Cloning And Characterization Of A Major QTL Conferring Resistance To Head Smut In Maize

Head smut is the soil-borne fungal disease caused by the pathogen Sporisorium reilianum. The disease occurs in most maize growing areas worldwide, and poses a great threat to maize production. Development and deployment of resistant maize hybrids is proved to be the most effective and economic methods to control head smut for sustainable maize productivity.Genetic analysis disclosed that maize resistance to head smut was a quantitative trait, controlled by multiple genes. Our previous study detected a major QTL-qHSR1in bin2.09of chromosome2, which could explain36%of the total phenotypic variation. By using the recombinant-derived progeny-test strategy, we conducted fine-mapping of this QTL successively and finally limited it to the interval flanked by molecular markers STS1M3and STS3M1. The positive BAC clones were screened from both resistant Mo17and susceptible Huangzao4libraries and subjected to sequencing. We found a147-kb DNA fragment in the qHSRl region was deleted from the susceptible Huangzao4as compared to the resistant Mo17. In this region, we found5genes that were reported to be involved in disease resistance, including ZmWAK, ZmHCH, ZmTIF, ZmXa21-1and ZmXa21-2. In this study, analysis of these5reistance genes for their RNA expressions confirmed ZmWAK was the candidate gene of qHSRl. We subcloned the ZmWAK gene from the Mo17BAC clone, and transformed it into maize hybrid Hill. The transgenic plants significantly enhanced head smut resistance compared to non-transgenic siblings. Similarly, RNAi interference assay confirmed decreased expression of ZmWAK reduced head smut resistance. Both transgenic complementation test and RNAi interference assay definitely indicated that ZmWAK is the gene for the major resistance QTL-qHSRl.ZmWAK encoded a wall-associated kinase, localized in plasma membrane, which potentially served as a receptor-like kinase to perceive extracellular signal. It expressed in various tissues in maize seedling and adult plant, with the highest level in seedling mesocotyl, first internode and toppest leave of adult plant. In addition, ZmWAK can be induced in coleoptile by S. reilianum inoculation. We used a pair of near isogenic lines, HZ4(susceptible) and HZA-qHSRl (resistance), to explore the molecular mechanism underlying ZmWAK-mediated resistance. S. reilianum invaded maize seedlings through the root after seed emergence and accumulated at the base of mesocotyl. In HZ4, S. reilianum could quickly pass through the mesocotyl to infect the above-ground tissue, thereby caused head smut symptoms in the filling stage of adult maize. By contrast, the upward growth of S. reilianum was arrested in the resistant HZA-qHSRl due to the high expression of ZmWAK in the mesocotyl, resulting in reduced disease incidence. Histological assay indicated that S. reilianum was mainly colonized in the phloem of meosoctyl, while RNA in-situ hybridization assay revealed that ZmWAK was expressed in the pericycle parenchyma cells and xylem parenchyma cells, which encircling the phloem. Moreover, highly expressed ZmWAK induced high expression of SA-dependent resistance genes, such as ZmPR-1and ZmPR5, in the resistant HZA-qHSRl, as compared with HZ4. We also analyed the causative variations of ZmWAK alleles, and found that resistance ZmWAK alleles showed higher expression levels in the mesocotyl that those of weak ZmWAK alleles; wheras, no any difference of expression levels was found in roots and leaves for all ZmWAK alleles. These findings indicated that impaired expression would compromise maize resistance to head smut.Analysis of PAV in the ZmWAK locus in522maize lines and184teosinte entries revealed that deleted ZmWAK locus was only detected in maize germplasm, indicating that the deletion may occur after maize domestication. We sequenced the transcribed and promoter regions for54maize lines and23teosinte entries. Analysis of nucleotide diversities among these lines indicated the exon3region encompassing the kinase domain of ZmWAK evolved under functional restriction to form a selective sweep region.All above results revealed that Zm WAK confers maize resistance to head smut, which triggers the high expression of SA-related resistance genes to arrested the growth of S. reilianum in the mesocotyl, thus reduced the disease incidence.