MicroRNA-based gene silencing is a functional genomics tool for a wide range of eukaryotes. As a basis for broader application of virus-induced gene silencing (VIGS) to photosynthesis research, we employed a tobacco rattle virus (TRV) vector to silence expression of the nuclear psbS gene in Nicotiana benthamiana. The 22-kiloDalton psbS protein is essential for xanthophyll- and H⁺-dependent thermal dissipation of excitation in higher plants widely known as nonphotochemical quenching (NPQ). Controls treated with the TRV-VIGS vector containing a bacterial chloramphenicol resistance gene as the silencing target were included to test for non-silencing effects of the viral vector system. PsbS protein was undetectable and both psbS mRNA transcript levels and NPQ capacity were dramatically reduced in new leaf tissue of VIGS-psbS plants only. Photosynthetic performance in TRV-VIGS-treated and uninfiltrated plants was assessed by application of CO₂ exchange, chlorophyll fluorescence, and in vivo absorbance changes at 810 nm. TRV-VIGS caused a mild stress based on pigment content and light absorption characteristics in some cases. To assess transient complementation of NPQ, the endogenous psbS gene was silenced using only the transit sequence in the TRV vector followed by Agrobacterium-mediated transient expression of a modified gene consisting of an altered transit sequence fused to the native mature protein sequence. Nevertheless, NPQ in infused fully expanded leaves that expressed this re-introduced form was not fully restored indicating the possible importance of psbS incorporation prior to formation of grana stacks.