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Rice stripe virus (RSV) is one of the most devastating viruses affecting rice production. During virus infection, ubiquitination plays an important role in the dynamic regulation of host defenses. We combined the ubiquitomics approach with the label-free quantitation proteomics approach to investigate potential ubiquitination status changes of Nicotiana benthamiana infected with RSV. Bioinformatics analyses were performed to elucidate potential associations between proteins with differentially ubiquitinated sites (DUSs) and various cellular components/pathways during virus infection. In total, 399 DUSs in 313 proteins were identified and quantified, among them 244 ubiquitinated lysine (Kub) sites in 186 proteins were up-regulated and 155 Kub sites in 127 proteins were down-regulated at 10 days after RSV infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that proteins with up-regulated Kub sites were significantly enriched in the ribosome. Silencing of 3-isopropylmalate dehydratase large subunit through virus-induced gene silencing delayed RSV infection, while silencing of mRNA-decapping enzyme-like protein promoted RSV symptom in the late stage of infection. Moreover, ubiquitination was observed in all seven RSV-encoded proteins. Our study supplied the comprehensive analysis of the ubiquitination changes in N. benthamiana after RSV infection, which is helpful for understanding RSV pathogenesis and RSV-host interactions. [ABSTRACT FROM AUTHOR]

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast. [ABSTRACT FROM AUTHOR]