Functional analysis of microRNA triggers of phased siRNA biogenesis in plants
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Small RNAs are a class of noncoding RNAs which are of great importance in gene expression regulatory networks. Different families of small RNAs are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary siRNAs (phasiRNAs); these require RDR6, DCL4, and (typically) a microRNA (miRNA) trigger for their biogenesis. Protein-encoding genes are an important source of phasiRNAs, and the model legume Medicago truncatula generates phasiRNAs from many PHAS loci. ☐ We aimed to investigate their biogenesis and mechanism by which miRNAs trigger these molecules. We modulated miRNA abundances in transgenic tissues showing that the abundance of phasiRNAs correlates with the levels of both miRNA triggers and the target, precursor transcripts, and identified sets of phasiRNAs or PHAS loci that predominantly and substantially increase in response to miRNA overexpression. In the process of validating targets from miRNA overexpression tissues, we found that in the miRNA-mRNA target pairing, the 3’ terminal nucleotide (the 22nd position), but not the 10th position, is important for phasiRNA production. Mutating the single 3’ terminal nucleotide dramatically diminishes phasiRNA production. Ectopic expression of Medicago NB-LRR -targeting miRNAs in Arabidopsis showed that only a few NB-LRRs are capable of phasiRNA production; our data indicate that this might be due to target inaccessibility determined by sequences flanking target sites. Our results suggest that target accessibility is an important component in miRNA-target interactions that could be utilized in target prediction, and the evolution of mRNA sequences flanking miRNA target sites may be impacted. ☐ CRISPR/Cas9 has become a powerful technique in genome editing. In my study, CRISPR/Cas9 was employed to edit the passenger strand of MIR160a to convert pre-miR160 into an asymmetric structure in Arabidopsis, because evidence has been shown to support that the length of 22-nt miRNAs is important to trigger phasiRNA production. In the mutant with a single nucleotide insertion on miR160a*, we found that target transcripts of miR160, including ARF10, ARF16 and ARF17, did not produce secondary siRNAs, suggesting that the asymmetric structure of miRNA might not be a determinant of phasiRNA production. Moreover, we tested the efficiency of fragment deletions in Arabidopsis MIR160a via the CRISPR/Cas9 vector with double guide RNAs, which would potentially generate the null mutant of miR160a. We found that CRISPR/Cas9 with a double guide RNA worked successfully by the floral dip method of transformation, reaching ~30% of fragment deletions (~50 bp) in the T1 generation. The mir160a mutants with a 47- or 48-bp fragment deletion showed severe pleotropic developmental defects, such as serrated leaf, inward-curled and thin petal, short siliques, reduced fertility and arrested embryo development. These results show that CRISPR/Cas9 is an efficient tool for functional studies of noncoding RNAs by fragment deletions in the plant genome. ☐ Studies in the past a few years have shown that miRNAs, together with phasiRNAs are important regulators of plant NB-LRRs. In the Chapter 4 of this dissertation, we integrated small RNAs into the classic “zig-zag-zig model” of plant defenses, highlighting the roles of small RNAs in the modulation of host immunity.