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Tomato leaf curl disease (ToLCD), caused by strains of Tomato leaf curl virus (ToLCV), is a major constraint to tomato production. To understand the molecular mechanism of virus tolerance, we studied the abundance of viral genomic replicative intermediate molecules and virus-derived siRNAs generated by host plant in naturally tolerant (H-88-78-1) and susceptible (Punjab Chhuhara) cultivars at different days-post-infection. We showed that both viral DNA methylation and siRNA-mediated degradation play an important role in conferring tolerance against ToLCNDV. Further, we functionally characterized a (i) 26S proteasomal subunit RPT4a (SlRPT4) gene and (ii) U-box type E3 ligase (SlARM18) gene, which were differentially expressed after ToLCNDV infection in tolerant cultivar. (i) The study showed that SlRPT4 protein binds to promoter region of ToLCNDV genome thereby hindering the expression of virus genes which subsequently reduces viral replication and infection in tolerant cultivar. Transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. (ii) An increase in viral load upon silencing SlARM18 in cv. H-88-78-1 was observed validating its role in tolerance. Upon virus infection, WRKY41 was found to regulate the expression of SlARM18 by binding onto the W-box elements present within the promoter of SlARM18. Further, downstream, SlARM18 interacts with viral AC4 protein and leads to its ubiquitinylation. Overall, present study highlights non-proteolytic function of SlRPT4 as well as SlARM18-mediated ubiquitination of viral AC4 protein and their participation in defense-pathway against virus infection in tomato |