Screening And Functional Analysis Of Small RNA Involved In Pathogenicity Of Fusarium Oxysporum F.sp. Cubense Race 4

Fusarium wilt caused by Fusarium oxysporum f.sp.cubense race 4(FOC4)has become one of the most destructive banana disease worldwide.Due to the extensive banana production and soil-borne character of FOC4,without effective prevention measures,the Fusarium wilts rapidly spreaded across the world.Therefore,the molecular mechanisms underlying the virulence and pathogenicity of this fungal pathogen could provide more evidence to control this disease.As information about function of small RNA(s RNA)from FOC was liminted,we conducted s RNA sequencing and get a large number of candidate s RNA through bioinformatics analysis.In order to identify the s RNA related to pathogenicity in FOC4,q RT-PCR was used for s RNA screening.And those RNAs with significantly up-regulated after inoculation were selected for further knock out and overexpress experiment.The main results are as follows:1.Two up-regulated milRNAs(milR70 and milR75)during infection were selected from 31 candidate s RNAs which were consistant with the results of s RNA sequencing.s RNA structure prediction showed that stem and loop structure is common exited.And the two s RNA are located in the intergenic region of the FOC4 genome.2.Via PEG-mediated protoplast transformation,milR70 precursor and milR75 precursor were knocked out from wild type XJZ2 by homologous recombination,respectively.Mil R70 deletion mutants and milR75 deletion mutants were obtained and examined by resistance screening and PCR detection.3.The phenotypic analysis of milR70 deletion mutants was conducted.The colony of milR70 deletion mutants showed thin aerial hypha,poor production of conidia comparing to wildtype.In assessment of stress tolerance,milR70 deletion mutant displayed less sensitive to the cell wall perturbing agents Congo red and Calcofluorwhite compared to wildtype strain.In virulence assays,milR70 deletion mutant showed virulence defective to the banana seedlings.4.The phenotypic analysis of milR75 deletion mutants was conducted.The colony of milR75 deletion mutants showed less aerial hypha and lower dry weight of mycelia than wildtype.The conidia production of milR75 deletion mutant was lower and conidia were less heat tolerant comparing to wildtype.In assessment of stress tolerance,milR75 deletion mutant displayed less sensitive to Congo red and Calcofluorwhite compared to wildtype strain.In virulence assays,milR75 deletion mutant showed virulence defective to the banana seedlings.Our data demonstrate that the two predicted micro RNA like RNA milR70 and milR75 probably involved in mycelia growth,conidia production and virulence to banana.5.In order to further verify the function of target small RNAs,according to their precursors,small RNA over expression vectors were constructed and transformed into wildtype strain XJZ2 genome,and the over expression mutants of milR70 and milR75 were verified by resistance screening and PCR detection.The phenotype of over expressed mutant was found to have no significant difference in spore yield,growth of aerophytic hyphae,cell wall sensitivity,and pathogenicity compared to wild type XJZ2.These results indicated that milR70 and milR75 precursors in FOC4 were involved in the growth,sporulation and pathogenicity of the host.