PMB Lab., International Centre for Genetic Engineering and Biotechnology, AAA Marg, New Delhi-110067, India.
Abstracts of the papers presented at the International Conference of Indian Virological Society on “Emerging and Re-emerging viral Diseases of the Tropics and Subtropics” at Indian Agricultural Research Institute, New Delhi, India, December 11–14, 2007.
Viruses encode RNAi-suppressors to ensure their growth and cause pathogenesis. So developing the techniques to detect the suppressors is an important determinant of RNAi related studies and we have developed three such detection methods. One of the techniques uses reversal of silencing in the GFP-silent tobacco lines (cultivar Xanthi), the other relies on enhancement of replication of an amplicon based on geminiviral DNA replicon while the third assay is carried out with insect Sf21 RNAi sensor lines. The evidences of functionalities of these techniques will be presented. We detected RNAi-suppressors from innumerable plant viruses along with few animal viruses using such techniques.
The ‘AC2’ protein of a begomovirus, MYMIV, and ‘B2’ protein of the insect virus, FHV, turned out to be potent suppressors and hence these were selected for mechanistic studies. The MYMIV-AC2 was also a transactivator but the domains of suppression and transactivation were separable. The suppressor was biochemically a dimmer, inhibited siRNA generation in plants and interacted with many Arabidopsis proteins including the silencing factors, namely, RDR6, AGO1, DCL-1, and ARF6 in the phage display as well as yeast two-hybrid assays. The AC2 tobacco transgenics were mostly stunted and showed a range of interesting phenotypes. On the other hand, the FHV-B2 protein acted like an inhibitor of siRNA biogenesis and interacted with the ‘PAZ’ domain of the DICER in the Sf21 RNAi sensor cell line based assay. The FHV-B2 rice transgenics were also stunted and surprisingly were salt tolerant. A number of genes responsible for salt tolerance were up regulated in the transgenics and a few of them are known to be controlled by the rice microRNAs. Currently we are studying the molecular controls underlying the above-mentioned phenotypic observations. We have also used the ‘AC2’ suppressor as an aid to the ribozyme technology directed to the replication initiation protein, MYMIV-‘Rep’ and were thus able to specifically down regulate the transient viral DNA replication in-planta. Thus the viral suppressors are very interesting molecules that control the host RNAi processes.
