Nuclear immune mechanisms associated with begomovirus infection

Abstract

Plants are constantly exposed to different biotic and abiotic stresses and, as sessile organisms, have evolved complex mechanisms to recognize and respond to adverse environmental conditions. Plant immunity involves different layers of recognition and signaling, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), which activate molecular and physiological responses that can limit infection. As obligate intracellular parasites, plant viruses face challenges because they depend on host machinery and host factors for replication, gene expression, and intracellular movement. Among plant viruses, begomoviruses, in the family Geminiviridae, possess single-stranded DNA genomes and depend on both viral and host proteins to complete their infection cycle. Following nuclear replication, viral DNA (vDNA) associates with the nuclear shuttle protein (NSP), which participates in the nucleocytoplasmic trafficking of vDNA in association with the NSP-interacting GTPase (NIG). The dependence of host factors involved in vDNA trafficking represents an important connection between viral infections and antiviral defense. In Arabidopsis thaliana, AtWWP1 contributes to antiviral defense by recruiting NIG into nuclear bodies and reducing its availability for vDNA movement. Although the formation of these nuclear bodies depends on the WW domains, here we showed that AtWWP1 is also associated with cyclin-dependent kinase 2 (CDKC2), which phosphorylates the C-terminal domain (CTD) of RNA polymerase II and is associated with nuclear speckles and spliceosome components, suggesting a possible connection between AtWWP1 nuclear bodies and transcriptional processes. However, the mechanisms that control the organization and composition of these nuclear bodies, and their contributions to antiviral defense, remain poorly understood. A methyl-CpG-binding domain, AtMBD2, was identified as an interactor of AtWWP1. AtMBD2 recognizes CpG-methylated regions and is associated with chromatin regulation, transcriptional repression, DNA demethylation, and gene expression, suggesting that its recruitment to AtWWP1 nuclear bodies may connect these structures with epigenetic and transcriptional mechanisms during infection. Here, we demonstrated that AtMBD2 interacts with AtWWP1 in vivo and is redirected from the nucleoplasm to AtWWP1-derived nuclear bodies. Domain-mapping analysis revealed that this interaction is independent of the AtWWP1 WW domain and instead requires its C-terminal region, spanning amino acids 273-463. Functional analysis further demonstrated that AtMBD2 modulates begomovirus infection, as its overexpression increases viral DNA accumulation, whereas genetic inactivation of AtMBD2 reduces viral load relative to the wild-type Col-0 line. Although these alterations were not fully correlated with disease symptoms, they were accompanied by differential expression of defense-related genes, indicating that AtMBD2 affects host responses during infection. Collectively, these results reveal a previously uncharacterized connection between an epigenetic regulator and AtWWP1-derived nuclear immune bodies and suggest that the AtMBD2-AtWWP1 interaction contributes to the regulation of begomovirus infection through mechanisms involving nuclear organization and defense-related gene expression. This study provides new insights into the molecular organization of nuclear antiviral defense and the complex interplay between viral infection, host nuclear processes, and epigenetic regulation. Keywords: AtWWP1; AtMBD2; nuclear bodies; antiviral defense