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

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

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

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 regions

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

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 interactions

Transcriptomic alterations in the sweet orange vasculature correlate with growth repression induced by a variant of citrus tristeza virus

Citrus tristeza virus (CTV, family Closteroviridae) is an economically important pathogen of citrus. CTV resides in the phloem of the infected plants and induces a range of disease phenotypes, including stem pitting and quick decline as well as a number of other deleterious syndromes. To uncover the biological processes underlying the poorly understood damaging symptoms of CTV, we profiled the transcriptome of sweet orange (Citrus sinensis) phloem-rich bark tissues of non-infected, mock-inoculated trees and trees singly infected with two distinct variants of CTV, T36 or T68-1. The T36 and T68-1 variants accumulated in the infected plants at similar titers. With that, young trees infected with T68-1 were markedly repressed in growth, while the growth rate of the trees infected with T36 was comparable to the mock-inoculated trees. Only a small number of differentially expressed genes (DEGs) were identified in the nearly asymptomatic T36-infected trees, whereas almost fourfold the number of DEGs were identified with the growth-restricting T68-1 infection. DEGs were validated using quantitative reverse transcription-PCR. While T36 did not induce many noteworthy changes, T68-1 altered the expression of numerous host mRNAs encoding proteins within significant biological pathways, including immunity and stress response proteins, papain-like cysteine proteases (PLCPs), cell-wall modifying enzymes, vascular development proteins and others. The transcriptomic alterations in the T68-1-infected trees, in particular, the strong and persistent increase in the expression levels of PLCPs, appear to contribute to the observed stem growth repression. On the other hand, analysis of the viral small interfering RNAs revealed that the host RNA silencing-based response to the infection by T36 and that by T68-1 was comparable, and thus, the induction of this antiviral mechanism may not contribute to the difference in the observed symptoms. The DEGs identified in this study promote our understanding of the underlying mechanisms of the yet unexplained growth repression induced by severe CTV isolates in sweet orange trees