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Chloroplast-bound vesicles are key components in viral replication complexes (VRCs) of potyviruses. The potyviral VRCs are induced by the second 6 kDa protein (6K2) and contain at least viral RNA and nuclear inclusion protein b. To date, no chloroplast protein has been identified to interact with 6K2 and involve in potyvirus replication. In this paper, we showed that the Photosystem II oxygen evolution complex protein of Nicotiana benthamiana (NbPsbO1) was a chloroplast protein interacting with 6K2 of Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) and present in the VRCs. The first 6 kDa protein (6K1) was recruited to VRCs by 6K2 but had no interaction with NbPSbO1. Knockdown of NbPsbO1 gene expression in N. benthamiana plants through virus-induced gene silencing significantly decreased the accumulation levels of TVBMV and another potyvirus Potato virus Y, but not Potato virus X of genus Potexvirus. Amino acid substitutions in 6K2 that disrupted its interaction with NbPsbO1 also affected the replication of TVBMV. NbPsbP1 and NbPsbQ1, two other components of the Photosystem II oxygen evolution complex had no interaction with 6K2 and no effect on TVBMV replication. To conclude, 6K2 recruits 6K1 to VRCs and hijacks chloroplast protein NbPsbO1 to regulate potyvirus replication.

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Tobacco vein banding mosaic virus (TVBMV) is a potyvirus which mainly infects solanaceous crops. The helper component proteinase (HCpro) of a potyvirus is an RNA silencing suppressor protein and determines the severity of disease symptoms caused by different potyviruses, including TVBMV. It has been shown that substitution mutations introduced into the HCpro open reading frame (ORF) in a TVBMV infectious clone result in changes of Asp to Lys or Ile -Gln to Asp-Glu (Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid; Ile, isoleucine). These amino acid changes eliminate the RNA silencing suppression activity of the mutant HCpro (HCm) and attenuate the disease symptoms caused by the mutant TVBMV (T-HCm) in Nicotiana benthamiana plants. Here, we used RNA-sequencing technology to compare gene expression in plants inoculated with the wild-type TVBMV (T-WT) with that in plants inoculated with T-HCm at 1, 2 and 10 days post-agroinfiltration (dpai). At 1 and 2 dpai, N. benthamiana genes related to the translation machinery were up-regulated, whereas genes related to lipid biosynthesis and metabolism or to responses to extracellular/external stimuli were down-regulated in leaves inoculated with T-WT or T-HCm. At 10 dpai, T-WT infection repressed photosynthesis-related genes. T-WT and T-HCm infections differentially perturbed the genes involved in the RNA silencing pathway. The salicylic acid and ethylene signalling pathways were induced, but the jasmonic acid signalling pathway was repressed after T-WT infection. Infections of T-WT and T-HCm also differentially regulated the genes involved in auxin signalling transduction, which is known to associate with the stunting phenotypes caused by TVBMV. These results illustrate the dynamic nature of TVBMV infection in N. benthamiana at the transcriptomic level.