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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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