Proteome Analysis for Defense Response of Apple Leaves Induced by Alternaria Blotch ,Alternaria alternata Apple Pathotype

摘要

Apple (Ma/us domestica) is considered a model fruit plant owing to its world-wide economic importance,and a large number of cultivars dominate world fruit production.To date,the cultivation of apple has been limited by many kinds of fungal diseases,and among the many fungal diseases affecting apple trees,Alternaria blotch,the disease caused by Alternaria alternata apple pathotype,is spreading worldwide and results in severe negative effect on apple production.Currently,management of a fungal disease such as Alternaria blotch mainly through traditional chemical control agents instead of using resistant cultivars.As in apple,the molecular mechanism of disease resistance against A.altemata apple pathotype has not been illustrated clearly,although most of the genes have been identified in apple,little is known about the biological roles for each protein in that plant.Considering that apple Altemaria blotch is the usual diseases influencing apple production,further studies about the interaction mechanism between the pathogen and its host,particularly some host self-defense response mechanisms which closely related to the anti-disease properties of apple should be performed.In this work we aimed to generate fundamental insights into the plant' s defense responses to infection with Altemaria blotch using a proteomics approach,to aid in the development and breeding of high-quality disease-resistant apple varieties.One seedlings which derived from the cross of ‘Huacui’ and ‘Golden Delicious’,with highly resistant,were chosen to be hosts for this study.The sample leaves were inoculated with spore suspension of A.altemata apple pathotype,and the control samples were treated with water instead of inoculums.Following inoculation,the samples were harvested 48 h after inoculation.The whole experiment was repeated three times in order to get a reliable result.The total soluble protein extracts prepared using modified phenol-precipitation procedures.Total protein extracts were focused in the first dimension using pH 4-7 immobilised ampholine strips (18 cm),and separated as according to size using SDS-PAGE on a 12.5％ cross-linked polyacrylamide gel in the second dimension.Spots were visualized using Coomassie Blue staining.The resultant gels were then scanned and digitized using the Image Master 2D Platinum Version 7.0 software (GE Healthcare).In total in this experiment,31 differentially-expressed proteins were obtained.After tryptic digestion,MALDI-TOF/MS MS analysis and database searching of some of the identified proteins indicated that the proteins are known to be involved in several biotic stress related functions as disease associate with pathogens.Mass spectrometry analysis allowed the identification of 16 differential expressed proteins with isoforms including well known biotic stress responsive proteins.7 of 16 were identified as biotic stress responses proteins directly coupled to disease and pathogen infection on apple.The identified proteins that associated with the antifungal mechanisms of host plant against A.altemata apple pathotype may be newly linked with the antifungal mechanisms of resistant apple.All in all,our studies provides new insights into identification of biotic stress responses protein in disease infected apple leaves under in vitro condition,and demonstrates the advantages of proteomic analysis.