The Role Of Several Antioxidant Stress Related Genes In Fusarium Oxysporum F. Sp. Cubense4(Foc4)

Fusarium wilt of banana (Panama disease), caused by Fusarium oxysporum f. sp. cubense (Foc), is a destructive disease that has caused the greatest damage to banana crops worldwide. The results to research the molecular basis of this disease not only help us to understand the pathogen-host plant interaction, but also provide theoretical guidance for cultivating new varieties for resistance to Fusarium wilt of banana and provide new targets for exploiting new fungicide and ultimately control of banana wilt disease hazards.During plant-pathogen interactions, host plants have to develop a broad range of defence responses to cope with pathogenic infections. The oxidative burst, a rapid, transient, production of huge amounts of reactive oxygen species (ROS), is one of the earliest observable aspects of a plant’s defence strategyIn this paper, the oxidative burst, an early plant response in roots of banana tissue culture plantlets by Fusarium oxysporum f. sp. cubense (Foc) was observed. Six genes (four Catalases, a Foatf1and a Foap1) in Foc4were cloned and their roles in growth, development and pathogenicity were partly analyzed by targeted gene replacement.The results are shown as follows:1. Six genes (four Catalases, a Foatf1and a Foap1) were identified by bioinformatics. The cDNA sequence of Foatfl, Foapl and four Catalases were obtained after cloning and sequencing using reverse transcription PCR. Genes structure of Catalase P2, Foatf1and Foap1were determined after sequences alignment between the genome sequences of Foc4and the cDNA sequences of these three genes.2. The deletion mutants of Foatf1, Catalase P2and Foap1were created using a gene-replacement approach, respectively, and were validated by PCR and southern blot analysis.3. The relative expression level of Catalase P2is the highest in comparsion with other three Catalases, however, both of△Catalase P2-1and△Catalase P2-2 (Catalase P2deletion mutants) showed no sensitivity to exogenous oxidative stress and not attenuated the virulence of Foc4;A. There were no significant differences in colonial morphology between the Foatfl mutant and wild-type B2strain. However, both spore quantity and growth ability were weaker than the wild-type B2strain with shaking condition. Foatfl deletion caused smaller spores, delayed spore germination. Pathogenicity of the Foatfl mutant were less than the wild-type B2strain both in roots and on leaves in vitro of banana tissue culture plantlets. The attenuated virulence of the Foatfl mutant strain could be complemented and full virulence restored by reintroducing the Foatfl gene. The Foatfl deletion mutant showed sensitivity to oxidative stress and osmotic stress. The results of real-time PCR showed that Foatf1deletion decreased the activity of Catalases;5. The H2O2and superoxide. anion (O2-) contents in roots of banana tissue culture plantlets infected by Foatfl mutant and wild-type B2strain were detected using titanium sulfate and hydroxylamine oxidation methods. DAB staining was used to examine H2O2accumulation in roots of banana tissue culture plantlets after infection by Foc4. The results showed that "oxidative burst" is an early plant response in roots of banana tissue culture plantlets6. There were no significant differences of the morphology of mycelium and conidia between the Foapl mutants and wild-type B2strain, but the aerial hyphae became rare. The pathogenicity detection both in roots and on leaves in vitro showed Foapl deletion attenuates the virulence of Foc4. However, the Foapl deletion mutants were not sensitivity to oxidative stresss suggested that Foapl was not involved in regulating Oxidative Stress Responses of host.The results substantiate the conclusion that the two genes(Foatfl and Foapl) are involved in the Pathogenesis in Foc4. And Foatfl, a bZIP transcription factor, related to resistance to environmental stress, plays a pivotal role in surviving in harsh environments produced by the roots of banana tissue culture plantlets during early plant response to pathogen(Foc4) infection and successfully invading host cells. Foapl is also a bZIP transcription factor involved in the Pathogenesis in Foc4, however, loss of Foapl function cann’t resulted in increased sensitivity to external oxidative stresses compared with Yap1in Saccharomyces cerevisiae and Moapl in Magnaporthe oryzae. In four Catalases, Catalase P2may play a major role in eliminating H2O2of ROS. Catalase P2deletion mutants showed no sensitivity to exogenous oxidative stress and were not attenuated the virulence of Foc4because of the existence of several isozymes.