Gene Function Of Predicted Programmed Cell Death Protein 5 In Fusarium Oxysporum F.sp. Cubense Race 4

Fusarium wilt caused by Fusarium oxysporum f.sp.cubense（Foc）is the most serious and most difficult devastating disease in China.It is called"banana cancer".According to the pathogenicity of Foc on different banana varieties,it can be divided into 4races,of which the physiological race 4（Foc4）is the most harmful,it can harm almost all kinds of bananas.In the previous work,our laboratory conducted research on the secreted proteome of Foc4 and found that the expression of Program Cell Death Protein 5（PDCD5）in Foc4 was significant up-regulated after the induction of banana root tissue extraction.;PDCD5 is an important factor involved in the apoptosis process in eukaryotes,so we speculate that it may be related to the pathogenicity of Foc4.At present,there is no report about PDCD5 in plant pathogenic fungi.In this paper,the function of PDCD5 gene in Foc4 was studied for the first time through methods such as gene cloning,gene knockout and gene complement.The specific results are as follows:（1）The FoPDCD5 protein consists of 129 amino acids and contains the ds DNA＿binding domain.The signal peptide prediction shows that the protein does not have a signal peptide and is a secreted protein that is neither a classical nor a n on-classical secretory pathway.Through q RT-PCR technology,the expression level of programmed cell death protein 5 gene（FoPDCD5）in Foc4 was analyzed.The results showed that under the conditions of banana root tissue extraction induction,the expression level of FoPDCD5 in Foc4 was significantly up-regulated,which was consistent with the results of t with the results of the previous secreted proteome.（2）Using PEG-Ca Cl₂ mediated protoplast transformation method,through hygromycin screening,PCR analysis,Southern blot identification and other methods,the Foc4 gene knockout mutant△Fopdcd5 and the complement mutation△Fopdcd5-com were obtained.The results of phenotypic analysis showed that compared with the wild type of Foc4,there was no significant difference in the morphology of colonies,hyphae and conidia of△Fopdcd5 and△Fopdcd5-com,and there was no significant difference in the growth rate of colonies.The results of spore production analysis showed that,compared with Foc4 wild type,the spore production of△Fopdcd5 was significantly reduced,and the spore production of△Fopdcd5-com was restored to the level of Foc4 wild type;The expression of four spore-forming genes（FOIG＿00370,FOIG＿02194,FOIG＿02194,and FOIG＿07247）were analyzed by q RT-PCR technology.The results showed that the expression levels of spore-forming genes in△Fopdcd5 were significantly lower than those of Foc4 wild type and△Fopdcd5-com.（4）The results of oxidative stress analysis showed that compared with Foc4 wild type,the sensitivity of△Fopdcd5 to hydrogen peroxide was significantly increased,while the sensitivity of△Fopdcd5-com to hydrogen peroxide was not significantly different from that of Foc4 wild type.The expressions of three peroxidase-related genes（FOIG＿04532,FOIG＿08821,and FOIG＿09161）were analyzed by using q RT-PCR technology.The results showed that the peroxidase gene in△Fopdcd5 was significantly down-regulated.Further measurement of intracellular reactive oxygen species showed that the intracellular reactive oxygen content of△Fopdcd5 was higher than that of Foc4 wild type;the apoptosis test results showed that the apoptosis rate of△Fopdcd5 after hydrogen peroxide induction was higher than that of Foc4 wild Type,but did not reach a significant level of difference.The results of DAB staining experiments showed that the burst of reactive oxygen in banana root cells infected with△Fopdcd5 was higher than that of Foc4 wild type and△Fopdcd5-com.And the active oxygen level of△Fopdcd5-com is similar to Foc4 wild type.（5）Using tomato and apple as materials,the invasion and colonization ability of△Fopdcd5 was tested;the results showed that the area of lesions inoculated with△Fopdcd5 was significantly smaller than that of Foc4 wild type;The area of the lesions also decreased significantly,and the mycelium on the surface of the lesions was significantly reduced.The symptoms after inoculation with△Fopdcd5-com were similar to those of the wild type Foc4,indicating that the knockout of FoPDCD5 affected the invasiveness and colonization ability of Foc4.（6）Using Brazil banana as material,the pathogenicity analysis of△Fopdcd5 was carried out.The results showed that the pathogenicity of△Fopdcd5 was significantly lower than that of Foc4 wild type,while the pathogenicity of△Fopdcd5-com returned to the level of Foc4 wild type.The expression of FOIG＿16453,a gene related to fusaric acid synthesis,was further analyzed using q RT-PCR technology.The results showed that the expression of FOIG＿16453 was significantly lower than that of Foc4 wild type.It is speculated that the knockout of FoPDCD5 may affect its pathogenicity by reducing the synthesis of fusaric acid.（6）Using q RT-PCR technology,the expression of Brazil banana defense related genes（Ma PR1,Ma PR3 and Ma PR4）after△Fopdcd5 inoculation was analyzed.The results showed that the banana seedlings infected by△Fopdcd5 were compared with wild type The expression levels of Ma PR1 and Ma PR4 genes were significantly increased.The expression of Ma PR3 was not significantly different between the wild type and△Fopdcd5.