Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-D2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-D2b requires SUP-PRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-D2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-D2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21-and 22-nucleotide secondary siRNAs are discussed.

Transgenic plants expressing artificial microRNAs (amiRNAs) have been shown to confer specific resistance to corresponding viruses. Here, we generated Nicotiana benthamiana transgenic lines containing Oryza sativa miR528 as backbone, expressing amiRNAs targeting RNA-dependent RNA polymerase (RdRp) gene of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). The amiRNA transgenic lines could express amiR-CymMV and confer high percentage resistance to CymMV, while lack of detectable level of amiR-ORSV expression in amiR-ORSV transgenic N. benthamiana plants led to weak resistance to ORSV infection. In this project, we provide the first report of CymMV-resistant transgenic N. benthamiana plants based on amiRNA strategy. We believe that this amiRNA approach can be extended to generate CymMV-resistant transgenic orchids.

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Perennial orchids worldwide are frequently infected with Cymbidium mosaic virus (CymMV). To date, no studies have described non-coding small RNA (sRNA) responses in the CymMV long-term-infected orchids. In this study, sRNA profiles of microRNAs (miRNA) and small virus-derived interfering RNAs (siRNA) in symptomless, long-term CymMV-infected Dendrobium and severe symptom, long-term CymMV-infected Ascocenda were identified in four sRNA libraries of virus-free and CymMV-infected orchids by Illumina high-throughput sequencing. The miRNA analysis revealed that the infected orchids had unique sRNA profiles that differed in size distribution, specially the presence of 21 nt sequences in severe symptom Ascocenda. Gel blot analysis showed a change in the miR528 expression which was at high level in virus free Ascocenda, on the other hand, only detected at low level in severe symptom Ascocenda. This expression alteration was also detected with the miR156 expression level in Dendrobium. Moreover, the characterization in polarity of CymMV-derived siRNAs in both infected orchids demonstrated that antisense polarity siRNAs accumulated in severe symptom Ascocenda, and the symptomless Dendrobium contained more sense polarity siRNAs. Here, we showed that miRNA expression was altered after long-term CymMV infection. Furthermore, a different pattern of siRNAs was observed between two orchid genera infected with the same virus and depended on the symptom severity.