Pathogenic Function Analysis Of Focfb1and Foccak1 From Fusarium Oxysporum F.sp. Cubense Race 4

Banana fusarium wilt is a kind of destructive soil borne fungal disease caused by Fusarium oxysporum f.sp.cubense,which result in destructive extermination of banana production and threat the development of banana industry.race 1 of the pathogen(Foc1)mainly wiped out“Gros Michel”,now which belongs to the world of distribution.Race 4(Foc4)can almost infests all banana species,which is the most dangerous disease.At present,there is a lack of effective control measures against Fusarium oxysporum f.sp.cubense.It is particularly important to study pathogenic genes from molecular level and reveal their pathogenic mechanism.In this study,The difference analysis of Foc1 and Foc4 transcripts of Banana Fusarium Wilt in 3 different carbon sources(host cell wall,glucose,pectin)was completed.On this basis,the results of the FOC transcriptome(Foc4_CCW vs Foc1_MCW)are compared with the PHI(The Pathogen-Host Interaction Database)database BLAST to select the genes with higher relative expression of Foc4 than Foc1,and predict the related genes.I pricked off four genes from them:comp18499_c0,comp14433_c0,comp 16617_c0,comp17473_c0.comp18499_c0 is a serine/threonine protein kinase gene.comp14433_c0programmed death associated protein.comp 17473_c0 is protein kinase gene.comp16617_c0 is F-box protein gene.The primers were designed to verify the expression of these 4 genes in the process of Banana Fusarium wilt infecting bananas by real-time fluorescence quantitative analysis,and selected three time points,which were 0h,48h and 96h respectively.The results showed that the expression of comp 16617_c0 gene was almost not expressed in Foc1,but the expression in Foc4 was high.Compared with 0h,the expression of 48h and 96h was significantly up,especially in 48h,the highest expression was 200times than 0h.Although the Foccak1 gene was expressed in Foc1,the expression was significantly lower than that in Foc4.Compared with 0h in Foc4,the expression of Foccak1was at a high level.Therefore,it is speculated that the comp 16617_c0 and comp 17473_c0genes are related to the pathogenicity of Fusarium oxysporum f.Foc4,and the pathogenicity function of these two genes is significant.The Foc4 total DNA was as template toclone,and the genes of comp 16617_c0 and comp 17473_c0were obtained,and sent to the biological company for sequencing,and their DNA and cDNA sequences were obtained.Bioinformatics analysis was carried out according to the sequence of target genes.The DNA amplification products of Foc1 and Foc4 of comp 16617_c0 were about1080bp,cDNA amplification products 1080bp and DNA encoded 358 amino acids.The results of comp 16617_c0 nucleotide analysis in 2 small species showed that there was no intron in Foc1-comp 16617_c0 and Foc4-comp 16617_c0,and the homology of the two small nucleotides was 99%.The encoding amino acid homology is 99.2%.The comp16617_c0gene has three differences at the amino acid level in the two races,which are at the positions 96,202,and 322.There are 11 differences in the encoded nucleotides.The BLAST alignment of the amino acid sequence of comp 16617_c0on NCBI showed that the gene did not detect putative conserved regions.The DNA amplification products of Foc1 and Foc4 of comp 17473_c0 were approximately 1444 bp and the amplified products cDNA were all 1332 bp.The nucleotide analysisof comp17473_c0 of the two races showed that Foc1-comp 17473_c0and Foc4-comp 17473_c0had one intron,Foc1-comp 17473_c0DNA encodes 460 amino acids and the Foc4-comp 17473_c0DNA encodes 462 amino acids.The homology of Foccak1 in two minor nucleotides is 99.2%.The encoded amino acid homology was 97.8%.comp17473_c0There are 10 differences between the two races at the amino acid level,and there are 12 differences in the encoded nucleotides.The amino acid sequence of Fusarium oxysporum comp 17473_c0was placed on NCBI for BLASTP alignment.The results showed that Foccak1 belongs to the CCL1 family protein and 100-281aa is its conserved region.The flanking fragments of Foc4-Focfb1 and Foc4-Foccak1 genes were obtained by TAIL-PCR,and the Foc4-ΔFocfb1 and Foc4-ΔFoccak1genedeletionmutant were obtained by ATMT method,and the pathogenicity of the mutants was determined.The results showed that knockout mutants of the Foc4-ΔFocfb1 and Foc4-ΔFoccak1 genes had almost no change in biological traits compared to wild-type Foc4.The wild-type Foc4 and knockout mutants Foc4-ΔFocfb1 and Foc4-ΔFoccak1 were inoculated to the Brazilian banana plants using potted root-injection and inoculation methods.The spore concentrations were 1.0×10~5cells/mL and 1.0×10~6 cells/mL,respectively.1.0×10~7 cells/mL were inoculated with 10strains per treatment,and each treatment was repeated 3 times.After 30 days of inoculation,the incidence was observed.The results showed that the disease index of Brazilian bananas vaccinated with Foc4-ΔFocfb1 was 10%lower than that of wild-type Foc4,indicating that the knock-out of Focfb1 had a reduced effect on the virulence;the Brazilian banana disease index of Foc4-ΔFoccak1 was inoculated.The disease index of wild-type Foc4 was lower,indicating that knockout of the Foccak1 gene,although not affecting the normal growth of Foc4,weakened the pathogenicity to some extent.In summary,this study enriches the molecular mechanism of interaction between Foc and host,which provide scientific basis for the selection of pathogenic genes related to Fusarium oxysporum f.