# Comprehensive Ubiquitome Analysis of Nicotiana benthamiana Leaves Infected with Tomato Brown Rugose Fruit Virus

## TL;DR

This study explores how Tomato Brown Rugose Fruit Virus affects protein ubiquitination in Nicotiana benthamiana leaves, revealing key changes in host proteins involved in stress responses and metabolism.

## Contribution

The first comprehensive ubiquitome analysis of ToBRFV-infected plants, identifying novel ubiquitination patterns and their functional implications.

## Key findings

- ToBRFV infection alters ubiquitination levels of 302 proteins, with 224 showing increased and 80 decreased ubiquitination.
- Differentially ubiquitinated proteins are mainly localized in the cytoplasm, nucleus, and plasma membrane.
- Functional analysis shows ubiquitination changes in proteins related to ion transport, signaling, and metabolism.

## Abstract

Ubiquitination is a crucial post-translational modification involving the covalent attachment of ubiquitin (a small 76-amino-acid protein) to the lysine residues of target proteins, which is catalyzed by ubiquitin ligases. This modification has been shown to regulate nearly all aspects of plant biology, including growth, development, and responses to both abiotic and biotic stresses. Tomato brown rugose fruit virus (ToBRFV), a newly emerging tobamovirus, has now been detected in over fifty countries worldwide. However, the global impact of ToBRFV infection on host protein ubiquitination profiles remains largely unexplored. In this study, we performed an integrated ubiquitinomics and proteomics analysis to identify differentially ubiquitinated proteins in Nicotiana benthamiana upon ToBRFV infection. Our findings provide a valuable foundation for further characterization of plant genes functionally involved in ToBRFV–host interactions.

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.