. acutatum , while virus re-isolated from C. acutatum was able to infect plants.…”

“… 21 The fluorescence was maintained for six passages, but was lost in most monoconidial cultures on the seventh passage. Furthermore, infection of C. acutatum expressing a transgenic GFP gene by TMV-GFP resulted in the silencing of both the GFP transgene and the virally-expressed GFP gene, which again persisted for six passages, with the transgene fluorescence regained in a few of the monoconidial cultures at the seventh passage 21 . TMV-GFP infectious to plants was not recoverable from the fungus at the seventh passage.…”

“…Thus, TMV and several other plant viruses, such as potato virus X and tomato bushy stunt virus, are referred to as being highly contact transmissible plant viruses, vs. those plant viruses that can be transmitted by rubbing high concentrations of inoculum onto leaves, but not by leaves contacting against each 23 . Infection of fungi by TMV was shown to be an efficient process, approaching nearly 100% 21 . Hence, it is likely that when hyphae grow in/through a medium, some cell membrane damage occurs and the virus enters the cell (Fig.…”