Molecular Mechanism Of WRKY26-bHLH3 Co-promoting Red Pear Anthocyanin Synthesis And Anthocyanin Inhibition Of Aspergillus Niger Infection

Aspergillus niger is a common fungus of the genus Aspergillus.As a typical pHytopathogenic microorganism,it is ubiquitous in various food products,horticultural fruits and vegetables and soil,and can cause mildew and rot in food,cereals and fruits,causing inestimable economic losses to countries all over the world every year.The pathogenic mechanism and prevention strategy of Aspergillus niger have also been the problems that researchers should continue to explore.In recent years,the role of anthocyanins in inhibiting pathogenic microorganisms has gradually aroused people's attention.Anthocyanins are common secondary metabolites in plants,which have strong antioxidant capacity,and are closely related to resistant to stress and resistance of plants,plant growth and development.And it has many benefits for human health.Relevant research reports that prove that anthocyanins can effectively resist the invasion of some pathogenic microorganisms.However,whether anthocyanins can inhibit Aspergillus niger and its mechanism is not yet clear.In addition,this paper explores the inhibitory effect of anthocyanins in red pears on the infection of Aspergillus niger,and further reveals the molecular mechanism of WRKY family transcription factor that regulates the synthesis of anthocyanins in red pear peels.The details are as follows:As Aspergillus niger is a major pathogenic microorganism that causes fruit rot,we used wild-type Aspergillus niger strains and their gene deletion mutants(including AcatA,AcpeB,AsodC;catA,cpeB and sodC genes were confirmed and Antioxidant metabolism of Aspergillus niger is directly related to the pathogenicity of Aspergillus niger).Inoculation experiments were carried out in pear fruits,and we found that the diameter of the lesions measured in red eggplant pears rich in anthocyanins were generally smaller than those on Dangshan Su pear.The diameter of the lesion is estimated by biological methods,and the size difference reaches 10%;it shows that the red pear fruit with more anthocyanin content is more resistant to infection by Aspergillus niger,which proves that anthocyanin play an active role in antagonizes microbial infection in the plant body.And this difference is particularly obvious in the infected parts of Aspergillus niger AcpeB deletion body,AsodC deletion body and AcpeB-AsodC double deletion body,the difference with wild type Aspergillus niger is up to 15%,which is significantly lower than AcatA and AsodC The diameter of the lesion caused by the missing body.It shows that these deletions are more sensitive to anthocyanins,indicating that the cpeB gene and sodC gene in Aspergillus niger play an important role in response to anthocyanin stress Then using red eggplant pear with high anthocyanin content as the material,the anthocyanin was isolated and purified in vitro,and further verified by plate bacteriostasis that anthocyanin can limit the normal growth of E.coli and Aspergillus niger.Finally,we proved by RT-qPCR analysis that compared with healthy peels,the genes related to anthocyanin synthesis in the infected skin,such as PyMYB10,PyMYB114,PybHLH3,PyDFR,PyANS,PyUFGT,PyWRKY26,PyWRKY31 and PyGST.Their gene expression level is significantly up-regulated,this result indicates that the infection of pathogenic microorganisms will cause the plant's own anthocyanin accumulation,a self-response response.In addition,we determined the transcription factors PyWRKY31 and PyWRKY26 are the key candidates genes involved in the regulation of anthocyanin biosynthesis through transcriptome data analysis and preliminary bioinformatics screening of the peels of"Starkrimson" and its green buds at different developmental stages gene.Through RT-qPCR experiments and correlation data analysis,it was found that in the red-skinned pear "Starkrimson",PyDFR,PyANS,PyUFGT,PyMYB10,PyMYB114,PybHLH3,PyWRKY26,PyWRKY31,PyGST gene expression levels were significantly higher than that of the green pear "JinZheng No.1".In order to further verify the regulatory relationship between these transcription factors and anthocyanin synthesis,we conducted transient expression experiments in tobacco leaves and strawberries and found that PyMYB10,PyMYB114 and PybHLH3 transcription factors were not observed in tobacco and strawberry fruits when transformed separately.Pigment accumulation pHenomenon;when PyMYB10+PybHLH3 or PyMYB114+PybHLH3 are co-transformed,a small amount of pigmentation can be observed;when PyMYB114,PyMYB10 and PybHLH3 are co-injected,there is a more obvious color change;and the transcription factor PyWRKY26 is selected When PyWRKY31 and PyMYB10,PyMYB114,and PybHLH3,respectively,were transformed into tobacco and strawberry,the accumulation of anthocyanins in tobacco and strawberry fruits was greatly enhanced.These evidences indicate that PyWRKY26/PyWRKY31 are involved in the biosynthesis of anthocyanins in pears.In order to further reveal how PyWRKY31/PyWRKY26 is involved in the regulation anthocyanin synthesis,we further confirmed by RT-qPCR analysis and dual luciferase reporting system that the four transcription factors,PyMYB10,PyMYB114,PybHLH3 and PyWRKY26/PyWRKY31 co-transformation can activate the expression of key genes PyDFR,PyANS and PyUFGT in the process of anthocyanin biosynthesis and the expression of PyGST genes in the anthocyanin transport,but there is no obvious activation effect on the PyABC transport protein and PyAVP involved in the transport process.In addition,the joint action of PyWRKY26 and PybHLH3 can bind to the PyMYB114 promoter,and PyWRKY26 directly activates its transcriptional expression by binding to the PyMYB114 promoter to participate in the regulation of anthocyanin biosynthesis.In addition,through luciferase complementation and yeast two-hybrid experiments,we find that PyWRKY26 can interact with PybHLH3This study confirmed that anthocyanins have a positive role in the resistance of Aspergillus niger infections in pear fruits.Red pear fruits co-target activation of the PyMYB114 promoter through the interaction of PyWRKY26 and PybHLH3,resulting in anthocyanins accumulation in red pear.This paper provides new insights into the pHysiological process of anthocyanin antagonizing microbial infection and the regulatory network of anthocyanin accumulation in pear fruits is expected to provide some theoretical support in the future work of plant products to control Aspergillus niger.