Cloning And Interacting Protein Screening Of An Apple Replant Disease Fungal Pathogen Resistance Associated Gene MdCERK1

Apple replant disease(ARD)is a historical problem which is complicated and hard to solve,at the same time,it is also a problem needed to be solved to ensure the sustainable and healthy development of China's apple industry.For now,screening of apple ARD resistant rootstocks is the most environmentally friendly and effective way to prevent ARD.It is of great significance to carry out research about selecting genes involved in ARD regulation and eluciadating their disease resistance mechanism.All these research work will contribute to providing theoretical basis for breeding ARD resistant apple rootstocks through genetic engineering methods.There is research results showed that fungi are the key disease factor for ARD,whose virulence is directly controlled by chitin.In model plants,chitin receptor proteins can recognize chitin,trigger immune responses and thus showing resistance to subsequent pathogen infections.Our research took model plants' research results as references,using bioimfomatic analysis and successfully obtained an ARD associated gene MdCERK1.qPCR,pull-down coupled with nanospray LC/MS/MS analysis were used for gene expression specificity identification and intereacting protein screening.The main results are as follows:1.After verifying that G.935 and B.9 have different resistance towards ARD,Pythium ultimum was used to inoculate the two cultivars.Then the RNA-seq was employed to screen the target genes functioned in responding to ARD through finding differential expressed genes.Among a series of candidate genes,the CERK(chitin elicitor receptor kinase)was picked.CERK contains an extracellular LysM(lysin motif)domain,which can specifically recognize chitin.Theoretically,CERK gene should be resistant to a board spectrum of fungal ARD pathogens,and thus has high research significance and application value.2.When using RT-PCR to study gene expression patterns,a reference gene is needed for normalization.While gene expression in apple roots in response to various stress conditions is a less-explored research subject,it is necessary to have an appropariate reference gene for relevant experiments to yield an accurate result.We took advantage of our RNA-seq data,a set of 10 apple genes was selected based on their low variance of gene expression in apple root tissues.Five previously reported apple reference genes from other tissue types were also included.Four methods,Delta Ct,geNorm,NormFinder and BestKeeper,were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions.A small panel of stably expressed genes,MDP0000095375,MDP0000147424,MDP0000233640,MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses.3.Based on apple genome database GDR and The Apple Genome and Epigenome,the LysM family genes were identified.A sum of 30 LysM genes was identified systematically in apple.ExPASy Proteomics predict Server was used for the prediction of the basic information of MdLysM proteins.According to the phylogenetic analysis,30 MdLysM proteins can be into three groups(group A,B and C),of which group C was divided into three subgroups according to the phylogeny relationship and gene structures;this implies that function differentiation may have happened.The predicted results of the protein domains were congruent with the clustering in phylogenetic tree.Sequence analysis on the genomic structure,domain composition and transcriptional response to exogenous chitin treatment indicated that MD09G1111800 is the ortholog AtCERK1 and was therefore named as MdCERK1.4.Tissue specific expression patterns indicated that MdCERK1 is primarily functional in vegetative tissues of leaf and root,rather than flower,fruit and seed of apple plant.The transcriptional regulation patterns in response to infection by Rhizoctonia solani demonstrated that MdCERK1 is a functional pattern recognition receptor protein(PRR)in apple root tissues.The ability of purified GST-MdCERK1 fusion protein to bind chitin molecules added biochemical evidences for its role as a chitin binding receptor.MdCERK1 can response to pathogen inoculation in all tested genotypes,but its regulation patterns are different.5.Non-target proteomic approach was also employed for identifying its putative in vivo interacting partners from apple plant cells,which sujgests the existence of a functional receptor complex between MdCERK1 and PR4.These data support the conclusion that MdCERK1 is a chitin binding receptor kinase functioning in apple vegetative tissues,which plays an important role in defense activation in response to pathogen infection.