Functional Analysis Of Three Responsive Genes Of Lasiodiplodia Theobromae During Infection

Peach?Prunus persica?gummosis is one of the most detrimental diseases to peaches in southern China,leading to enormous economic losses to peach production.Lasiodiplodia theobromae is one of the important causal agents for fungal gummosis in peach.So far,the molecular mechanism underlying the pathogenic gummosis remains unclear.Reactive oxygen species?ROS?play pivotal roles in plant-pathogen interactions;however,the changes of ROS production-scavenging systems and the role of ROS in the pathogenesis of peach gummosis,are largely unknown.In addition,host plants generally produce large amounts of ROS in response to foreign pathogen invasion that will threaten pathogens.In turn,pathogens have evolved sophisticated ROS scavenging systems to minidish and avoid excess oxidative damage.AP1?Activating protein 1?,a b ZIP transcription factor,is one of the main redox sensors in response to oxidative stress and modulates a number of antioxidation genes to neutralize excess ROS.In many fungal phytopathogens,AP1 mediated ROS detoxification plays a crucial role in pathogenicity.Therefore,functional characterization of Lt AP1 in ROS detoxification is of great importance to better understand the pathogenic mode of L.theobromae-induced peach gummosis.This study investigated the role of ROS in L.theobromae-induced peach gummosis through an integrative analysis from physiological and biochemical changes,ROS metabolism and gene expression.Then,transgenic and biochemical approaches were used to functionally characterize Lt AP1 and the genes regulated by Lt AP1.In the end,the work aims to provide a new way for the effective control against peach gummosis.The main results are as follows:1. The infection by L.theobromae led to a ROS burst and activated antioxidant enzyme-dependent scavenging system in host plant.With disease progression,the capacity of plant antioxidant machinery declined,and allowed for ROS accumulation and eventual malondialdehyde production.As for the fungus L.theobromae,the transcripts of genes related to ROS production were significantly repressed,and concomitantly the expression of genes related to antioxidant systems and oxidative stress resistance was markedly up-regulated.Moreover,genes involved in phytohormones biosynthesis and pathogenesis-related proteins in peach shoots were all markedly higher than the control.Altogether,ROS production and accumulation may play an important role in peach gummosis development.2. A b ZIP transcription factor,Lt AP1,in L.theobromae,a homolog of Saccharomyces cerevisiae YAP1 that regulates the oxidative stress response,was functionally characterized.The transcripts of Lt AP1 were significantly induced by hydrogen peroxide?H₂O₂?or tert-butyl hydroperoxide?t-BHP?exposure.Deletion of Lt AP1??Lt AP1?caused retarded vegetative growth of mycelia,and exhibited higher sensitivity to oxidative and nitrosative stresses than the wild-type strain.The?Lt AP1mutant was more tolerant to cell wall damaging stress and high osmotic stress than the wild-type strain.The?Lt AP1 mutant had dramatically reduced transcription levels of genes in glutaredoxin and thioredoxin systems as compared to the wild-type strain,particularly in the presence of H₂O₂ or during infection.Moreover,the?Lt AP1 mutant showed significantly reduced virulence on peach shoots as compared to the wild-type strain.The ROS contents and transcripts of plant defense-related genes were strongly enhanced in plant shoots infected by the?Lt AP1 mutant strain when compared to the wild-type strain.In a word,the transcription factor Lt AP1 involved in oxidative stress responses,and could regulate several antioxidation genes to neutralize ROS and then affect pathogenicity of L.theobromae.3. Gene Lt GPX3 encoding glutathione peroxidase for redox homeostasis,was down-regulated in the?Lt AP1 mutant and functionally characterized in this study.Lt GPX3 encodes a protein with an open reading frame of 167 amino acids interrupted by one intron.Lt GPX3 transcripts in the wild-type strain were highly induced by H₂O₂,t-BHP,or Cumene H₂O₂ treatment than the wild-type strain.Deletion of Lt GPX3??Lt GPX3?resulted in more sensitive to H₂O₂,t-BHP,Cumene H₂O₂,or SNP?Sodium nitroferricyanide dihydrate?than the wild-type strain.Again,the?Lt GPX3 mutant was more sensitive to high salt stress than the wild-type strain.However,the?Lt GPX3 mutant was more tolerant to high osmotic stress and cell wall damaging stress than the wild-type strain.Pathogenicity assay indicated that the deletion of Lt GPX3 severely attenuated the virulence of L.theobromae on peach shoots.In addition,ROS production and transcripts of plant defense-related genes were strongly enhanced in peach shoots infected by the?Lt GPX3 strain when compared to the wild-type strain.In summary,Lt GPX3 acting as glutathione peroxidase involved in ROS scavenge and affected the virulence of L.theobromae on peach shoots.4. Gene Lt TRX2 encoding thioredoxin protein was down-regulated in?Lt AP1 mutant through profiling of gene expression and functionally identified in this study.Lt TRX2encodes a protein with an open reading frame of 156 amino acids interrupted by two introns.The expression of Lt TRX2 was highly induced by ROS-induced agents in relation to the wild-type strain.The?Lt TRX2 mutant was more sensitive to cell wall damaging stress and cell membrane damaging stress than the wild-type.However,?Lt TRX2 was more tolerant to high osmotic stress and high salt stress than the wild-type.?Lt TRX2mutant displayed enhanced sensitivity to oxidative stress when compared with the wild-type strain.Pathogenicity assays revealed that the?Lt TRX2 strain induced smaller brown necrotic lesions and less fungal biomass at the site of?Lt TRX2 inoculation on peach shoots than those induced by the wild-type.The transcripts of Pp RBOHD/F,H₂O₂contents and superoxide anion in inoculated peach shoots were significantly increased by?Lt TRX2 mutant as compared to those in the wild-type.The transcripts of plant defense-related genes were significantly up-regulated in the?Lt TRX2 mutant in relation to the wild-type.Taken together,the results demonstrated that Lt TRX2 acting as thioredoxin was required for ROS detoxification,plant defense suppression and L.theobromae virulence.