Virus-Induced Silencing of a Sequence Coding for Loricrin-like Protein in Phytophthora infestans upon Infection of a Recombinant Vector Based on Tobacco Mosaic Virus

Abstract

Phytophthora infestans is the oomycete responsible for late blight disease of Solanaceae that causes both yield and economic losses. With the aim of reducing plant wilt and high management costs mainly due to wide fungicide applications, alternative eco-sustainable control strategies are needed. RNA interference (RNAi) is a powerful tool for gene function studies that can be accomplished by constitutive transformation or transient expression such as virus-induced gene silencing (VIGS) experiments. VIGS makes use of viruses to deliver sequences homologous to a target gene fragment and trigger RNAi. Indeed, a P. infestans ortholog of plant loricrin-like protein (LLP), named PiLLP, has been silenced using the direct infection of a recombinant vector based on the plant virus tobacco mosaic virus (TMV-PiLLP-1056), aiming to reduce the oomycete sexual reproduction. For this purpose, the gene coding for the green fluorescent protein (GFP) present in the TMV-GFP-1056 vector has been replaced with an antisense construct obtained by fusion PCR of the PiLLP 5′-UTR and 3′-UTR sequences. Here, we show that RNAi can be expressed in the A1 mating type of P. infestans strain 96.9.5.1 by VIGS using the direct infection of TMV-PiLLP-1056. We provide evidence that the recombinant vector can enter, replicate, and persist in mycelia of P. infestans where it induces the partial downregulation of the PiLLP transcript. Compared with the wild-type, the PiLLP-silenced A1 mating type had slower colony growth and a diminished virulence in detached tomato leaflets. This seems to be the first evidence of a constitutive gene downregulation of P. infestans using a recombinant vector based on a plus-sense RNA plant virus.