fIdentification of B. napus small RNAs responsive to infection by a necrotrophic pathogen.

BACKGROUND: Small RNAs are short non-coding RNAs that are key gene regulators controlling various biological processes in eukaryotes. Plants may regulate discrete sets of sRNAs in response to pathogen attack. Sclerotinia sclerotiorum is an economically important pathogen affecting hundreds of plant species, including the economically important oilseed B. napus. However, there are limited studies on how regulation of sRNAs occurs in the S. sclerotiorum and B. napus pathosystem. RESULTS: We identified different classes of sRNAs from B. napus using high throughput sequencing of replicated mock and infected samples at 24 h post-inoculation (HPI). Overall, 3999 sRNA loci were highly expressed, of which 730 were significantly upregulated during infection. These 730 up-regulated sRNAs targeted 64 genes, including disease resistance proteins and transcriptional regulators. A total of 73 conserved miRNA families were identified in our dataset. Degradome sequencing identified 2124 cleaved mRNA products from these miRNAs from combined mock and infected samples. Among these, 50 genes were specific to infection. Altogether, 20 conserved miRNAs were differentially expressed and 8 transcripts were cleaved by the differentially expressed miRNAs miR159, miR5139, and miR390, suggesting they may have a role in the S. sclerotiorum response. A miR1885-triggered disease resistance gene-derived secondary sRNA locus was also identified and verified with degradome sequencing. We also found further evidence for silencing of a plant immunity related ethylene response factor gene by a novel sRNA using 5'-RACE and RT-qPCR. CONCLUSIONS: The findings in this study expand the framework for understanding the molecular mechanisms of the S. sclerotiorum and B. napus pathosystem at the sRNA level.

Coupling of microRNA-directed phased small interfering RNA generation from long noncoding genes with alternative splicing and alternative polyadenylation in small RNA-mediated gene silencing.

MicroRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) play vital regulatory roles in plant growth and development. Little is known about these small RNAs in litchi (Litchi chinensis), an economically important fruit crop widely cultivated in Southeast Asia. We profiled the litchi small RNA population with various deep-sequencing techniques and in-depth bioinformatic analyses. The genome-wide identification of miRNAs, their target genes, and phasiRNA-generating (PHAS) genes/loci showed that the function of miR482/2118 has expanded, relative to its canonical function. We also discovered that, for 29 PHAS loci, miRNA-mediated phasiRNA production was coupled with alternative splicing (AS) and alternative polyadenylation (APA). Most of these loci encoded long noncoding RNAs. An miR482/2118 targeted locus gave rise to four main transcript isoforms through AS/APA, and diverse phasiRNAs generated from these isoforms appeared to target long terminal repeat (LTR) retrotransposons and other unrelated genes. This coupling enables phasiRNA production from different exons of noncoding PHAS genes and yields diverse phasiRNA populations, both broadening and altering the range of downstream phasiRNA-regulated genes. Our results reveal the diversity of miRNA and phasiRNA in litchi, and demonstrate AS/APA as a new layer of regulation in small RNA-mediated gene silencing.