Identification Of Disease Resistance Of SA-Specific Inducible Gene MdWRKY40 Stimulated By Botryosphaeria Dothidea

Botryosphaeria dothidea is one of the biological stresses that affect the development of apple branches and fruits.According to statistics,the annual loss caused by Botryosphaeria dothidea in Chinese apple producing areas is as high as 5 billion yuan.WRKY transcription factors are involved in various physiological processes of plants.Among them,WRKY transcription factor plays a key role in plant disease resistance.Although WRKY transcription factors have been well studied in model plants,the function of WRKY TFs against disease in apple is still unclear.Therefore,the apple‘Orin’callus and Arabidopsis thaliana were used as materials,and identify the biological function of MdWRKY40 in plant defense disease pathway through using protoplast transformation,inoculation and other techniques.The main findings are as follows:1.The full-length CDS sequence of MdWRKY40 gene was cloned from apple’Fuji’fruit.The phylogenetic tree analysis showed that the MdWRKY40 was closely related to PbWRKY40.Protein sequence alignment analysis revealed that MdWRKY40 protein and the AtWRKY4 and AtWRKY40 proteins all contained fiveβ-strands,a C₂H₂ zinc finger structure and a WRKYGQK conserved domain,and this conserved domain spans the entireβ2 strand.2.The promoter sequence of MdWRKY40 was obtained by segmental cloning,transformed into apple protoplasts and treated with SA for luciferase activity analysis.The promoter sequence was specifically responsive to SA,and different lengths responded differently to SA.When the sequence is increased to 2000 bp,the response to SA is the strongest,which is 11.5 times that of non-SA treatment,but the specific cis-acting elements that respond to SA need to be further explored.3.In the roots,stems and leaves of the’Gala’tissue culture seedling,0.5 mM SA strongly induced the expression of MdWRKY40 gene,and both of them increased first and then decreased,and the expression level was the highest at 6 h.Exogenous SA treatment can increase the resistance of apple leaves to Botryosphaeria dothidea,the incidence rate decreased from 92.59%to 79.26%,and the expression of pathogenesis-related protein genes MdPR2 and MdPR5 was significantly higher than that of the control.4.Overexpression of MdWRKY40 gene apple callus inoculated with Botryosphaeria dothidea for 3 days,the lesion area was significantly lower than the control,from 26.44cm² to8.68cm²,the expression of disease-related protein genes MdPR2,MdPR5,MdPR8 was significantly higher than ordinary’Wang Lin’callus,increased the resistance of apple callus to Botryosphaeria dothidea.After silencing the MdWRKY40 gene in apple callus,there was no significant effect on the resistance of Botryosphaeria dothidea,but the expression levels of MdPR2,MdPR4,MdPR5 and MdPR8 were decreased.5.The Arabidopsis thaliana leaves heterologously expressing the MdWRKY40 gene were inoculated with Botryosphaeria dothidea,and the incidence was low compared with the control,and the incidence was light.The incidence of wild-type Arabidopsis reached 77.5%,while the incidence of the two transgenic lines was only 21.5%and 17.4%,the expression levels of the pathogenesis-related protein genes AtPR1,AtPR3 and AtPR4 increased,increased the resistance of Arabidopsis to Botryosphaeria dothidea.6.The heterologous expression of MdWRKY40 in Arabidopsis thaliana significantly inhibited the elongation of roots.After 7 days of culture,the main root lengths of the two transgenic lines were 39.9%and 43.1%of wild-type Arabidopsis,respectively.After 10 days of culture,the main root length was 58.5%and 55.4%of wild-type Arabidopsis,respectively.The expression levels of the auxin synthesis-related gene AtTAA1 and the auxin transport-related genes AtPIN1 and AtPIN2 were significantly reduced.7.Overexpression of apple callus with MdMPK6 and induction of purification of MdWRKY40 protein.The results of pull down assay indicated that MdWRKY40 protein interacted with MdMPK6 protein.