Mechanism For Suppression Of Post-transcriptional Gene Silencing By A Calmodulin-related Protein Nbrgs-CaM In Nicotiana Benthamiana

Post transcriptional gene silencing (PTGS) is an important plant immune defense mechanism against viral infection. However, most plant viruses have evolved viral suppressors of RNA silencing (VSRs) to counteract the host’s antiviral silencing activity. Tomato yellow leaf curl China betasatellite (TYLCCNB) is a DNA molecule which is associated with Tomato yellow leaf curl China virus (TYLCCNV). The TYLCCNB contains a single open reading frame (βC1) that plays important role in symptom induction. The βC1 protein is a PTGS suppressor which can increase Nbrgs-CaM mRNA levels to inhibit the expression of NbRDR6. Nbrgs-CaM has been previously identified as an endogenous regulator of gene silencing, which is also required for efficient virus infection. However, the precise mechanism of rgs-CaM-mediated PTGS suppression is yet to be understood.In this study, we focus on the relationship between Nbrgs-CaM and NbSGS3. To determine the key region of NbSGS3 involved in its intracellular localization, the three recombinant vectors pCHF3-NbSGS3-dZF-GFP, pCHF3-NbSGS3-dXS-GFP, pCHF3-NbSGS3-d2CC-GFP, in which ZF, XS or 2CC domain was deleted, respectively, were constructed. Our results showed that the ZF and 2CC domains of NbSGS3 were important for its intracellular localization. To investigate whether the ZF and 2CC domains were also important for the interaction with Nbrgs-CaM, we constructed the BiFC expression vectors 2YN/2YC-NbSGS3-dZF,2YN/2YC-NbSGS3-dXS, 2YN/2YC-NbSGS3-d2CC. The results from confocal imagings showed that the ZF and 2CC domains of NbSGS3 were equally important for interaction with the Nbrgs-CaM.Nbrgs-CaM includes four domains, to explore which domain is involved in its interaction with NbSGS3, we constructed BiFC expression vectors 2YN/2YC-NbCaM-dX,2YN/2YC-NbCaM-dEFⅠ,2YN/2YC-NbCaM-dEFⅡ,2YN/2YC-NbCaM-dEFⅣ. The results showed that the EFⅠ and EFⅡ domains of Nbrgs-CaM were required for the interaction between Nbrgs-CaM and NbSGS3. We next tested whether domains essential for the interaction between Nbrgs-CaM and NbSGS3 were also required for the PTGS suppressor activity of Nbrgs-CaM, four Nbrgs-CaM deletion mutants, pCambia2300-NbCaM-dX-Flag, pCambia2300-NbCaM-dEFI-Flag pCambia2300-NbCaM-dEFⅡ-Flag and pCambia2300-NbCaM-dEFIV-Flag were Constructed using the pCambia2300-Flag recombinant expression vector. Our date demonstrated that domains essential for the interaction between Nbrgs-CaM and NbSGS3 (EFⅠ and EFⅡ domains) were also indispensable for Nbrgs-CaM suppressor activity.We also we found that overexpression Nbrgs-CaM can reduce the amount of accumulated NbSGS3 protein. These results indicate that Nbrgs-CaM can interact with NbSGS3 and degrade the NbSGS3 protein to inhibit RNA silencing.