Functional investigation of the role of BYVMV (begomovirus) proteins in epigenetic modulation.

Bhendi, or okra (Abelmoschus esculentus, family Malvaceae), has numerous nutritional benefits. In India, which is its largest producer, the prevalence of begomoviruses causes enation leaf curl (ELCu), yellow vein mosaic (YVM) disease, or a combination of both, resulting in huge losses in cultivation. Previously, we have shown that BYVMV C2, C4, and βC1 act as suppressors of PTGS and TGS, with pronounced TGS inhibition arising from the former two proteins. The current research intends to uncover the mechanism behind the TGS hindrance induced by C4 and C2. Initially, to screen for host interactions with C2 or C4, the viral proteins were fused with His-tag, followed by agroinfiltration into N. benthamiana leaves in various combinations, including three negative controls. The extracted proteins were subjected to pull-down. We subsequently performed LC-MS and a meticulous data analysis which revealed a preliminary interacting partner for C2 or C4. Intriguingly, C4 was found to interact, either directly or indirectly, with potential TGS suppressors such as S-adenosyl homocysteine hydrolase (SAHH), S-adenosyl methionine synthetase (SAMS), cystathionine beta synthetase domain containing protein (CBS), nuclear importin (NbNUP50a), heterochromatin formation (NbFDM4), and chromatin remodeling (NbBRM). Initially, we selected the first three candidates and performed bioinformatics analysis by modeling the host proteins to look for their interaction potential. By confirming interactions in silico, we cloned SAHH, SAMS, and CBS from N. benthamiana and fused them with the Streptavidin Tag, which aided in confirming the interaction through pull-down and Co-IP. Virus-induced gene silencing (VIGS) was used to silence SAMS and CBS. Their impact on viral DNA accumulation and phenotypic differences will be discussed during the presentation.