Pathogenic Mechanism In Cercospora And Cloning And Functional Verification Of Resistance Gene ZmPMT1 To Gray Leaf Spot In Maize

Maize（Zea mays L.）gray leaf spot（GLS）caused by Cercospora zeae-maydis and Cercospora zeina has seriously affected the yield of maize.Parsing the genomic variation and pathogenesis of Cercospora spp.,cloning disease-resistant genes,developing germplasm innovation and new varieties of maize are effective means to control GLS.In this study,genomes of Cercospora zeae-maydis and Cercospora zeina were assembled via Nanopore sequencing technology,and the genome structure and pathogenicity variation between two species were preliminarily analyzed.Resistant QTL q GLS1.02 to GLS in maize was finely mapped and the resistant gene ZmPMT1 was cloned by using chromosome segment substitution lines constructed by resistant inbred line Qi319 and susceptible inbred line Ye478.Three kinase genes Zm CERK1,Zm NIK1 and Zm BAK1 interacting with ZmPMT1 were screened though yeast two-hybrid,and the function of Zm CERK1 was preliminarily verified.Using Qi319 as the disease-resistant donor,the susceptible inbred lines Zheng 58 and Chang 7-2 were improved by marker-assisted selection,which provided technical and material support for the improvement of maize germplasm to GLS resistance.The main results are as follows:1.The reference genomes of Cercospora zeae-maydis-LN（collected from Liaoning,LN）and Cercospora zeina-YN（collected from Yunnan,YN）with different pathogenicity were obtained by Nanopore sequencing,and the genome size of the two species is 45.08 Mb and 42.18 Mb,and contains10,839 and 10,867 genes,respectively,sharing 86.58%of the homologous genes.Long terminal repeat（LTR）retrotransposons of 3.8 Mb in total length may partly contribute to the larger genome of C.zeae-maydis-LN.A higher number of Carbohydrate-binding modules（CBMs）families（33.33%）found in C.zeae-maydis-LN,with unique CBM4,CBM37,and CBM66 in particular,may contribute to variation in pathogenicity between the two fungus as the carbohydrate-binding modules genes are known to encode cell wall degrading enzymes.Besides sharing 61 potential effectors,C.zeae-maydis-LN and C.zeina-YN contain 47 and 46 unique effectors,respectively.Five of eight randomly selected unique effectors could inhibit cell death induced by Bcl2-associated X（BAX）protein by developing assay of inhibiting tobacco cell death.2.q GLS1.02 was located within the physical distance of 148 kb between marker COP-M1 and SNP1502 on chromosome 1 by means of genotypes and phenotypes of progeny,and the physical distance contains two genes,Zm00001eb008060（Zm COP9）and Zm00001eb008070（ZmPMT1）.After artificial inoculation of C.zeae-maydis-LN,compared to Ye478,B73 and B104,ZmPMT1 in disease-resistant near isogenic line NIL-R showed more high expression level,however,no obvious change in that of Zm COP9 was found.Compared to Ye478,B73 and B104,there were more variations in the 1500 bp promoter region of ATG upstream in Qi319,including single nucleotide polymorphisms（SNPs）and insertion deletion（Insertion-deletion,Indel）and twenty of these variants are specific in the ZmPMT1^Qi319promoter.Compared to Ye478,B73 and B104,there were variations in the protein sequence encoded by ZmPMT1 in Qi319,and these variations include amino acid conversion and insertion,and 8 variants are specific in the ZmPMT1^Qi319protein.3.EMS mutants zmpmt1^B73in B73 and gene knockout mutants zmpmt1^B104in B104 showed that mutants in two genetic backgrounds were more susceptible to GLS than the control by artificial inoculation.The disease scores of transgenic plants overexpressing haplotype of ZmPMT1 coming from Qi319 and Ye478 are 6,which is more resistant than that of control B104（disease scores 8）（P<0.01）.Therefore,ZmPMT1 is the GLS resistance gene in QTL q GLS1.02.ZmPMT1 encodes a sugar alcohol transporter with 12 transmembrane-spanning domains.ZmPMT1 was located on the cell membrane by expressing ZmPMT1 in maize protoplasts and tobacco leaves.Yeast two-hybrid and bimolecular fluorescence complementation assays showed that ZmPMT1^Qi319and ZmPMT1^Qi319,ZmPMT1^Qi319and ZmPMT1^Ye478or ZmPMT1^Ye478and ZmPMT1^Ye478can interact with each other and form oligomers,indicating that the amino acid variation coded by two different haplotypes of ZmPMT1 did not affect the formation of oligomers.4.In order to further explore the interacting protein with ZmPMT1,three proteins,including Zm CERK1,Zm NIK1 and Zm BAK1 were screened by yeast two-hybrid.The average disease scores of EMS mutant zmcerk1^B73-1 and Mu transposon insertion mutant zmcerk1^B73-2 in B73 genetic backgrounds were 8 by artificial inoculation of C.zeae-maydis-LN,which were more susceptible than the wild type（disease scores 6,P<0.01）.5.Two molecular markers COP-M1 and SLY-80 closely-linked with ZmPMT1 were used to improve the susceptible inbred line,Zheng58 and Chang7-2 to GLS resistance by marker-assisted selection with Qi319 as disease-resistant donor.The average disease scores of the Zhengdan958-R,the cross produced by Zheng58-R and Chang7-2-R,were 4,significantly lower than that of Zhengdan958（disease scores 7,P<0.01）by artificial inoculation of C.zeae-maydis-LN.6.Taken together,the difference in pathogenicity between the two Cercospora based on genomic information was analyzed.The GLS resistance gene ZmPMT1 was cloned and its function was verified.Three protein kinase genes interacting with ZmPMT1 were cloned by yeast two-hybrid.Zhengdan958with significantly improved resistance to GLS was created by marker-assisted selection.The results provide material and technical support into development of resistant breeding to gray leaf spot.