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

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′-overhangs

Small RNA-Omics for Plant Virus Identification, Virome Reconstruction, and Antiviral Defense Characterization

RNA interference (RNAi)-based antiviral defense generates small interfering RNAs that represent the entire genome sequences of both RNA and DNA viruses as well as viroids and viral satellites. Therefore, deep sequencing and bioinformatics analysis of small RNA population (small RNA-ome) allows not only for universal virus detection and genome reconstruction but also for complete virome reconstruction in mixed infections. Viral infections (like other stress factors) can also perturb the RNAi and gene silencing pathways regulating endogenous gene expression and repressing transposons and host genome-integrated endogenous viral elements which can potentially be released from the genome and contribute to disease. This review describes the application of small RNA-omics for virus detection, virome reconstruction and antiviral defense characterization in cultivated and non-cultivated plants. Reviewing available evidence from a large and ever growing number of studies of naturally or experimentally infected hosts revealed that all families of land plant viruses, their satellites and viroids spawn characteristic small RNAs which can be assembled into contigs of sufficient length for virus, satellite or viroid identification and for exhaustive reconstruction of complex viromes. Moreover, the small RNA size, polarity and hotspot profiles reflect virome interactions with the plant RNAi machinery and allow to distinguish between silent endogenous viral elements and their replicating episomal counterparts. Models for the biogenesis and functions of small interfering RNAs derived from all types of RNA and DNA viruses, satellites and viroids as well as endogenous viral elements are presented and discussed

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

Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease

Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence

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