Identification And Function Analysis Of Apple Genes Involved In Defense Responses To Botryosphaeria Dothidea

Ring rot (Botryosphaeria dothidea) is one of the highly devastating diseases of apple in China. Understanding the defense responses of host to B. dothidea is the base of effectively managing the disease and breeding disease-resistant cultivar. In the present study, defense-related genes involved in the defense against B. dothidea were identified and their function were characterized for understanding the defense mechanism of apple plant against B. dothidea.Apple genome was analyzed using bioinformatical approach for identifying the immune-related genes. The expression of six genes including MdMBP2、MdPR-4-1、MdPR-4-2、 MdCBiP1、MdCf3and MdACBP2demostrated significant changes in response to B. dothidea challenge as revealed by qRT-PCR and semi-qRT-PCR. Among them, MdMBP2、MdPR-4-1、 MdPR-4-2、MdCBiPl and MdCf3were up-regulated by inoculation, while MdACBP2was significantly downregulated. MdMBP2. MdPR-4-1and MdACBP2were chosen for further function analysis.MdPR-4-1encodes a PR-4protein which belongs to class Ⅱ of PR-4family. The high-level expression of MdPR-4-1was observed in flowers and leaves as revealed by quantitative real time PCR. The temporal expression analysis demonstrated that MdPR-4-1expression could be up-regulated by B. dothidea infection and salicylic acid (SA) or methyljasmonate (MeJA) treatment, but suppressed by diethyldithiocarbamic acid (DIECA). In vitro assays, recombinant MdPR-4-1protein exhibited ribonuclease activity specific for single strand RNA and significant inhibition to hyphal growth of three apple pathogenic fungi B. dothidea, Valsa ceratosperma and Glomerella cingulata. Moreover, the inhibition was reduced by the presence of5’-ADP.MdMBP2encodes a protein containing a B-lectin domain and PAN-apple domain, and has high identity to epidermis-specific secreted glycoprotein from Vitis vinifera (66%) and Glycine max (42%), as well as mannose binding protein from Arabidopsis thaliana (57%). Quantitative real time PCR revealed that the expression of MdMBP2gene was observed in all tissues examined here, and the highest expression of MdMBP2gene was found in bark and leaf, and the lowest in root. MdMBP2expression was significantly enhanced by B. dothidea infection, and the temporal expression was related with the course of disease, which suggested that MdMBP2might be involved in the defense responses against B. dothidea infection. The recombinant protein of MdMBP2exhibited a strong affinity toward oligomannosides and high-Man N-glycans, but weakly interacted with D-Mannose, indicating that MdMBP2gene might be involved in the recognition of fungal pathogen.MdACBP2encodes an Acyl-CoA-binding protein which belonges to Class I of ACBP family. MdACBP2was expressed in root, leaf, flower, young fruit, bark and bud as revealed by Qrt-PCR. The highest mRNA expression was found in leaf, and the lowest in young fruit. The responses of MdACBP2expression to biotic or abiotic stress were also investigated using quantitative real-time PCR. The results showed that MdACBP2expression was inhibited by osmotic stress and B. dothidea infection, but was enhanced by cold stress. There was no significant changes among the apple seedlings treated with1mmol·L-1Pb(NO3)2. It was speculated that MdACBP2gene might be involved in the defense of apple seedling against cold stress.In summary, the data presented here indicated that MdPR-4-1and MdMBP2are involved in the defense responses of apple plant against B. dothidea. MdPR-4-1encodes an antifungal protein which contributes to the defense against B. dothidea by inhibiting the growth of pathogenic fungi; MdMBP2is involved in the recognition of pathogen fungi, and may be contribute to the defense against B. dothidea as a potential PRR. The correlation between MdACBP2and defense responses of apple against B. dothidea need more detailed data, but its expression can be up-regulated by low temperature and down-regulated by challenge with B. dothidea or PEG suggesting that MdACBP2is involved in the resistance of apple against bio-stress or abio-stress.