The reliability of virus-induced gene silencing experiments using tobacco rattle virus in tomato is influenced by the size of the vector control

Abstract

The tobacco rattle virus (TRV)-based vector pYL156 is the most widely used vector for virus-induced gene silencing (VIGS) in solanaceous plants. We show here that vacuum agroinfiltration of the pYL156 empty vector causes extensive stem lesions, foliar necrosis, stunted growth and delayed flowering in tomato, whereas plants infiltrated with pYL156-based silencing constructs that carry inserts of ∼250 bp or more show few symptoms of virus infection. This difference in plant health influences plant susceptibility to insects, a trait measured in many VIGS experiments, and could potentially bias the results of other bioassays designed to assess the phenotypic consequences of VIGS. When plants were infiltrated with the pYL156:GUS construct, which carries a 396-bp fragment of the β-glucuronidase gene that has no significant homology with any endogenous gene in tomato, they showed similar growth and development as buffer-infiltrated controls, and exhibited significantly fewer viral symptoms than the empty vector. Furthermore, pYL156:GUS did not influence plant susceptibility to aphids. Reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the GUS insert may cause a modest delay in virus movement within the plant, but does not limit TRV replication in infected tissue. Therefore, the pYL156:GUS construct represents an improved negative control for TRV-induced gene silencing.