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Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana.

ABSTRACT: Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.

SUBMITTER: Andika IB

PROVIDER: S-EPMC4623235 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Project description:Using a transient plant system, it was previously found that the suppression of Cucumber mosaic virus (CMV) 2b protein relies on its double-strand (ds) RNA binding capacity, but it is independent of its interaction with ARGONAUTE (AGO) proteins. Thus, the biological meaning of the 2b-AGO interaction in the context of virus infection remains elusive. In this study, we created infectious clones of CMV mutants that expressed the 2b functional domains of dsRNA or AGO binding and tested the effect of these CMV mutants on viral pathogenicity. We found that the mutant CMV2b(1-76) expressing the 2b dsRNA-binding domain exhibited the same virulence as wild-type CMV in infection with either wild-type Arabidopsis or rdr1/6 plants with RDR1- and RDR6-deficient mutations. However, remarkably reduced viral RNA levels and increased virus (v)siRNAs were detected in CMV2b(1-76)-infected Arabidopsis in comparison to CMV infection, which demonstrated that the 2b(1-76) deleted AGO-binding domain failed to suppress the RDR1/RDR6-dependent degradation of viral RNAs. The mutant CMV2b(8-111) expressing mutant 2b, in which the N-terminal 7 amino acid (aa) was deleted, exhibited slightly reduced virulence, but not viral RNA levels, in both wild-type and rdr1/6 plants, which indicated that 2b retained the AGO-binding activity acquired the counter-RDRs degradation of viral RNAs. The deletion of the N-terminal 7 aa of 2b affected virulence due to the reduced affinity for long dsRNA. The mutant CMV2b(18-111) expressing mutant 2b lacked the N-terminal 17 aa but retained its AGO-binding activity greatly reduced virulence and viral RNA level. Together with the instability of both 2b(18-111)-EGFP and RFP-AGO4 proteins when co-expressed in Nicotiana benthamiana leaves, our data demonstrates that the effect of 2b-AGO interaction on counter-RDRs antiviral defense required the presence of 2b dsRNA-binding activity. Taken together, our findings demonstrate that the dsRNA-binding activity of the 2b was essential for virulence, whereas the 2b-AGO interaction was necessary for interference with RDR1/6-dependent antiviral silencing in Arabidopsis.

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Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana.

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