Endogenous and antiviral RNA silencing pathways in Arabidopsis Public Deposited



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  • RNA silencing pathways are required for a wide variety of processes in most eucaryotes. In plants, small-interfering RNA (siRNA) arising from transposons and other repetitive sequences is associated with heterochromatin formation and maintenance. MicroRNAs and trans-acting siRNAs encoded at discrete loci function as negative regulators of gene expression by triggering cleavage or translational repression of mRNA transcripts with base complementarity to the small RNA. siRNA processed from viral RNA directs antiviral silencing that represses virus accumulation in plants and other organisms. Together, these pathways serve numerous functions in plants including genome maintenance, developmental timing and patterning and antiviral defense. Virus-encoded RNA silencing suppressor proteins are viral pathogenicity factors and inhibit the antiviral silencing response through interaction with small RNA intermediates. In this work, small RNA duplex binding was demonstrated for unrelated suppressors from multiple viruses using molecular biology and biochemistry techniques. Sequestration of virus-derived siRNA and microRNA/microRNA* duplexes, inhibition of microRNA methylation, and perturbation of Arabidopsis development was demonstrated for several suppressors using transgenic approaches and molecular techniques. Suppressor inhibition of antiviral RNA silencing and endogenous microRNA pathways indicates that small RNA binding is a common strategy used by unrelated viruses, and suggests that interference with miRNA-directed processes may be a general feature contributing to pathogenicity of many viruses. Finally, small RNA preparation and high-throughput sequencing procedures were developed for profiling small RNA populations in Arabidopsis. Genome-wide profiles of small RNA from wild-type Arabidopsis thaliana and silencing pathway mutants revealed dynamic changes in expression of some microRNA families as well as genome-wide distribution patterns of small RNA in plants. These results establish high-throughput sequencing as a small RNA profiling tool and provide a comprehensive description of the major small RNA pathways in Arabidopsis. Together, the results presented here provide a basic understanding of the breadth of small RNA pathways in plants and show how interference with these pathways by viruses contributes to virus disease.
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