Chili pepper (Capsicum annuum L.) is a very important vegetable crop, commonly used as a spice or seasoning in various dishes. With the growth of the global population, the demand for chili peppers has also increased exponentially. Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that has spread to dozens of countries worldwide. Its infection in chili peppers can severely impact yield and quality, posing a significant threat to the chili pepper industry. However, the transcriptional response of chili peppers to ToBRFV infection has not been studied yet. This research utilized RNA-Seq technology to analyze the transcriptional profiles of chili pepper leaves (‘Haonong 11’) 13 days post-infection with ToBRFV or mock treatment, identifying a total of 1468 differentially expressed genes (DEGs), of which 1366 were upregulated and 102 were downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the DEGs were involved in biological processes such as defense response, response to reactive oxygen species, protein folding, and plant-pathogen interaction. Twelve DEGs were validated by RT-qPCR, with their expression trends consistent with the transcriptome data, indicating the reliability of the high-throughput data. Our systematic analysis provides a molecular basis for the response of chili pepper leaves to ToBRFV infection at the transcriptomic level and offers potential candidate genes for further research into the interaction mechanisms between ToBRFV and plant hosts.

Tomato brown rugose fruit virus (ToBRFV) is an important emerging virus that poses a serious threat to the global agricultural economy. Ubiquitination is one of the key post-translational protein modification types in plant responses to biotic stress, but the extent to which ToBRFV infection alters the overall ubiquitination status has not been reported. This study conducted integrated ubiquitome and proteome analyses of Nicotiana benthamiana leaves infected with ToBRFV and identified differentially ubiquitinated proteins. A total of 346 lysine sites on 302 identified proteins were found to be affected, with 260 sites exhibiting upregulated ubiquitination levels in 224 proteins and 86 sites showing downregulated ubiquitination levels in 80 proteins. The differentially ubiquitinated proteins were primarily localized in the cytoplasm (29%), nucleus (18%), plasma membrane (8.9%), mitochondria (5.1%), and chloroplasts (4.6%). Fourteen conserved ubiquitination motifs, including ENNNK, ENNK, SK, and KNG, were identified. Furthermore, enrichment analysis indicated that ToBRFV infection induces an increase in the ubiquitination levels of proteins associated with ion transport, MAPK signaling pathways, and plant hormone signal transduction, while the ubiquitination levels of proteins related to carbon metabolism and secondary metabolite synthesis decreased. Functional analysis of the three differentially ubiquitinated proteins revealed that a RING/U-box superfamily protein negatively regulates ToBRFV infection. Our work provides the first systematic analysis of the ubiquitination profile in N. benthamiana leaves following ToBRFV infection, providing important resources for further studies on the regulatory mechanisms of ubiquitination in plant responses to ToBRFV.