Map-based Cloning And Functional Analysis Of Multiple Disease Resistance Locus QLMchr7 In Maize

Maize is one of the three major food crops around the world.Also,it is the most productive grain in China.The yield and quality of maize is essential for Chinese food security and national economic development.However,the epidemic of northern leaf blight in Northeast area,southern corn rust in Huang Huai-hai area and grey leaf spot in Southwest area,threatens the maize production and the development of maize industry.Nowadays,identification and application of resistance alleles,especially multiple disease resistance alleles,is one of the most efficient and economic mothed to control maize diseases.To address this issue,we selected a teosinte introgression line C117,which exhibits resistance related lesion mimic phenotype,to clone multiple disease resistance genes in maize Through map-based cloning and functional analysis,we investigated the genetic bases and mechanism of a multiple disease resistance allele.The major results are as follows1.By analysis the F2 population developed by C117 and its background inbred line Mo 17,we identified three QTLs controlling lesion mimic phenotype in C117.The major QTL,named qLMchr7,is on chromosome 7 and positively regulated lesion mimic phenotype2.Fine mapping process narrowed down qLMchr7 into a 1-kb region,lied on the 3'UTR of ZmMM1 gene.By crossing zmmml-1 mutant in B73 background and C117,we confirmed that qLMchr7 regulates ZmMM1 to form lesion mimic.The transient expression assay in N.benthamiana also proved that ZmMM1 promotes cell death3.qPCR,WB and Ribosome profiling were used to evaluate the variation of ZmMM1 genes between two different NILs.The results showed that qLMchr7 affects the translation efficiency of ZmMM1 mRNA,resulting in the difference on ZmMMl protein level.qLMchr 7C117 up-regulates ZmMM1 protein level4.Multiple sequence alignment analysis revealed the functional site of qLMchr7,which was formed by 2 SNP and 1 InDel variations and was named SR.We first confirmed the qLMchr7 regulation element could also be used in N.benthamiana,and illustrated the C117 derived SR raises ZmMM1 protein level5.KASP marker was used to detect the distribution of qLMchr7C117 in maize,landrace and teosinte population.We confirmed that qLMchr 7C117 is a rare haplotype in landrace and teosinte population,and it is lost in maize6.Through natural inoculation,we comparing the resistance between different lines in field.The disease assays results showed that ZmMM1 positively regulates the resistance to northern leaf blight,southern corn rust and grey leaf spot.And qLMchr 7C117 also contributes resistance to these three diseases7.Through detecting the ROS production after PAMPs treatment in different lines,we confirmed that ZmMM1 positively regulates ROS accumulation,and qLMchr7C117 enhances this process.Under pathogen infection,ZmMM1 protein is accumulated slowly,resulting in ROS accumulation to fight against pathogens.The regulation element qLMchr7C117 can further increase the content of ZmMM1 protein,so as to enhance the production of ROS and mediate stronger resistance8.We turned out ZmMM1,containing MYB DNA binding domain,is a transcription repressor,and it mainly regulates the ncRNA process in disease resistance.ZmMT3,one of the four targets of ZmMM1 protein was annotated as a LncRNA,and shows the most powerful effect in ROS accumulation and disease resistance.The phenotype of ZmMT3 transgenic lines indicated ZmMT3 can suppress ROS accumulation,and negatively regulates plant resistance9.Combined the phenotype and ZmMM1 haplotype in maize association mapping population,we figured out the variation of ZmMM1 genomic sequences.The coding region of ZmMM1 is very conserved,and the variations do not influence the cell death function of ZmMM1.The regulation elements of ZmMM1 varied much in maize,and these variants can influence resistance,nor lesion mimic phenotype.These results indicated ZmMM1 is well-regulated by new regulation elements in maize population10.We showed the application of qLMchr7 through two different NILs.We figured out that qLMchr7C117 does not cost yield lost under non-disease conditions,indicating the qLMchr7C117 mediated lesion mimic do not fitness trade-off.Whether qLMchr7C117 improve the production under disease condition is remained to be solved.