Proteomic Analysis Of Response To Alternaria Alternata Apple Pathotype Infection In Apple Leaves

Apple(Malus × dqmestica Borkh.)is one of the most widely cultivated tree fruits in the world,and the majority of apple cultivars currently grown in the major production areas are susceptible to alternaria blotch diseases.Alternaria blotch caused by Alternaria alternata apple pathotype(A.Alternata AP).Alternaria blotch is one of the severest fungus disease for apple production Severe infection can result in 60%～80%defoliation.However,the mechanism of host response has not been fully revealed.In this study,we assayed the changes of physiological parameters,proteomics and pathogenesis-related gene expression following A.alternata AP infection.We expect to gain more knowledge and data about the interaction between apple and the pathogen for the further related study in future.The results are as follows:1.Dynamic changes in the activities of superoxide dismutase(SOD),catalase(CAT), peroxidase(POD),phenylalanine ammonia-lyase(PAL)and H2O2 accumulation in compatible interaction,contrasted with those of incompatible interactions,between apple leaves and A.alternata AP were studied.The results showed that H2O2 was detectable on apple leaves following pathogen infection,especially in resistant interaction.The activities of SOD,CAT,POD and PAL increased in both the susceptible cultivar and resistant cultivar,but the activities of different enzymes in the same cultivar or the same enzyme activity in different cultivar are distinguished.2.In this study,we conducted protein expression analyses of interactions between ’Starking Delicious’ apples and A.alternata AP.Total of 45 differentially expressed proteins(DEPs)were detected.Functional classifications analysis of DEPs showed that the molecular response occurring inside the host was extremely complex.Several factors play positive roles against pathogen infection,including phytohormone signaling,pathogenesis-related(PR)proteins(PR2,PR5 and chitinases),transcription factors.Meanwhile,several loopholes within the defense mechanism have also been found,including a physical barrier,ROS generation and a scavenging system and HSPs.3.In order to better understand the molecular mechanisms that underlie the defence responses of apple resistance to alternaria blotch disease,we conducted a comparative proteomic approach to analyse the response of compatible and incompatible interactions between apple and A.alternata AP using iTRAQ technique.Total of 4225 proteins were identified and 1226 proteins were quantified.Of the quantified proteins,280 and 34 expressed differentially(fold change>1.5)at 72 HPI(hours post-infection)in the susceptible and resistant apple cultivars respectively,compared with the mock-inoculated controls.Most of the differentially expressed proteins(DEPs)were associated with host plant resistance to pathogens,including signal transduction,stress and defense,photosynthesis metabolism.Among these proteins,beta-1,3-glucanase(PR2),thaumatin-like protein(PR5),lipoxygenase were found in both the susceptible and resistant hosts.However,endochitinase,(+)-neomenthol dehydrogenase were only detected in the resistant variety and increased in abundance in response to the pathogen attack.The above results suggested that endochitinase and(+)-neomenthol dehydrogenase play important roles in apple against A.alternata AP infestation.4.The real-time quantitative PCR technology was used to analysis the expression of PRs gene in leaves of the susceptible cultivar ’Starking Delicious’ and resistant cultivar ’Jonathan’under the stress of A.alternata AP.At the same time we detected the expression of PR2,PR5 in apple leaves by western blot analysis.The results showed that PR responded to the pathogen infection earlier in the resistant leaves than in the susceptible ones,which suggested that PR exerted important function at early stage in resistant variety and played important roles in apple against A.alternata AP infestation.