Silencing Defense Pathways in Arabidopsis by Heterologous Gene Sequences from Brassica oleracea Enhances the Performance of a Specialist and a Generalist Herbivorous Insect

Abstract

The jasmonic acid (JA) signaling pathway and defensive secondary metabolites such as glucosinolates are generally considered to play central roles in the defense of brassicaceous plants against herbivorous insects. To determine the function of specific plant genes in plant-insect interactions, signaling or biosynthetic mutants are needed. However, mutants are not yet available for brassicaceous plants other than Arabidopsis thaliana, e.g., cabbage (Brassica oleracea). We employed virus-induced gene silencing (VIGS) by using tobacco rattle virus (TRV) to knock down the endogenous expression of lipoxygenase (LOX), an upstream enzyme of the JA pathway and thioglucoside glucohydrolase: myrosinase (TGG1/TGG2), a hydrolytic enzyme that catalyzes the release of defensive volatile products originating from glucosinolates, in Arabidopsis thaliana. This was done by using the heterologous gene sequences from B. oleracea. Silencing these genes in A. thaliana plants is efficient and specific. Only 18 nucleotides with 100% identity between the trigger (BoMYR) and the target (AtTGG1/2) sequence are sufficient to achieve gene silencing. LOX-silenced plants showed significantly reduced AtLOX2 transcript accumulation after Pieris rapae larval feeding. TGG-silenced plants exhibited significantly lower TGG1/TGG2 transcript levels only after shorter larval feeding. The inhibition of TGG1/TGG2 transcript accumulation via gene silencing may be overruled by longer larval feeding. Specialist P. rapae larvae developed significantly better on both types of silenced plants than on empty vector (EV) control plants, while generalist Mamestra brassicae larvae developed significantly better on the TGG1/TGG2 silenced plants than on EV control plants. This shows that not only the generalist herbivore but also the Brassicaceae-specialist P. rapae is negatively affected by the ability of brassicaceous plants to produce their specific secondary metabolites, i.e., glucosinolates. Our results demonstrate the important roles of AtLOX2 and AtTGG1/TGG2 genes, which were silenced by heterologous gene sequences from B. oleracea BoLOX and BoMYR, in A. thaliana resistance to the specialist P. rapae and the generalist M. brassicae.