Identification Of MicroRNA-like RNAs In The Filamentous Fungus Fusarium Oxysporum F.Sp.Cubense

Banana Fusarium wilt disease is a typical soil-borne fungal disease caused by Fusarium oxysporum f. sp. cubense (Foc), which can spread rapidly and seriously threat the normal production of banana cultivation in China. Foc has long-term infection ability and complex pathogenic processes, which made it difficult to control effectively by traditional control methods, such as, biological control, soil treatment and crop rotation. Therefore, cultivating disease-resistant varieties is one of the fundamental way to solve banana fusarium wilt.MicroRNA-like RNA (milRNA) is one kind of non-coding small RNA in fungi,which can induce gene silencing and inhibit the immune system of host plants through RNAi pathway, playing an important role in the regulation of fungal pathogenesis and immune process of plant.Based on the long-term studies on Foc, this study sequenced and analyzed four samples of Foc1 conidia, Foc1 mycelial, Foc4 conidia and Foc4 mycelial using Solexa sequencing technology. Tobacco instantaneous expression method was applied to improve the expression of milRNAs in low abundance of Foc, and the candidate milRNAs were further experimentally validated by Northern blot. The above studies lay a solid foundation for the further research of the Foc infection related milRNAs’biological function during the infection process, and they are also of great significance to the subsequent construction of transgenic plants with milRNAs as RNAi gene silencing target. The main results of this study are as follows:1. The four samples of Foc1 conidia, Foc1 mycelial, Foc4 conidia and Foc4 mycelial were sequenced by high-throughput sequencing technology, and a total of 93,204,898 small RNA were finally excavated. The sequence and base distribution of small molecular RNAs in the four samples had no significant difference, and the four samples in the sequencing had good consistency, indicating that the sequencing data were reliable enough to be used for follow-up experimental analysis. The proportion of the species and the quantities of small RNAs in the four samples were the lowest among all the samples, and the mainly length distribution of candidate milRNAs predicted by sequencing was 19-22 nt, Among the candidate milRNAs of the four samples, the first nucleotide was usually uracil, which almost don’t appear in the 2nd-4th position, and the preference of the tenth base to adenine was extremely high.2. A total of 354 candidate small RNAs were predicted by comparison with the database, 221 of which were known candidate small RNAs, including 133 small RNAs known in plants and 88 small RNAs known in fungi, 133 for new candidate small RNA. All sorts of small RNA among the four samples were different, showing that small RNA may be involved in the control of different physiological races and growth stages of Foc. A total of 2186 target genes were predicted from the 354 candidate small RNAs using the miRNA target genes forecast software TargetFinder.As for GO function category, most of the above target genes were classified to biological process. A total of 1261 target genes were annotated into 214 KEGG metabolic pathways, of which 1193 target genes were annotated into the metabolic pathway, regulating the amino acids, various enzymes and secondary metabolites in the metabolic process,thus realizing the control of the overall growth of fungi.3. It was verified that 20 small RNAs existed in Foc, using Northern hybridization technique, two of which were high-abundance expressing small RNA,and could be directly verified by Northern blotting, and tobacco instantaneous expression method was applied to improve the expression of the remaining 18 milRNAs in low abundance, which could further be verified by Northern blotting. The identification results showed that among the 20 small RNAs, 16 were known small RNAs and the other four were newly found small RNAs, they are novel-milR16,novel-milR31, novel-milR19 and novel-milR168. In subsequent work, these 20 small RNAs will be functionally analyzed, and the remaining 300 candidate small RNAs will be identified.