Screening And Functional Analysis Of MicroRNA Involved In Pathogenicity Of Fusarium Oxysporum F.sp.cubense

MicroRNA(miRNA)is a kind of single-stranded non-coding small RNA(s RNA),which plays an important role in the growth,development and various stress-resistant responses of eukaryotes.Although a class of s RNAs produced in fungi has been found to be very similar to micro RNAs in plants and animals,they are called micro RNA-like RNAs(milRNAs),the research on their functions,especially on pathogenicity,is very limited.Banana wilt caused by Fusarium oxysporum f.sp.cubense(Foc)is a destructive disease that restricts banana production worldwide.Some progress has been made on pathogenesis of the pathogen,but the regulatory role of miRNAs in Foc when infecting host banana during the pathogenic process has not been reported.The purpose of this study was to screen and identify milRNAs related to pathogenicity in Foc,and to clarify the functions of the candidate milRNAs.The results are as follows: 1.High-throughput sequencing of s RNA in Foc race 4 at the cultural condition and 36 hours post inoculation,as well as its bioinformatic data analysis.The s RNA database of Foc was obtained by high-throughput sequencing at cultural condition and 36 hours post inoculation.The results of bioinformatic analysis showed that there were 1,280 differentially expressed s RNAs sequences when 36 h post inoculation compared with cultural condition.Among them,953 s RNAs sequences were up-regulated significantly.Through prediction of stem and ring structure of s RNA precursor,108 candidate milRNAs with high expression after infection were screened and their expression were detected by q RT-PCR.2.Validation and synthesis pathway analysis of up-regulated milRNAs after inoculation.The candidate 108 milRNAs were validated by q RT-PCR.Eleven milRNAs with stable and high expression after inoculation were screened by q RT-PCR.Three milRNAs with stem and ring secondary structure in their precursors were cloned by reverse transcription PCR.Small RNAs milR87 and milR106 were 22 nt in length and were belong to typical milRNA,while s RNA milR72 was 107 nt in length and were belong to presumably long non-coding RNA(lnc RNA).It was found that the synthesis of milR87 depends on Argonaute and Dicer,while the synthesis of milR106 depends on Dicer itself.The synthesis of milR72 is related to Argonaute.3.Screening and identification of the knockout and over-expression mutants of target milRNA in Foc.The homologous recombination of precursor fragments of the target milR87,milR72,and milR106 were carried out by PEG mediated protoplast transformation.The knockout mutants of milR87,milR106,and milR72 were screened by PCR analysis.The expression of the target milRNAs were not tested in their corresponding mutants by q RT-PCR.In order to study the functions of milRNAs,over-expression vectors were constructed.The over-expression transformants of milR87,milR72,and milR106 were also obtained by The PEG mediated protoplast transformation.The q RT-PCR results showed that the relative expression levels of the milRNAs in these transfotmants were significantly higher than that of wild type strain XJZ2.4.Phenotype analysis of the target milRNAs mutants.There was no significant difference in colony morphology,growth rate and mycelial growth between knockout and overexpression mutants of milR87,milR106 and the WT strain.However,milR72 mutants grew slower than the WT strain,and the growth rate of milR72 over-expression transformant was the same as that of the WT.The conidiation of milRNAs knockout mutants(ΔmilR87,ΔmilR72,and ΔmilR106)were significantly decreased compared to the WT,but increased in the over-expression transformants.Under the condition of hydrogen peroxide,the growth of the knockout mutants was significantly inhibited and was restored to the level of the WT after over-expression of corresponding milRNAs.There was no significant difference between the knockout mutants and the WT strain when cultured on PDA supplemented with cell wall inhibitor Congo Red and Calcofluor White.5.Pathogenicity of the target milRNA mutants.Pathogenicity tests showed that the target milRNAs knockout mutants(ΔmilR87,ΔmilR72,and ΔmilR106)almost couldn’t penetrate through cellophane membrane on MM,failed to produce remarkable macerated symptoms on tomato fruits,and showed a dramaticlly reduced virulence to host banana seedling compared with the WT.While the virulence of the over-expressed tansformants was restored to the level of WT.The above results show that milR87,milR106 and milR72 in Foc are all involved in the growth,development,conidiation,and stress responses.They also participate in the regulation of pathogenicity during the early infection process.