Transcriptome Of Response Of Apple To Podosphaera Leucotricha And Molecular Mechanism Of MdWRKY40b Regulating Resistance

Apple powdery mildew is one of the main diseases in apple production.It is a fungal disease caused by Podospharea leucotricha,which seriously affects the tree potential and the yield and quality of fruit.Breeding rootstocks with resistance to powdery mildew is an important way to enhance apple resistance to powdery mildew.Malus hupehensis is one of the excellent rootstocks with high resistance to powdery mildew cultivated in China.’Qingzhen1’ is a rootstock variety and it is susceptible to powdery mildew in the whole growing season.In addition,the molecular biology of powdery mildew resistance is also focused on the discovery of plant disease resistance genes.Research shows that WRKY family genes are a kind of regulatory factors involved in plant defense to pathogens infection and immune response.MdWRKY40 b is a transcription factor responds to a variety of pathogens infection and regulates the resistance response to different diseases.The study used the BGISEQ-500 sequencing technology and denovo transcriptome assembly to gain a comprehensive overview of the transcriptome of the leaves of ‘Qingzhen 1’,‘Qingzhen1’/Malus hupehensis and M.hupehensis after the outbreak of powdery mildew in the same period.To explore the molecular mechanism of stronger resistance to powdery mildew of M.hupehensis than‘Qingzhen 1’ rootstock,and to screen differentially expressed genes response to the powdery mildew infection.’Columbia’,At WRKY18/40 double mutant and At WRKY18/40/60 triple mutant Arabidopsis thaliana were used as materials to analysis the mechanism of MdWRKY40 b response to powdery mildew infection by overexpressing MdWRKY40 b gene.Apple ’Gala’ as experimental material,RNAi-MdWRKY40 b gene silencing test was carried out to further explore the mechanism of MdWRKY40 b response to apple powdery mildew infection.The main results are as follows:1.The resistance to powdery mildew of ‘Qingzhen 1’,‘Qingzhen 1’/Malus hupehensis and M.hupehensis three materials increased in turn.Gene Ontology(GO)enrichment showed significant enrichment of GO term associated with Metabolic process and Catalytic activity in ‘Qingzhen 1’,‘Qingzhen 1’/Malus hupehensis and M.hupehensis.KEGG analysis showed Linoleic acid metabolism,Plant-pathogen interaction,Inositol phosphate metabolism,Brassinosteroid biosynthesis and Phosphatidylinositol signaling system etc.were significantly enriched in ‘Qingzhen 1’/Malus hupehensis and M.hupehensis,which have medium resistance to powdery mildew and high resistance to powdery mildew,respectively.And the Carbon metabolism pathway was only significantly enriched in M.hupehensis.Expression analysis of genes related to the resistance to powdery mildew show that WRKY70、WRKY33 、 NPR3 and WRKY40 was significantly up-regulated in ‘Qingzhen 1’/Malus hupehensis and M.hupehensis compared to ‘Qingzhen 1’.The accuracy of sequencing were confirmed by q PCR experiment.2.’Columbia’,At WRKY18/40 double mutant and At WRKY18/40/60 triple mutant Arabidopsis thaliana were used as materials to carry out experiment of overexpression MdWRKY40 b gene.Disease resistance of transgenic lines to powdery mildew were lower than control plants.Compared to control plants,the expression level of disease resistance related genes such as PR1/3/4 and NPR1 were significantly reduced and the expression of gene PDF1.2 was up-regulated in transgenic lines after powdery mildew infection.These results indicated that MdWRKY40 b negatively regulates the resistance of Arabidopsis thaliana to powdery mildew.2.Gene silencing experiment of MdWRKY40 b by RNA interference in Apple ‘Gala’material was carried out.Compared with control plants,the area of mildew spot in transgenic lines leves reduced significantly at 25 d after powdery mildew infection and the expression of disease resistance related genes such as PR1/2/5/8 and EDS1 etc.were up-regulated significantly at 10 h and 24 h after powdery mildew infection.Upon P.leucotricha inoculation,accumulation of reactive oxygen species and hydrogen peroxide was less in transgenic lines compared with control plants in whole time by histochemical staining,and the activity of antioxidant enzymes was stronger compared with control plants in whole time.These results indicated that the resistance of transgenic plants to powdery mildew was vigorous compared with control plants,MdWRKY40 b involved in the response of apple to powdery mildew infection by as a negative regulator to regulate the expression of disease resistance related genes or the activity of antioxidant enzymes.