Final Report Summary - SUPRA (Suppression of RNA silencing by plant pararetroviruses: protein versus RNA-based)
Plants use ribonucleic acid (RNA) silencing as key mechanism of defence against viruses and the viruses counteract this defence by expression of suppressor proteins. The main goal of our project was to test the hypothesis that a viral RNA can also serve as silencing suppressor. Cauliflower mosaic virus (CaMV) is a pararetrovirus with 8 000-nucleotide double-stranded deoxyribonucleic acid (DNA) genome that replicates through reverse transcription of pregenomic RNA (35S RNA). CaMV encodes a transactivator protein (TAV) that acts as a weak suppressor of silencing. Analysis of Arabidopsis plants infected with CaMV showed accumulation of massive amounts of virus-derived, short-interfering RNAs (siRNAs), comparable to the entire complement of endogenous microRNAs (miRNAs) and siRNAs which direct silencing of plant genes. The bulk of viral siRNAs (65 to 82 %) is derived from a highly structured, 600-nucleotide leader region. Mapping of CaMV transcripts showed that the leader region is transcribed not only as a part of the pregenomic 35S RNA but also as a separate 8S transcript containing just the leader sequence. The 8S RNA was also found to be copied to antisense RNA to form ca. 600 bp double-stranded RNA (dsRNA). In most eukaryotes, dsRNA is processed by Dicer into siRNAs that trigger silencing and RNA interference (RNAi). Our genetic analysis and short RNA deep-sequencing revealed that 8S dsRNA is processed by four Dicer-like enzymes into 21, 22 and 24 nt viral siRNAs. High titer of replicating virus in the plants which accumulate massive amounts of 21-24 nt vsRNAs suggests that leader-derived vsRNAs may not have antiviral activity. Indeed, testing CaMV-infected dcl1 dcl2 dcl3 dcl4 quadruple mutant plants, in which DCL-mediated production of viral siRNAs was nearly abolished, revealed no increase in the viral titer compared to the wild type plants. These findings and other lines of experimental evidence (which we published in Blevins, Rajeswaran et al. 2011, Rajeswaran and Pooggin 2012, Rajeswaran et al. 2012) led us to conclude that CaMV is deliberately producing leader-derived 8S dsRNA and vsRNAs to act as a decoy and divert the silencing machinery from other regions of the viral genome. Thus, not only proteins but also RNA can act as a suppressor of anti-viral silencing. To evaluate if other pararetroviruses also use RNA as a decoy, we extended this study in Rice tungro bacilliform virus (RTBV), a pararetrovirus which infects rice. Similar to CaMV, the 35S RNA of RTBV also has a long and highly structured leader. Deep sequencing of viral siRNAs and mapping of RTBV transcripts showed that RTBV may also use an RNA-based decoy to suppress RNA silencing (Rajeswaran et al. in preparation). RTBV in combination with Rice tungro spherical virus (RTSV) causes a severe rice tungro disease. We found that, similar to RTBV, RTSV genomic RNA has a long and highly structured leader which is bypassed by ribosome shunt during translation initiation (Pooggin, Rajeswaran et al. 2012). A remarkable structural similarity of shunt configurations in RTBV and RTSV suggests co-evolution of these viruses in the disease complex. It will be interesting to investigate whether RTSV has also co-evolved a decoy strategy of silencing evasion or exploits an 8S RNA decoy of the partner RTBV. Our findings add substantially to the existing basic knowledge of silencing and suppression mechanisms and have implications for designing RNA-based strategies to control viral pathogens of crop plants.
The knowledge and the expertise gained by the researcher through this project and the publications in many reputed journals will be very instrumental in his career development. The collaborations which were continued and initiated during this project will also be useful for the researcher for future scientific interactions. The host group has long-term collaboration with prof. Veluthambi group at Madurai Kamaraj University (India) through Indo-Swiss Collaboration in Biotechnology (ISCB). The researcher, who was introduced to the host group through ISCB, has enhanced the collaboration between the groups. The researcher initiated collaboration with Dr Il-Ryong Choi, International Rice Research Institute (Philippines) to study the rice tungro disease complex. Furthermore, the researcher collaborated with Dr Marie-line Iskra-Caruana group at CIRAD (Montpellier, France) to study silencing and its suppression in Banana streak virus, a pararetrovirus which infects banana. There was good exchange of information and visits including a short term scientific mission of the researcher to Cirad supported by European Cooperation and Science and Technology (COST) in the frame of the COST action FA0806. The researcher has taken part in training undergraduate and graduate students in the host lab and actively participated in the teaching programme in plant physiology and plant molecular biology at the host institute. Taken together, the experience gained by the researcher during his stay at the host institute should be useful for establishing his independent research group back in India.
The knowledge and the expertise gained by the researcher through this project and the publications in many reputed journals will be very instrumental in his career development. The collaborations which were continued and initiated during this project will also be useful for the researcher for future scientific interactions. The host group has long-term collaboration with prof. Veluthambi group at Madurai Kamaraj University (India) through Indo-Swiss Collaboration in Biotechnology (ISCB). The researcher, who was introduced to the host group through ISCB, has enhanced the collaboration between the groups. The researcher initiated collaboration with Dr Il-Ryong Choi, International Rice Research Institute (Philippines) to study the rice tungro disease complex. Furthermore, the researcher collaborated with Dr Marie-line Iskra-Caruana group at CIRAD (Montpellier, France) to study silencing and its suppression in Banana streak virus, a pararetrovirus which infects banana. There was good exchange of information and visits including a short term scientific mission of the researcher to Cirad supported by European Cooperation and Science and Technology (COST) in the frame of the COST action FA0806. The researcher has taken part in training undergraduate and graduate students in the host lab and actively participated in the teaching programme in plant physiology and plant molecular biology at the host institute. Taken together, the experience gained by the researcher during his stay at the host institute should be useful for establishing his independent research group back in India.