The Research On The Mechanism Of MdWRKY33 Mediated Disease Resistance Against The Apple Ring Rot Pathogenic Fungi Botryosphaeria Dothidea

Apple ring rot caused by Botryosphaeria dothidea can harm both apple stems and fruits. Recent years this disease occurred in almost all the major apple producing areas of China and greatly reduced both the quantity and the quality of apple production. At the same time, due to the characteristics of latent infection, the pathogen has become one of the most serious diseases during fruit storage period which lead to the huge economic loss and greatly limits the development of apple industry in China.Plants have formed a very complex mechanism of immune responses during the long evolutionary process with pathogens, which can fight against different types of pathogens in activing corresponding resistance response. Generally, the SA-mediated pathway was utilized to fight against biotrophs and the JA-/ET-mediated pathway was used to fight against necrotroph. As a pseudomomonas, the infected apple fruit released a lot of ethylene with Botryosphaeria dothidea. However, the role and the mechanism of ethylene in the interaction between the pathogen and apple fruit were seldom reported. The study on the molecular mechanism of interaction between apple fruit and B. dothidea is significant to explore the apple resistance genes, to enrich the prevention mechanism of B. dothidea, and to culvate the new resistant varieties.In this study,’Fuji’was selected as the research material, as it was susceptible to the disease. The changes of cell ultrastructure of apple fruit after inoculation with the pathogen were studied, and then we speculate the extension mode of B. dothidea hypha in apple fruit and the mechanism of B. dothidea pathogenesis, as well as the changes of endogenous ethylene synthesis and lesion size of different physiological state and maturity fruits inoculated with B. dothidea after treatment with 1-MCP, the antagonist of ethylene, was studied in order to illustrate the relationship between ethylene and apple resistance. Through the analysis of the influence of 1-MCP to inoculated fruits and different hormones, hydrogen peroxide and other abiotic stress to cultured in vitro seedlings on the expression of ethylene biosynthesis and signal transduction pathway related genes and transcription factors and pathogenesis related (PR) genes, transcription factors ERFs and MdWRKY33 which might be involved in the regulation of MdPRs induction were screened out. The research results of apple protoplast transformation further verified the regulating function of ERFs and MdWRKY33 on MdPRs. The main results are shown as follows:1. With the light and electronic microscopy methods, we found that the hypha of B. dothidea mainly grows in the extracellular space between cell wall and intercellular spaces of infected apple fruits. The cell wall degrading was a typical symptom of infected apple fruit cells; the specific damage on mitochondrion was triggered by the pathogen infection which occurred at the early infection time when the cell wall was complete without plasmolysis. Pathogen infection also induced cell apoptosis in certain fruit cells, which was demonstrated by the aggregation of condensed chromatin to intact membrane of nuclear even in the severely destroyed cells with the obvious degradation of cell wall.2. Ethylene promoted the extension of B. dothidea in apple fruit but 1-MCP inhibited the process. Ethylene biosynthesis of fruits was strongly induced and ring rot lesion expanded after infection and the treatment of exogenous ethylene promoted the size of ring rot lesion; Ethylene biosynthesis and lesion size of fruits were suppressed by the treatment of different concentrations of ethylene inhibitor 1-MCP after inoculation and the suppression of 1-MCP strengthened with concentration increased. At the same concentration of 1-MCP, inhibitory effect on mature fruit was the best.3. Suppressing ethylene biosynthesis and signaling by 1-MCP further promoted the pathogen-induced expression of MdPRs in apple fruits. MdPR-4, MdPR-5 and MdPR-8 could be drastically induced by the pathogen infection of immature fruit and mature fruit, however suppressing ethylene biosynthesis and signaling drastically promoted the induction magnitude of those MdPRs under the pathogen infection. But the pathogen infection suppressed the expression of these MdPRs in mature fruit stored several months while this suppression was abolished by suppressing ethylene biosynthesis and signaling. The expression of the chitinase encoding gene MdChiB-1 in inoculated-fruit was significantly improved when ethylene biosynthesis and signaling were inhibited, indicating that MdPRs were negatively regulated by ethylene. MdPRs in the apple seedlings were differentially regulated by the stress-related plant hormones and the expression of those MdPRs could also be elicited by hydrogen peroxide, suggesting that multiple signaling pathways were involved in regulating the expression of those MdPRs and the implicated interaction existed in the manipulation of those MdPRs under stresses.4. The highly correlated expression of MdWRKY33-l (MDP0000296025) with up-regulated MdPRs under ethylene inhibition. The RT-PCR analysis of 19 transcription factors showed that MdERF2 could be induced by B. dothidea in immature fruit but not in mature fruit and the induction of MdERF3, MdERF98-3, MdERF98-6 and MdERF98-7 in immature fruit was more significant than mature fruit and the pathogen-induced expression could be suppressed by 1-MCP, Only the changing pattern of MdWRKY33-l showed the highest consistence with that of MdPRs highlighted by its further enhanced expression in freshly harvested mature fruit and the non-influenced expression in immature fruit and the prevented suppression of its expression in stored mature fruit upon ethylene biosynthesis and signaling inhibition. The high consistence with MdPRs was also found in the apple seedlings with the treatment of a variety of plant hormones.5. The expression of those MdPRs was significantly up-regulated by the overexpression of MdWRKY33-1 in apple protoplast cells but not influenced by the overexpression of ERFs, indicating that the transcription factor MdWRKY33-1 was involved in regulating the expression of those MdPRs.6. Botryosphaeria dothidea induced the ethylene synthesis of fruits by regulating the expression of MdACS5a and MdACS5b, with different pathway in the process of the fruit ripening (MdACSl, MdACS3), but ethylene signal transduction in the process of apple resistance associated with fruit ripening process. The RT-PCR analysis showed that the expression of MdACSl and MdACS3, two genes responsible for vegetative ethylene biosynthesis and climacteric ethylene biosynthes is respectively in apple fruit was not induced by the pathogen infection while the expression of MdACS5a and MdACS5b, two genes mediated wounding-induced ethylene biosynthesis in apple fruit was strongly induced by the pathogen infection, indicating that wounding-induced signaling rather than the fruit ripening controlled tunnel contributed to the pathogen-induced ethylene biosynthesis in apple fruit. The expression of ethylene receptor genes MdERSl, MdERS2 was induced by B. dothidea in immature fruit but not in mature fruit, and the pathogen-induced expression could be suppressed by 1-MCP; Similarly, the induction of MdETR2 and MdERF98 in immature fruit was more significant than mature fruit and the treatment of 1-MCP could also inhibited the expression of these genes, suggesting the regulation of ethylene response to B. dothidea may be related to fruit ripening process. MdPG1 gene was strictly dependent on ethylene could also be up-regulated by B. dothidea which further confirmed that apple resistance to the pathogen associated with fruit ripening process.