RDR6-mediated synthesis of complementary RNA is terminated by miRNA stably bound to template RNA

RNA-dependent RNA polymerase RDR6 is involved in the biogenesis of plant trans-acting siRNAs. This process is initiated by miRNA-directed and Argonaute (AGO) protein-mediated cleavage of TAS gene transcripts. One of the cleavage products is converted by RDR6 to double-stranded (ds)RNA, the substrate for Dicer-like 4 (DCL4). Interestingly, TAS3 transcript contains two target sites for miR390::AGO7 complexes, 5′-non-cleavable and 3′-cleavable. Here we show that RDR6-mediated synthesis of complementary RNA starts at a third nucleotide of the cleaved TAS3 transcript and is terminated by the miR390::AGO7 complex stably bound to the non-cleavable site. Thus, the resulting dsRNA has a short, 2-nt, 3′-overhang and a long, 220-nt, 5′-overhang of the template strand. The short, but not long, overhang is optimal for DCL4 binding, which ensures dsRNA processing from one end into phased siRNA duplexes with 2-nt 3′-overhang

Endogenous banana streak virus sequences (eBSV) are likely transcriptionally silenced in the resistant seedy diploid Musa balbisiana Pisang Klutuk Wulung (PKW). [P.50]

The B genome of banana (Musa sp.) harbours integrations of Banana streak virus (eBSV) for at least three BSV species, whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses contexts. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated, more or less extensively rearranged containing at least one full length viral genome. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSIMV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. Using deep sequencing of total siRNAs of PKW we underlined the presence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSIMV by blasting sequences against the 3 BSV species genomes. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. vsRNA are enriched in 24-nt class which represent about 75% of the total 21-24nt siRNA matching eBSV. We also demonstrated that eBSV are highly methylated in the three different sequence contexts (CG, CHH and CHG) throughout the whole sequence of eBSVs with no difference in methylation profile between siRNA producing and non producing areas. Interestingly, methylation patterns of all three eBSV are similar whereas they are located in different genomic context; eBSOLV being in a TE rich area whereas eBSIMV and eBSGFV are in genes rich region. It seems that eBSV are controlled mainly by epigenetic mechanisms similar to those described for transposable elements (TE). All together, our data indicate that eBSVs in PKW genome are likely silenced at the transcriptional level and this is probably responsible for the natural resistance of this genotype to the activation of such infectious eBSV as well as infection by external BSV particles. (Résumé d'auteur

Natural resistance of banana genotypes to banana streak virus is probably driven by transcriptional gene silencing

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (BSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. We postulated these eBSV induced a natural resistance driven by gene silencing mechanisms based on their complex molecular re-arranged structure which could lead to dsRNA hairpins formation. In collaboration with the group headed by M. Pooggin (Basel, Switzerland), a deep sequencing of total siRNAs of PKW was performed using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from BSOLV, BSGFV and BSImV by blasting sequences against the 3 BSV species genomes. vsRNA are enriched in 24-nt class thus eBSV in PKW genome are likely silenced at the transcriptional level. A repartition of the vsRNA population matching eBSV will be also presented in order to determine hot and cold spots of vsRNA generation. (Texte intégral

Banana plants use post-transcriptional gene silencing to control banana streak virus infection

Banana streak virus (BSV), the causative agent of banana streak disease, is a plant pararetrovirus belonging to the family Caulimoviridae, genus Badnavirus. The genome of BSV is a circular double-stranded DNA of 7.4 kbp made of three ORFs and like other pararetroviruses replicates via reverse transcription of viral pregenomic RNA (Lockhart, 1990). While the first two ORFs encode two small proteins of unknown function, the third ORF (~210 kD) encodes a polyprotein that can be cleaved to yield the viral coat protein and proteins with homology to aspartic protease, reverse transcriptase and RNaseH. Little information is available about antiviral defense response of the host plant on BSV or other members of Caulimoviridae. RNA silencing, also known as RNA interference (RNAi), is an ancient gene regulation and cell defense mechanism, which exists in most eukaryotes (Xie and Qi, 2008). Plants have adapted the RNA silencing machinery into an antiviral defense system. Interestingly, Arabidopsis plants infected with Cauliflower mosaic virus (CaMV), a type member of the genus Caulimovirus in the family Caulimoviridae, accumulate siRNAs of 21, 22 and 24 nt size classes, where the 24 nt species are the most predominant ones (Blevins et al., 2006; Moissiard and Voinnet, 2006). Further analysis showed that, the leader region (600 nt) of CaMV pregenomic RNA produces massive amounts of siRNAs with several hot and cold spots of siRNA generation (Blevins et al., 2011) to function as a decoy for the RNA silencing defense system of the plant. To determine whether the viral decoy strategy was universally used among viruses belonging to the family Caulimoviridae, we have performed a deep sequencing of total siRNAs of 6 Cavendish banana plants infected independently with one of the 6 BSV species we own in the laboratory. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from those 6 BSV species by blasting sequencing data against the 6 BSV species genomes. vsRNA are enriched in 21-nt class thus BSV are likely silenced at the post-transcriptional level. Besides, our data unequivocally show that the decoy strategy used by the CaMV is not employed by the BSV since most of the hot spots of siRNA production are located in ORF1 and 2. Information generated about siRNAs derived from BSV genome could help us to design silencing-based transgenic and non-transgenic (RNA vaccination) approaches to obtain BSV resistance in banana crop. (Texte intégral

Natural resistance of the diploid Musa balbisiana Pisang Klutuk Wulung (PKW) to banana streak virus is probably driven by transcriptional gene silencing

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (eBSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. In collaboration with the group headed by M. Pooggin (Basel, Switzerland), a deep sequencing of total siRNAs of PKW was performed using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSImV by blasting sequences against the 3 BSV species genomes. vsRNA are enriched in 24-nt class thus eBSV in PKW genome are likely silenced at the transcriptional level. Interestingly, we show that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. All together, those data seem indicate these eBSV induce a natural resistance driven by gene silencing mechanisms based on their complex molecular re-arranged structure which could lead to dsRNA formation. (Texte intégral

Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21-24nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common regio

Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis

Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA. This tasiRNA and miR173 guide Argonaute 1 complexes to excise the segments from TAS2 and three TAS1 transcripts including TAS1c itself to be converted to dsRNAs, which restricts siRNA production to a region between the two cleavage sites. TAS1c is also feedback regulated by a cis-acting siRNA. We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation. TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNA

Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding region

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interaction