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MicroRNAs (miRNAs) processed from nuclear-encoded transcripts control expression of target transcripts by directing cleavage or translational inhibition. Artificial miRNAs (amiRNAs) that exploit this endogenous gene silencing mechanism can be designed to target any gene of interest and provide a highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants. Here, using Arabidopsis thaliana miR159a, miR167b and miR171a precursors as backbones, we designed two types of amiRNA targeting sequence that encode the silencing suppressor HC-Pro of Potato virus Y (PVY) and the TGBp1/p25 (p25) of Potato virus X (PVX). The detected amiRNAs efficiently inhibited HC-Pro and p25 gene expression and conferred highly specific resistance against PVY or PVX infection in transgenic Nicotiana tabacum; this resistance was also maintained under conditions of increased viral pressure. Moreover, resistance was strongly influenced by the complementarity between the target sequence and amiRNA, and was well correlated to amiRNA expression level; the expression level of amiRNAs was also well related to the precursor backbones. We further showed that transgenic N. tabacum developed highly effective resistance to both PVY and PVX through expression of a dimeric amiRNA precursor. Together, our findings indicate that transgenic plants with multiple virus-specific resistance can be obtained through co-expression of several amiRNAs targeting multiple viruses.

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Plant microRNAs (miRNAs) act as negative regulators of gene expression by slicing target transcripts or inhibiting translation. A number of miRNAs play important roles in development. In order to investigate the potential function of miRNAs during male gametogenesis in rice, we obtained both gene and small RNA expression profiles by combining microarray and high-throughput sequencing technologies. From the microarray datasets, 2,925 male gametophyte-specific genes were identified, including 107 transcription factors and three significant Argonaute genes (AGO12, AGO13, and AGO17). From the sRNA-Seq datasets, 104 unique miRNAs (miRus) were identified, including 47 known miRus and 57 novel miRus; interestingly, most of the new miRus are pollen-specific and not conserved among species. Furthermore, an interactive network of miRNA-target was constructed based on the two datasets. By employing enrichment analysis, the miRNA-regulated targets were found to be involved in both the up and down pathways, but predominantly in the down pathways, including 37 GO biological processes and 32 KEGG pathways. These findings indicate that miRNAs play a broad regulatory role during male gametophyte development in rice.

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