Pathogenesis Of Fusaric Acid And Effector FocCP1 Byfusarium Oxysporum F. Sp. Cubense

Banana(Musa spp.)is one of the most important economic crops for many Asian and African countries.Fusarium oxysporum f.sp.cubense(Foc)causes Fusarium wilt(Panama disease),one of the most destructive diseases of banana trees.Foc tropical race 4(TR4)is highly virulent on almost all banana cultivars,whereas efficient strategies for its management are not available to date,and the understanding of its pathogenesis is still rudimentary.Foc TR4 applies several strategies to overcome plant immue systems,such as producing toxic secondary metabolites and effectors.Studies on pathogenesis mechanism of Foc TR4 within its virulence factors could provide a therotical basis for preventing and controlling the disease.Here,we first analyzed the evolution charactersistics of the FSA biosynthetic genes,which could help us understand how Fo isolates have evolved to accommodate different host types and enviroments;then we investigated the underlying mechanism of the virulence factors,FSA and FocCP1,in Foc TR4 pathogensis.The detailed results are as follows:1.Genetic divestiy in FUB genes of Foc suggest horizontal gene transferTo investigate the evolution characteristics of the 12 Foc FSA biosynthetic genes(FUB),coding sequences of the 12 FUB genes and 3 housekeeping genes,EF-1?/RPB1/RPB2(translation elongation factor-1?/RNA polymerase II subunit I/RNA polymerase II subunit II),were subjected to genetic diversity analysis,phylogenetic analysis,recombination detection and selective pressure analysis.The results of selective pressure analysis showed that the 15 genes were mainly subjected to negative selection.However,significantly higher number of silent mutations,which could not be simply explained by selective pressure difference,was observed in the 12 FUB genes in Foc than in the three housekeeping genes.Infraspecies phylogeny and recombination detection analysis showed that significantly more horizontal gene transfer(HGT)events(normalized)had occurred in the FUB genes than in the three housekeeping genes.In addition,many of these events involved outgroup isolates and significantly increased the genetic diversity of FUB genes in Foc.The infraspecies phylogenetic analysis suggested that the polyphyletic phylogeny proposed for Foc requires further discussion,and the divergence of race 1,race 4 and the common ancestor of several Fusarium oxysporum(Fo)isolates pathogenic to nonbanana plants should have diverged over a short period.Finally,our results suggest that the FUB genes in Fo should have benefited from HGT to gain a relatively high genetic diversity to respond to different host plants and environments despite mainly being subject to negative selection.2.FSA instigates the invation of banana by Foc TR4This study revealed that FSA is dispersed into the host plant tissue before the arrival of the invading fungal hyphae.Mutation of key genes in the FSA bio-synthetic gene(FUB)cluster in Foc TR4 abolished the FSA production and resulted in decreased disease symptoms and reduced fungal biomass in the host banana plants.When pretreated with FSA,both banana leaves and pseudostems exhibited increased sensitivity to Foc TR4 invasion.Banana embryogenic cell suspensions(ECSs)treated with FSA exhibited a lower rate of O2 uptake,loss of mitochondrial membrane potential,increased rates of reactive oxygen species(ROS)accumulation,nuclear condensation and cell death.Overall,our results demonstrated that the FSA from Foc TR4 acts as a positive virulence factor that functions at the forefront of the disease movement ahead of actual fungal invasion.3.FocCP1 is essential for the penetration and virulence of Foc TR4We demonstrate that a cerato-platanin(CP)protein,FocCP1,functions as an effector that is required by Foc TR4 for penetration and full virulence.The FocCP1 gene was expressed in every condition studied,showing the high transcript level in planta at the early stage of infection.Infiltration of recombinant FocCP1 protein induced significant cell death and upregulated defence-related gene expression.FocCP1 knock-out strains showed a significantly decrease on aerial growth rather than aqueous growth,which is reminiscent of hydrophobins.Furthermore,deletion of FocCP1 significantly reduced virulence and dramatically reduced infective growth in banana roots,likely resulting from a defective penetration ability.Taken together,the results of this study provide novel insight into the function of the CP protein,a newly identified effector in Foc TR4.