Proteomic analysis of pathogen-responsive proteins from the chloroplast of apple leaves induced by Botryosphaeria dothidea

Abstract:【Objective】At present, apple ring rot is one of the three most serious apple diseases in the major apple producing areas in China. The apple ring rot seriously affects yield and quality. Proteome is the direct embodiment of life activities in plants, and proteomic study is a powerful tool to reveal the mechanisms of life activities. It is an important research approach to reveal the mechanisms of plant-disease interactions by examining the proteomic changes after the host plants are infected by diseases. In this study,a series of experiments were set up with excised apple leaves subject to the infection by the pathogenic fungus causing apple ring rot. Chloroplasts and chloroplast proteins were extracted from the leaves. With two-dimensional electrophoresis(2-DE) and mass spectrometry(MS), differentially expressed chloroplast proteins in apple leaves after infection by apple ring rot were screened out so as to reveal proteomic response in chloroplasts to the biotic stress of apple ring rot.【Methods】The experimental materials were the apple leaves infected by apple ring rot pathogenic fungus and the uninfected healthy leaves. The leaf samples were collected from the apple breeding nursery of Institute of Pomology of Chinese Academy of Agricultural Sciences(CAAS) in Xingcheng, Liaoning province, China. The activated pathogenic fungus of apple ring rot were inoculated in PDA medium and cultured in an incubator to produce spores at 75% humidity, 25 ℃ and 24 h light. The spore suspension was prepared with the conidiospore collected from the surface of the PDA medium. Healthy young apple leaves were collected and washed with sterilized water. After 2 days of incubation in aseptic conditions at 75% humidity, 16 ℃ and 12 h light, the leaves showing diseases were discarded, and those with no diseases were chosen as the experimental materials for the infection experiments below. Some of the leaf materials were inoculated with the spore suspension using sterilized cotton(as shown in Fig. 1), and the rest materials were wetted by sterilized water serving as the control. Five days after inoculation, the two groups of leaves were separately homogenized and filtrated. Then, chloroplasts were extracted from the filtrates with Percoll density gradient centrifugation method. The chloroplast samples were observed under optical microscope, and the purity and intact chloroplast rate of the extracts were tested. The chloroplast suspensions were frozen and grinded, and chloroplast proteins were extracted using the modified HB-phenol extraction method. After the protein extracts were dissolved with protein lysis buffer, the concentrations of the protein were tested using a 2-DE protein quantitative kit(2-D QUANT KIT, GE Healthcare). Then the protein samples were separated by 2-DE. The 2-DE gels were scanned by an Image Scanner to obtain the 2-DE maps of proteins, which were analyzed with the software Image Master 2D Platinum 7.0. Twenty one differentially expressed protein spots were selected and analyzed with mass spectrometry(MS). Through searching databases and related literatures, the functional information of the 21 differential proteins was obtained.【Results】Obvious ring spots caused by apple ring rot were observed in the surface of the leaf samples 5 days after the inoculation of the with pathogen. There was no scab in the control leaves. The results of 5 repeated infection experiment were the same. The chloroplasts extracted from the tested materials using Percoll density gradient centrifugation method were intact without contamination by other subcellular structures and impurities.And the intact rate of chloroplast sample reached(90.33±0.847)%, which met the requirements for chloroplast protein extraction. The chloroplast proteins extracted were separated by a 2-DE operating system.The result showed that there were no obvious lateral or vertical stripes in the clear background of the maps. The protein spots separated were clear and full. More than 400 protein spots were separated. 21 differentially expressed proteins were obtained from the two groups of chloroplast protein samples. All of the21 differentially proteins, including 9 up-regulated proteins and 12 down-regulated proteins, were identified by means of MS. They were classified into 6 functional groups, including photosynthesis, energy metabolism, protein metabolism, amino acid synthesis, antioxidation and unknown functions. 【Conclusion】The operating system with excised leaves is simple, and the repeatability of the infection system is good,and therefore the system is suitable for the research. In response to the infection of ring rot pathogen, multiple pathways in apple chloroplasts were activated to achieve resistance to the pathogen. The pathways involve photosynthesis, energy metabolism, protein metabolism, and oxidation reactions. The study brought a preliminary understanding about the response of apple chloroplast to the apple ring rot disease. The understanding of disease-response and defense mechanisms will provide reference for the development of new strategies for germplasm screening and genetic improvement for resistance to apple ring rot disease.