Exploring microRNA-like small RNAs in the filamentous fungus Fusarium oxysporum

Abstract

RNA silencing such as quelling and meiotic silencing by unpaired DNA (MSUD) and several other classes of special small RNAs have been discovered in filamentous fungi recently. More than four different mechanisms of microRNA-like RNAs (milRNAs) production have been illustrated in the model fungus Neurospora crassa including a dicer-independent pathway. To date, very little work focusing on small RNAs in fungi has been reported and no universal or particular characteristic of milRNAs were defined clearly. In this study, small RNA and degradome libraries were constructed and subsequently deep sequenced for investigating milRNAs and their potential cleavage targets on the genome level in the filamentous fungus F. oxysporum f. sp. lycopersici. As a result, there is no intersection of conserved miRNAs found by BLASTing against the miRBase. Further analysis showed that the small RNA population of F. oxysporum shared many common features with the small RNAs from N. crassa and other fungi. According to the known standards of miRNA prediction in plants and animals, milRNA candidates from 8 families (comprising 19 members) were screened out and identified. However, none of them could trigger target cleavage based on the degradome data. Moreover, most major signals of cleavage in transcripts could not match appropriate complementary small RNAs, suggesting that other predominant modes for milRNA-mediated gene regulation could exist in F. oxysporum. In addition, the PAREsnip program was utilized for comprehensive analysis and 3 families of small RNAs leading to transcript cleavage were experimentally validated. Altogether, our findings provided valuable information and important hints for better understanding the functions of the small RNAs and milRNAs in the fungal kingdom.

Figure 1. Characterization of small RNAs in F. oxysporum.

A: Size distribution of small RNAs. White and black columns represent unique and total reads of the small RNAs, respectively. B: Annotation of small RNA loci. Pie graphs show the proportions of small RNAs located in intergenic, exonic and intronic regions, respectively. C: Small RNA distribution on both strands of chromosomes.

Figure 2. milRNAs identified in this study.

A: Multiple sequence alignment of fox-milRNA precursors. Red sequences represent mature milRNAs and gray parts indicate low similarity. B: Secondary structures of fox-milRNA precursors. The red and blue sequences represent mature and star (milRNA*) sequences, respectively. C: Expression analysis of fox-milRNAs and small RNAs by RT-PCR experiments. Total RNA was extracted for seven-day-old mycelia and directly ligated with universal oligos at the 3′ end. After reverse transcription, specific forward primer and common reverse primer could detect a given small RNA. Small RNAs with high abundance are more apt to get positive results. Two bands of fox-milR-1 suggested that the precursor and mature sequences were both amplified and detected.

Figure 3. Scatter plot diagrams of degradome data on transcripts.

The 16 most abundant signals with high peak-to-total ratio (>0.8) were selected and illustrated. The x and y axis of each diagram represent position of transcripts and the frequency of tags, respectively. Each tag perfectly matching the transcripts was plotted. However, none of these peak signals correspond to predicted cleavage sites of any small RNAs of F. oxysporum. These truncated transcripts might be particularly stable decay intermediates or products of special endonuclease.

Figure 4. Illustration of three pairs of small RNAs and target transcripts.

Scatter plot diagrams show the frequency of tags and their positions on transcripts. The inferred cleavage sites were indicated by the asterisks and arrows. Although the abundance of tags and small RNAs was relative low, the corresponding small RNAs were experimentally validated by RT-PCR ( Fig. 2C ).