Xiaoming Zhang

MicroRNAs (miRNAs) are key regulators of gene expression in development and stress responses in most eukaryotes. We globally profiled plant miRNAs in response to infection of bacterial pathogen Pseudomonas syringae pv. tomato (Pst). We sequenced 13 small-RNA libraries constructed from Arabidopsis at 6 and 14 h post infection of non-pathogenic, virulent and avirulent strains of Pst. We identified 15, 27 and 20 miRNA families being differentially expressed upon Pst DC3000 hrcC, Pst DC3000 EV and Pst DC3000 avrRpt2 infections, respectively. In particular, a group of bacteria-regulated miRNAs targets protein-coding genes that are involved in plant hormone biosynthesis and signaling pathways, including those in auxin, abscisic acid, and jasmonic acid pathways. Our results suggest important roles of miRNAs in plant defense signaling by regulating and fine-tuning multiple plant hormone pathways. In addition, we compared the results from sequencing-based profiling of a small set of miRNAs with the results from small RNA Northern blot and that from miRNA quantitative RT-PCR. Our results showed that although the deep-sequencing profiling results are highly reproducible across technical and biological replicates, the results from deep sequencing may not always be consistent with the results from Northern blot or miRNA quantitative RT-PCR. We discussed the procedural differences between these techniques that may cause the inconsistency.

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.

BACKGROUND: MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes. RESULTS: We report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes. CONCLUSIONS: We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.