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Summary Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial–temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive.
Here, through histological and high‐throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis.
AGO1d preferentially binds to miR2118‐triggered 21‐nucleotide (nt) phasiRNAs with a 5′‐terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21‐ and 24‐nt phasiRNAs and temperature‐sensitive male sterility. At lower temperatures, anthers of the
ago1d mutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism.These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.
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Summary MicroRNAs (miRNAs) are 20‐24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta‐siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator‐like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants,
mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild‐typeMIR390 . Small RNA sequencing showed the two base pair deletion inmir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9‐mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double‐guide RNAs successfully generatedmir160a null mutants with fragment deletions, at a higher efficiency than a single‐guide RNA. The difference between the phenotypic severity ofmiR160a mutants in Col‐0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.
