Cloning And Functional Analysis Of Powdery Mildew Resistance Regulated Gene TaWAK6 In Wheat

In the process of wheat growth and development,powdery mildew is one of the most common diseases.It occurs frequently in various wheat planting areas and seriously affects the yield and quality of wheat.Exploring the powdery mildew resistance genes in wheat and cultivating powdery mildew resistant wheat varieties are the most economical and effective strategies to prevent and control powdery mildew.Previously,the powdery mildew resistance gene Pm21 and several defense related genes were cloned,such as Stkp1-Ⅴ encoding a serine threonine kinase,CMPG1-Ⅴencoding an U-Box type E3 ubiquitin ligase,receptor-like kinase gene RLK-Ⅴ encoding a LRR/Malectin kinase and transcription factor gene TaNAC6,etc.These disease resistance-related genes play important roles in the resistance pathway to powdery mildew of wheat.Transgenic plants over-expressing of these genes can significantly increase the resistance of wheat to powdery mildew,and have huge potentials in the breeding using genetic biotechnology.The wall associated kinase gene WAK is widely involved in the regulation of plant disease resistance.Arabidopsis AtWAKL22 resistant to verticillium wilt and fusarium wilt,tomato SlWAK1 resistant to Pseudomonas syringae,corn ZmWAK resistant to smut and ZmWAK-RLK1 resistant to large leaf spot,rice Xa4 resistant to bacterial blight and wheat Stb6 resistant to leaf blight all encode WAKs,which participate in the transmission of disease resistance signals by combining with pectin or pectin degradation products-oligogalacturonic acid(OGs).A susceptible mutant of the broad-spectrum powdery mildew resistance gene Pm21 was obtained previously in our laboratory.Through transcriptome analysis,it was found that the expression of a WAK gene in the mutant decreased significantly.So,it was speculated that this gene was involved in the regulation of powdery mildew resistance mediated by Pm21.In this study,WAK family genes of wheat crops and gramineous model crops were explored using various bioinformatics websites,and evolutionary analysis of these genes was performed,including characterization of the gene number,protein structure,chromosome distribution and expression patterns.In addition,the structure,expression and function of TaWAK6 were analyzed in detail,and its function in the resistance to powdery mildew was preliminarily clarified.The main research results obtained are as follows:1.Identification of WAK gene family in gramineous plantsFrom the Ensemble plants database,the protein sequences of 7 species were downloaded,including Triticum aestivum,Triticum.dicoccoides,Aegilops tauschii,Triticum urartu,Hordeum vulgare,Brachypodium diatachyon,and Oryza sativa.The Pfamscan program was used to analyze the domains of all protein sequences.From the annotated data,the proteins with the conserved domains of WAK(GUB_WAK_bind,EGF,Pkinase)were screened,and the proteins containing GUB_WAK_bind/Pkinase or EGF/Pkinase were further screened.The results showed that there were 1032 WAK family genes in the 7 species mentioned above.In Triticeae species,the number of WAK genes were 414,234,59,96,47 in Triticum aestivum,Triticum.dicoccoides,Aegilops tauschii,Triticum urartu,Hordeum vulgare,respectively.In the model crops,the number of WAK genes ranged 113,69 in Brachypodium diatachyon and Oryza sativa.2.Chromosome location,phylogenetic analysis and expression analysis of WAK family genes in common wheatThe WAK family gene of common wheat have 397 members distributed on 7 homeologous chromosomes.The chromosome distribution information of 17 WAK genes is unknown.The homeologous group 3,6,5,2 and 7 harbers more members,while the homeologous group 1 and 4 have less members.The homeologous group 3 chromosomes has the most members,with a total of 90 WAK genes;the homeologous group 6 chromosomes and the homeologous group 4 chromosomes have more,with 81 and 76 WAK genes respectively.There are 52,43,and 36 WAK genes in the homeologous group 2,7 and 1 chromosomes,respectively.The homeologous group 2 chromosomes has only 19 WAK genes.In the D genome,4D only has one WAK gene.Phylogenetic evolution and gene domain analysis showed that WAK family genes can be divided into 4 subclasses,Clade Ⅰ-CladeⅣ.The protein structure of Clade Ⅰmembers are characterized by two WAK-related domains and one serine/threonine kinase domain,Clade Ⅱ members are characterized by a WAK-related domain and a serine/threonine kinase domain,CladeⅢ members are characterized by two WAKrelated domains,one EGF/EGF_CA domain and one serine/threonine kinase domain,and CladeⅣ members are characterized by two WAK-related domains,one EGF/EGF_CA domain and one serine/threonine kinase domain.The number of the four subtypes is uneven.Clade Ⅰ has 59 members,Clade Ⅱ has 40 members,and Clade Ⅲhas 59 members.The WAK gene of Clade Ⅳ has the largest number of members,with 234 members.Using the gene expression database of Chinese Spring,the expression level of the WAK gene family in common wheat was investigated,it was found that WAK genes are widely involved in the regulation of wheat growth and development.Wheat WAK genes are expressed in many tissues,mainly in leaf tissues during the vegetative and reproductive growth phases,indicating that different WAK genes may play roles in different tissues of wheat.In addition,WAK family genes also respond to plant biological stress.For example,a large number of WAK genes are expressed in response to powdery mildew infection,and some WAK genes are expressed in response to stripe rust infection.At the same time,the cluster analysis of expression heat maps and evolutionary relationships also found that those genetically-clustered members tend to show similar expression patterns.3.Expression regulation and functional analysis of TaWAK6 gene in Nannong 9918The TaWAK6 gene,which was significantly down-regulated in susceptible mutants,was screened previously.In Triticum aestivum cv.Chinese Spring,Lancer,Norin61,Triticum turgidum,Triticum urartu and Aegilops tauschii,the TaWAK6 orthologous genes on each species were discovered through Blast search.It was found that TaWAK6 has the highest similarity with the orthologous gene on the 2A chromosome,so it was speculated that the TaWAK6 originated from 2A and named as TaWAK6-2A.According to the SNPs among TaWAK6-2A,TaWAK6-2B and TaWAK6-2D,specific primers that can distinguish the copies of TaWAK6-2A,TaWAK6-2B and TaWAK6-2D were designed,and the universal primers common to the three copies were also designed.The specific copies corresponding to TaWAK6-2A,TaWAK6-2B and TaWAK6-2D were cloned in Nannong 9918.The results showed that 94 single clones were obtained produced by amplification using the universal primer,and it was found that all the clones could produce the expected band specific to TaWAK6-2A while not to TaWAK6-2B and TaWAK6-2D.It was speculated that only TaWAK6-2A was expressed i,but TaWAK6-2B and TaWAK6-2D were not expressed.qRT-PCR was used for expression amalysis,and it was found that TaWAK6-2A was significantly upregulated by Bgt in Nannong 9918,however,TaWAK6-2B and TaWAK6-2D were not induced by Bgt.The function of TaWAK6-2A was analyzed by gene silencing and over-expression.It was found that the powdery midldew resistance of Nannong 9918 was compromised dramatically,while overexpressing of TaWAK6-2A could improve the powdery midldew resistance of Yangmai158.It indicated that TaWAK6-2A played a positive regulatory role in the powdery mildew resistance.The yeast two-hybrid system was used to screen the interaction proteins of TaWAK6-2A.A total of 53 single clones grown on the four-deficient medium SD/-AdeTrp-Leu-His/X-α-GAL were analyzed,and 45 non-redundant interacting proteins were obtained.Among them,6 proteins contain CBS domains,which play an important role in the process of wheat cold resistance;4 proteins contain serine/threonine kinase domains,which play an important role in various signal transmission processes;3 proteins are involved in cellular metabolism,including photosynthesis and respiration,which are DUF1230,PSI_PsaH Superfamily,and NADB_Rossmann super family;2 proteins contain the TIM super family triose phosphate isomerase domain,which participates in the process of mitochondrial transport proteins;2 proteins contain the SET super family,lysine transferase,which is closely related to methylation.It needs to be further studied about these interacted proteins to reveal the mechanism mediated by TaWAK6-2A.