Lipid transfer protein VAS inhibits the hypersensitive response via reactive oxygen species signaling in Nicotiana benthamiana.

Lipid transfer proteins (LTPs) are small cysteine-rich soluble proteins that affect flower and seed development, cuticular wax deposition, and biotic and abiotic stress responses. We isolated an LTP-encoding gene homologous to LTPVAS in Nicotiana benthamiana and designated it LTP-VASCULAR TISSUE SIZE (NbLTPVAS). This gene was expressed in seeds, leaves, roots, and stems. Additionally, NbLTPVAS expression was induced by hypersensitive response (HR)-inducing agents. Cell death was accelerated and the phytopathogenic bacterial population decreased significantly in NbLTPVAS-silenced plants infected with the incompatible Ralstonia solanacearum strain 8107. The expression of HR marker gene hin1 in NbLTPVAS-silenced plants was markedly induced by R. solanacearum 8107, indicative of the acceleration of HR. HR cell death in NbLTPVAS-silenced plants was also promoted by the Agrobacterium-mediated expression of HR-inducing proteins including INF1, AvrA, and PopP1. Excessive production of reactive oxygen species (ROS) was detected in NbLTPVAS-silenced plants. The expression of NbrbohB (encoding a ROS-generating enzyme) also increased in NbLTPVAS-silenced plants, but the expression of the antioxidant enzyme-encoding genes NbSOD and NbAPX decreased. The silencing of both NbLTPVAS and NbrbohB adversely affected HR induction. Moreover, NbLTPVAS was secreted into the intercellular washing fluid. The transient expression of the full-length NbLTPVAS induced the expression of antioxidant genes, attenuated ROS production, and suppressed the induction of HR cell death. This is the first functional analysis of LTPVAS in plant-microbe interactions. Our study provides novel insights into the role of NbLTPVAS as a negative regulator of HR via ROS homeostasis in N. benthamiana.