| In this dissertation work, I applied the NGS (Next-Generation Sequencing) technology to analyze sRNAs from a wide variety of plants as well as from different genotypes, to characterize sRNA function and biogenesis. Additionally, I developed novel bioinformatics tools that take advantage of the huge amount of sequencing data that has been generated.;1) MicroRNAs as Master Regulators of the Plant NB-LRR (Nucleotide-Binding site Leucine-Rich Repeat) Defense Gene Family via the Production of Phased, Trans-acting siRNAs.;Legumes and many non-leguminous plants enter symbiotic interactions with microbes, and it's poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt miRNA families that target conserved domains in the RNA from these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of sRNAs were matched to over 60% of all ~540 encoded Medicago NB-LRRs; in potato, a model for mycorrhizal interactions, phased siRNAs were also produced from NB-LRRs. Cleavage of DCL2 and SGS3 transcripts guided by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a second example of "two-hit" (two miRNAs targeting the same transcript at the same time, please see introduction for more details) phasiRNA processing was identified, utilizing miR156-miR172 sites. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions, and demonstrate miRNAs as master regulators of a large gene family, a new paradigm for miRNA function.;2) Plant MicroRNAs Display Differential 3'- Truncation and Tailing, Modifications Which Are ARGONAUTE1-Dependent and Conserved Across Species.;Plant small RNAs are 3' methylated by the methyltransferase HEN1. In plant hen1 mutants, 3' modifications of small RNAs, including oligo-uridylation ("tailing"), are associated with accelerated degradation of miRNAs. By sequencing small RNAs of wildtype and hen1 mutants from Arabidopsis, rice and maize, we found 3' truncation prior to tailing is widespread in these mutants. Moreover, the patterns of miRNA truncation and tailing differ substantially among miRNA families but are conserved across species. The same patterns are also observable in wildtype libraries from a broad range of species, only at lower abundances. ARGONAUTE (AGO1), even with defective slicer activity, can bind these truncated and tailed variants of miRNAs. An ago1 mutation in hen1 suppressed such 3' modifications, indicating that they occur while miRNAs are in association with AGO1, either during or after RISC assembly. Our results showed AGO1-bound miRNAs are actively 3' truncated and tailed, possibly reflecting the activity of co-factors acting in conserved patterns in miRNA degradation. (Abstract shortened by UMI.). |
