Alternative Splicing And Functional Analysis Of FoYjeF And FoCaMK Genes Of Fusarium Oxysporum

Crop wilt caused by Fusarium oxysporum is a typical soil-borne vascular disease.On the one hand,diseases occur frequently and cause serious damage,which is difficult to control due to its factors such as a wide range of pathogenic bacteria hosts,obvious pathogenicity differentiation,long survival time of primary infection sources in the soil and widespread crop continuous cropping.On the other hand,the pathogenic mechanism of F.oxysporum is complex and the interaction mechanism between F.oxysporum and host plants in different disease systems is still unclear,which further increases the difficulty of disease-resistant breeding against Fusarium wilt and microecological control based on pathogenic bacteria-host plants.In this study,F.oxysporum f.sp.momordicae strain was used as material to perform transcriptome sequencing on the highly pathogenic strain and the weakly pathogenic strain carrying the virus Fo Ou LV1.And we have analyzed the alternative splicing events that occurred in the transcriptome sequencing.Two of the genes FoYjeF and FoCaMK differentially expressed in alternative splicing patterns were selected for functional analysis,and the results are as follows:1.4009 alternative splicing events were identified from the transcriptome sequencing of F.oxysporum f.sp.momordicae strains with different pathogenicity,accounting for 7.4% and 15.5% of the detected genes and transcripts,respectively.Alternative splicing events include five types,among which intron retention(RI)and exon skipping(SE)account for the highest proportion;among 4009 alternative splicing events,1465 are newly discovered alternative splicing events.The SE type found the most new alternative splicing events,with 1163.On the whole of the alternative splicing events found,there were 3.31 alternative splicing events per gene on average,and the average values of alternative splicing events of RI and SE types were the highest.2.The full length of the FoYjeF gene is 1268 bp,and there is an alternative splicing pattern in the sixth intron,which is significantly reduced in F.oxysporum strain carrying the virus Fo Ou LV1.The results of gene function analysis showed that after the FoYjeF gene was knocked out,the growth rate and sporulation of the mutant increased significantly,the sensitivity to carbendazim and oxidative stress increased significantly,and its pathogenicity was significantly enhanced compared with the wild-type strain and the onset time of plants was advanced;the results of ROS accumulation effect test showed that the peroxidase activity of the FoYjeF gene knock-out mutant was significantly increased,and the ROS burst reaction at the infection site of the treated bitter gourd leaves was significantly reduced;the results of q RT-PCR assay results showed that the sporulation and pathogenicity-related homologous genes Type II myosin and WOR1 were significantly up-regulated in the FoYjeF knock-out mutant,the expression of the ROS-related gene Nox R was significantly down-regulated,and the expression of Nox A was significantly up-regulated.It shows that the FoYjeF gene participates in the regulation of physiological and biochemical processes such as vegetative growth,sporulation and ROS of F.oxysporum,and thus negatively regulates its pathogenicity.3.The full length of the FoCaMK gene is 862 bp,and there are two intron-reserved alternative splicing patterns,which are significantly enhanced in the weakly pathogenic F.oxysporum strain carrying the virus Fo Ou LV1.The results of gene function analysis showed that after the FoCaMK gene was knocked out,the growth rate and colony morphology of the mutant were not significantly different from those of the wild-type strain,but the amount of sporulation was significantly reduced,the tolerance to the cell membrane damage disruptor(SDS)was significantly reduced,and the pathogenicity was significantly reduced;FoCaMK gene had four spliced transcripts in F.oxysporum,the analysis of the spliced sequence suggests that GATGTA is the splice recognition site;signal peptide test and tobacco transformation tests showed that when the first intron next to the signal peptide region of the gene was spliced,FoCaMK could be secreted out of the cell,and could cause immune necrosis in tobacco leaves,which indicates that FoCaMK gene might be an effector protein.;after overexpressing the four splicing forms of the FoCaMK gene,the pathogenicity test shows that when FoCaMK was secreted out of the cell,the pathogenicity was enhanced.It can be seen that the FoCaMK gene is involved in the regulation of the sporulation process of F.oxysporum,thus positively regulates its pathogenicity,and the secretion function of FoCaMK is related to the pathogenicity of F.oxysporum.