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Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana , where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection—a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26—a necrosis-inducing pathogenicity determinant of ToTV—nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana. [ABSTRACT FROM AUTHOR]

To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant’s growth, as was illustrated by the plant’s increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant’s susceptibility to viroid infection.

RNA-dependent RNA polymerases (RDRs) play an important role in RNA silencing, antiviral and developmental progress. Here, we firstly isolated the full-length cDNA, genomic DNA and 5′-flanking region of RDR6 from Nicotiana glutinosa ( NgRDR6). Sequences analysis revealed that the cDNA of NgRDR6 was 3,921 bp in length, and the deduced protein consisted of 1,197 amino acids, containing all highly conserved sequence motifs that are present among all RDRs families. Moreover, two introns were detected in the genomic sequences. We also firstly investigated the expression profiles of plant RDR6 under the treatments of gibberellin A (GA), HO methyl jasmonate (MeJA), Potato virus Y (PVY), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Rhizoctonia Solani and Colletotrichum nicotianae. In addition, the expression patterns of RDR6 in Nicotiana glutinosa under the treatments of salicylic acid (SA) and abscisic acid (ABA) were also been analyzed. The results indicated that the NgRDR6 mRNA accumulation could be induced by ABA, GA, MeJA, CMV, Rhizoctonia Solani and Colletotrichum nicotianae. In contrast, the expression level of NgRDR6 exhibited no remarkable difference under the treatments of PVY, TMV, HO and SA. Further investigation suggested several potential cis-acting elements were found in the 5′-flanking sequence of NgRDR6, which might be responsible for the enhanced response to phytohormones. [ABSTRACT FROM AUTHOR]

Abstract: Suppressor of gene silencing 3 (SGS3) is involved in RNA-dependent RNA polymerase 6 (RDR6)-dependent small-interfering RNA (siRNA) pathways in Arabidopsis. However, the roles of SGS3 in those pathways are unclear. Here, we show that SGS3 interacts and colocalizes with RDR6 in cytoplasmic granules. Interestingly, the granules containing SGS3 and RDR6 (named SGS3/RDR6-bodies) were distinct from the processing bodies where mRNAs are decayed and/or stored. Microscopic analyses and complementation experiments using SGS3-deletion mutants suggested that proper localization of SGS3 is important for its function. These results provide novel insights into RDR6-dependent siRNA formation in plants. Structured summary: MINT-7014710: SGS3 (uniprotkb:Q9LDX1) and RDR6 (uniprotkb:Q9SG02) physically interact (MI:0218) by bimolecular fluorescence complementation (MI:0809) MINT-7014697: RDR6 (uniprotkb:Q9SG02) and SGS3 (uniprotkb:Q9LDX1) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [Copyright &y& Elsevier]

To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant's growth, as was illustrated by the plant's increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant's susceptibility to viroid infection. [ABSTRACT FROM AUTHOR]