either the bi-directional promoter or the AC2 coding region of MYMV-Vig (Pooggin et al., 2003 and unpublished results reported by T. Hohn; see. Figure 1).
Plant Molecular Biology 55: 149–152, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Meeting report
Fighting geminiviruses by RNAi and vice versaw Mikhail M. Pooggin and Thomas Hohn* Institute of Botany, University of Basel, Scho¨nbeinstrasse 6, CH-4056 Basel, Switzerland (*author for correspondence) Received 2 April 2004; accepted in revised form 5 April 2004
Key words: begomovirus, geminivirus, meeting, RNAi, silencing, silencing suppression, ssDNA
Abstract Geminiviruses have recently emerged not only as the cause of devastating diseases of important crop plants but also as a tool to study fundamental aspects of RNA interference (RNAi) and virus-induced gene silencing. RNA silencing is an evolutionary conserved mechanism protecting cell from pathogenic RNA and DNA, which is increasingly viewed as an adaptive immune system of plants against viruses. Here we summarize recent developments in the field of geminivirology presented by several leading groups at the Meeting ‘‘Gemini2004!!!’’ (a total of 85 participants from all over the world) with the main focus on the anti-viral strategies that exploit RNAi and related silencing phenomena.
(RNAi) and virus-induced gene silencing (Turnage et al., 2002). RNA silencing is an evolutionary conserved mechanism protecting cells from pathogenic RNA and DNA, which is increasingly viewed as an adaptive immune system of plants against viruses (Voinnet, 2001). Here we summarize recent developments in the field of geminivirology presented by several leading groups at the Meeting ‘‘Gemini2004!!!’’ (a total of 85 participants from all over the world) with the main focus on the anti-viral strategies that exploit RNAi and related silencing phenomena. Introduction Geminiviruses have recently emerged not only as the cause of devastating diseases of important crop plants (Mansoor et al., 2003) but also as a tool to study fundamental aspects of RNA interference w
The 4th International Geminivirus Symposium ‘‘Gemini2004!!!’’ was held at University of Cape Town in Cape Town, South Africa between February 15 and 20, 2004. The Meeting was sponsored by Beckman and Merck and was organized by E. Rybicki.
RNAi and virus-induced silencing Proof of the concept that RNAi can be engineered to effectively target geminiviruses has recently been documented in transient assays, initially for Mungbean yellow mosaic virus-Vigna (MYMVVig; Pooggin et al., 2003) and later for African cassava mosaic virus (ACMV; Vanitharani et al., 2003). Following this lead, several groups attempted to produce stable RNAi transgenic
150 plants resistant to begomoviruses, the major genus of geminiviruses. In some cases, these attempts proved to be successful, while in a few other cases no resistance, or at best only a delay with symptom development, could be achieved. S. Mansoor (Faisalabad, Pakistan) produced transgenic tobacco plants constitutively expressing double-stranded (ds)RNA cognate to coding and non-coding regions of DNA b from Cotton leaf curl virus (CLCuV). These plants were resistant to CLCuV when challenged either by agroinoculation or whitefly. Interestingly, the potent RNAi constructs were initially selected by using a transient assay, in which they were co-agroinoculated onto non-transgenic plants together with the CLCuV infectious clone. In a transient assay, we ourselves achieved high incidence recovery from the virus infection in blackgram plants treated biolistically with the RNAi constructs targeting either the bi-directional promoter or the AC2 coding region of MYMV-Vig (Pooggin et al., 2003 and unpublished results reported by T. Hohn; see Figure 1). S. Ribeiro (Brasilia, Brazil) in collaboration with M. Prins (Wageningen, Netherlands) produced transgenic N. benthamiana plants carrying two different 35S promoter-driven RNAi constructs designed to target Tomato chlorotic mottle virus (ToCMV-[BR]). One construct contained inverted repeat (IR) of the bi-directional DNA A promoter flanked with the 5¢-parts of Rep (AC1) and CP (AV1) coding regions, while the other carried IR covering the entire Rep, TrAP (AC2) and Ren (AC3) coding regions. These transgenic plants (R1 generation) were agroinoculated with
ToCMV. In both cases, significant delays in symptom development were observed. However, none of the several transgenic lines tested was immune to ToCMV. Also there was no correlation between the timing of delayed symptom appearance and the levels of small interfering RNA (siRNAs) accumulation (see below). A. Rezaian (Canberra, Australia) constructed transgenic RNAi tomato plants with a Tomato leaf curl virus (TLCV) C2 intron-spliced hairpin construct that accumulated high levels of siRNAs cognate to the C2 ORF. Inoculation of these plants with TLCV resulted in delayed accumulation of viral DNA, correlating with high level of methylation of a complementary-sense strand of the dsDNA form. However, complete resistance (immunity) to the virus was not achieved. In the RNA silencing process, siRNAs are generated by Dicer-mediated cleavage of long dsRNA, the key trigger of silencing. After incorporation into RISC (RNA-induced silencing complex) and RISC-like complexes, siRNAs guide these complexes to target homologous (viral) RNA for degradation in cytoplasm and, most likely, (viral) DNA for methylation in the nucleus (Matzke et al., 2004). Both A. Rezaian and M. Tavazza (Roma, Italy) obtained evidence that siRNAs corresponding to both coding and noncoding regions of TLCV and Tomato leaf curl Sardinia virus (TYLCSV) accumulate during normal virus infection in tomato and tobacco plants. We also made similar observations in a model ACMV-N. benthamiana system (A. Dodar, A. SeAmmour, F. Meins, M.M.P. and T.H., unpublished). These findings indicate that RNA silencing
Figure 1. Blackgram plants infected with MYMV-Vig (left) versus recovered from initial infection following transient treatment by particle bombardment with an RNAi construct targeting the MYMV-Vig bi-directional promoter (right).
151 is induced by begomoviruses, but apparently this natural anti-viral mechanism cannot totally abolish viral replication and spread. A. Rezaian suggested that geminivirus is able to evade silencing due to existence of multiple single-stranded viral DNA molecules and/or replicative DNA intermediates in the nucleus, which might escape siRNAdirected DNA methylation. Alternatively, geminivirus protein(s) might suppress RNA silencing. Indeed, it has been reported for ACMV (Voinnet et al., 1999) and also found for MYMVVig (Trinks et al., 2004; reported by M. Pooggin) and Tomato golden mosaic virus (TGMV; reported by D. Bisaro, Columbus, OH) that begomovirus AC2 can function as a suppressor of transgeneinduced RNA silencing in a model plant system. Interestingly, the mechanisms of silencing suppression might be different for old world and new world begomoviruses, because in the one case (M. Pooggin) a transactivator domain sequence of AC2 is essential, whereas in the other case (D. Bisaro) not. Both Ali Rezaian and M. Tavazza described a phenomenon, in which transgenes carrying different viral sequences [promoter (Seemanpillai et al., 2003) or coding regions] could become silenced upon infection by corresponding viruses (TLCV and TYLCSV). In these cases, transgene silencing correlated with both reduced levels of the transgene transcript and hypermethylation of its DNA. At the same time, virus itself was able to evade silencing. M. Tavazza suggested a model, in which siRNAs do interfere with viral infection but as soon as viral replication/expression exceeds a certain threshold, virus spreading in the silenced tissue cannot be prevented any longer. R. Rievera-Bustamante (Irapuato, Mexico) described a recovery phenomenon, in which pepper plants, originally infected with Pepper golden mosaic virus (PepGMV), under certain conditions exhibited decrease in symptom severity in the newly developing leaves. The recovered plants were resistant to re-inoculation with PepGMV, but could be re-infected when challenged with another begomovirus (PHYVV), suggesting that a sequence-specific RNA silencing mechanism is induced and maintained in the recovering tissues. Interestingly, siRNAs corresponding to coding and non-coding regions of PepGMV were detected in both symptomatic and recovered tissues, thus prompting the authors to rule out the RNA silencing-mediated mechanism. However, in a view
of the above-described findings of siRNAs associated with normal geminivirus infection, RNA silencing can still account for the observed recovery phenomenon. Some as yet unknown quality of siRNAs and/or the activity of siRNA-guided RISC and RISC-like complexes might be responsible for the recovery, even though the observed siRNA accumulation levels are indistinguishable between the recovering and severely infected tissues. C. Fauquet (Saint Louis, MO) reported successful generation of transgenic cassava (line Y85) resistant to ACMV as well as two other related cassava begomoviruses. Detection of the transgene-derived siRNAs and extremely low levels of the transgene product (the truncated Rep protein from ACMV) in this line suggested that RNA silencing is a mechanism responsible for the wide range resistance. Field trials are currently being planned in Africa.
Competitive inhibition In several reports, transgenic plants with different transdominant mutated versions of the begomovirus Rep (replication protein) gene, either truncated or inactivated by point mutations– alone or in a combination with Ren (replication enhancer) – were described. Overall, such an approach also proved to be successful in generating resistance against begomoviruses. However, in some cases, it was observed that resistance could be broken due to virus-induced, homology-dependent silencing of the transgene (see above). L. Hanley-Bowdoin (Raleigh, NC) reported a success story, in which transgenic N. benthamiana plants were generated to carry a bicistronic expression cassette for Rep (AL1) and Ren (AL3) mutants of TGMV. The AL1 contained two mutations that confer replication interference and enhanced transcription repression phenotypes and impair interaction with the retinoblastoma-related protein (RBR), respectively. Because RBR is a host factor that negatively regulates cell division cycle and facilitates differentiation, AL1–RBR interaction must be avoided in transgenic plants. AL3 contained a mutation that blocks replication enhancement and several protein interactions, most importantly, including the host protein PCNA (proliferating cell nuclear antigen), essential for
152 DNA replication. Two transgenic lines showed resistance to TGMV and the phenotypes were stable up to T2 generation. One line exhibited immunity to the virus, which correlated with high levels of transgenic RNA (and presumably mutant proteins), thus suggesting competitive inhibition as the underlying mechanism of immunity. In contrast, the second line accumulated very low amounts of the RNA, suggesting that a mechanism related to RNA silencing might interfere with the viral infection. M. Tavazza (Roma, Italy) produced N. benthamiana (Lucioli et al., 2003) and tomato transgenic plants expressing a truncated version of Rep from TYLCSV (Rep-210). In both plant species, Rep210-mediated resistance was overcome by TYLCSV due to shutting-down of transgene expression by a post-transcriptional homology-dependent mechanism (i.e. RNA silencing). One idea how to avoid this type of silencing would be to reduce homology between transgene and virus sequences. In the case of Rep, a synthetic Rep transgene could be designed, in which Rep sequence would be modified without changing the encoded amino acids (M. Tavazza, personal communication).
Conclusions and implications for the anti-viral approaches Despite apparent failure in some cases to generate resistance by RNAi, several groups proved the potency of this approach against geminiviruses and even reported immune transgenic lines. Two major targets of RNAi should be considered in the future, which would comprise the viral bi-directional promoter and the AC2/C2 coding region, because targeting of viral promoter in the nucleus will ensure the loss of viral transcription, while targeting the AC2/C2 mRNA in the cytoplasm will prevent accumulation of AC2/C2 protein that may potentially interfere with RNAi. Using a mutated and truncated version of MYMV-Vig Rep as a competitive inhibitor should also be considered a promising approach. However, to avoid virus-induced silencing of the transgene, which can break the resistance, a synthetic gene coding for (truncated) Rep, with the key amino acids substituted and about 50% codons mutated silently, should be designed. The new findings reported at the Meeting indicate that there is a complex relationship be-
tween geminiviruses and an RNA silencing-based plant defense system. Very potent RNA silencing is induced by the virus, which is able to target cognate RNA for complete destruction and DNA sequences for hypermethylation, thus leading to post-transcriptional and transcriptional silencing of any homologous (trans)gene. At the same time, the virus itself is able to evade or suppress silencing and spread systemically in the whole plant. The role of geminivirus suppressor AC2 and perhaps other viral proteins and/or replicative DNA intermediates in this counter-defense process remains to be investigated.
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