| Apple as one of the main cash crops in China has been receiving a lot of people’s attention,with the development of science and technology and the improvement of people’s quality of life,everyone’s demand for high-quality apples has gradually risen.Apple ring rot is one of the culprits that endanger the quality of apple fruit and seriously affect apple yield.Apple picking period and storage period is the peak period of apple ring rot,once the disease occurs,the edible value and nutritional value of apple fruit will be damaged,and the manpower and material resources invested in the early stage will also be wasted,and the fruit disease during the storage period will even lead to greater waste of resources.And if we can know how Botryosphaeria dothidea infect apple cells and their activities inside the cells,this will play a great role in the research against apple ring rot.In this study,we inoculated the B.dothidea in ‘Fuji’ apple fruit and ‘Orin’ apple calli,respectively,and then used transmission electron microscopy（TEM）to observe and compare the ultrastructure of infected and control cells,explored the infection pattern of pathogens in apple cells and callus cells,as well as the relationship between cell death and disease resistance in the apple/B.dothidea interaction pathosystem.We also explored the changes in the expression of different genes before and after the infection of apple cells and callus cells,and screened MdVDAC2（MDP0000271281）,a gene that encodes anion channel proteins in the mitochondrial outer membrane,and found that overexpression MdVDAC2 can improve the resistant of calli to B.dothidea.In addition,we also found that Md PIP1-4（MDP0000479436）interact with Md SYP121（MDP0000709455）in calli.Calli that cooverexpress the above two genes also exhibited a phenotype that predisposes to B.dothidea,and the main findings are as follows:1.Transmission electron microscopy results show that B.dothidea infects apple cells both intracellularly and extracellularly.Pathogens that roam around the cells can secrete related substances to degrade the host cell wall,and pathogens that invade the inside of the cell directly penetrate the host cell,causing less damage to its cell wall.We also found that the ratio of hyphae inside mature apple cells was significantly higher than that inside immature cells by comparing the position of pathogens in host cells,above results suggesting that penetration resistance mediated by the cell wall plays an important role in apples’ fight against B.dothidea.The infection of B.dothidea will also cause apple fruit cells and apple callus cells plasmolysis,damaging the mitochondria,plastid and other organelles inside the cells,sometimes resulting in the appearance of programmed cell death in some cells.2.Bd Catalase（KAF4307763）,a cytochrome P450 family protein Bd CYP52A4（KAF4300696）,and subtilisin-domain containing proteins were identified from B.dothideasecreted proteins,which suggested the potential involvement of active oxygen species and phytoalexins in B.dothidea infection and triggering or dampening apple resistance.3.MdVDAC2 is a membrane protein on the mitochondrial outer membrane,which is involved in regulating the programmed death of cells in plant cells.Overexpression of MdVDAC2 significantly promoted cell death under B.dothidea infection and simultaneously inhibited pathogen infection,suggesting that promoting the death of infected cells is also a disease resistance mechanism in apple against B.dothidea infection.The interaction between Md SYP121 and Md PIP1-4 was verified by bimolecular fluorescence（BIFC）and CO-IP experiments,and the transgenic calli of these two genes showed predispose to B.dothidea.Collectively,our research suggested that cell wall-mediated penetration resistance,programmed cell death machinery and microbial effector-interrelated signaling were among the strategies recruited in apple to combat B.dothidea.The current research laid the foundation for further investigations into resistance mechanisms in apple. |
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