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.

Cross-protection is a promising measure to control plant viral diseases. Reverse genetics had been recently adopted to generate attenuated mutants that have potential in crossprotection. But studies on the variability of the progeny viruses of the attenuated mutants are scarce. Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is the prevalent virus inducing maize dwarf mosaic disease in China. Here, we showed that the substitution of arginine with isoleucine in the FRNK motif at position 184 of helper component-proteinase (HC-Pro) abolished its RNA silencing suppression (RSS) activity, drastically reduced the virulence and accumulation level of SCMV, and impaired the synergism between SCMV and maize chlorotic mottle virus. The attenuated mutant could protect maize plants from a severe infection of SCMV. However, a spontaneous mutation of glycine at position 440 to arginine in HC-Pro rescued the virulence and synergism with maize chlorotic mottle virus of SCMV and the RSS activity of HC-Pro. Similar results were obtained with tobacco vein banding mosaic virus and watermelon mosaic virus. These results provide novel evidence for the complementary mutation of potyviruses in maintaining the HC-Pro RSS activity and potyviral virulence and remind us of evaluating the potential risk of attenuated mutants thoroughly before applying for the control of plant viral diseases via cross-protection.

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.

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