Cloning And Resistance Mechanism Of Genes For Gray Leaf Spot And Stalk Rot Resistance In Maize

Gray leaf spot and stalk rot are two devastating fungal diseases in maize worldwide.In our previous study,four QTLs have been identified in a segregation population derived from the resistant line Y32 and the susceptible line Q11.One of the major QTL-qRgls1 located on chromosome 8 was mapped to a 1 Mb region.For stalk rot,the laboratory used the resistant line 1145 and the susceptible line Y331 to construct a segregating population(F2,BC1:2).Two QTLs were identified by artificial inoculation of stalk rot,the minor efficient QTL-qRfg2 located on chromosome 1 was mapped into a 2.5 kb region and contained only one candidate gene,ZmAuxRP1.In this study,we report on fine-mapping and gene cloning of QTL-qRglsl and functional identification and resistance mechanism of QTL-qRfg2.1.Developing new molecular markers in the major QTL-qRgls1 region(IDP2-SNP2)to screen new recombinant plants for fine mapping.The progeny-test mapping strategy was used to fine mapping the QTL-qRgls1 in two years.15 BC4F7 and 24 BC6F7 recombinants were used to narrow down QTL-qRgls1 within 120 kb flanked by markers IDP2 and M2.Near-isogenic lines(NIL-R and NIL-S)were developed within the BC7F7 population.2.QTL-qRglsl could steadily decrease the field disease index by 12.2-29%among different backcross populations in continuous five years from 2013 to 2018,except 2017.It's suggested that the QTL-qRgls1 has potential to enhance maize resistance against Gray leaf spot.3.The positive BAC clones were screened from resistant line Y32 BAC library.In the QTL-qRgls1 region,we found 8 ORFs and 6 of them have functional annotation.RNA expressions confirmed ZmWAK-RLK was the candidate gene of qRgls1.Both transgenic complementation test and overexpression assay definitely indicated that ZmWAK-RLK is the gene for the major resistance QTL-qRgls1.4.ZmWAK-RLK encoded a wall-associated kinase,located in plasma membrane,which potentially served as a receptor-like kinase to perceive extracellular signal.The major difference of allele is also located in extracellular domain.A membrane protein ZmHR-like can combine with resistant ZmWAK-RLK in yeast and tobacco,but not with susceptible protein?5.Both enhance expression test and RNAi assay indicated that ZmAuxRPl can improve maize resistance to gray leaf spot.There was a negative correlation between resistance and ZmAuxRP1 expression.6.In the seedling,the resistance of Y331-R was significantly higher than Y331-S,but the root length of Y331-S was significantly longer than Y331-R.The root length and lateral root number of EE+ were significantly higher than EE-,while the root length and lateral root number of RNAi+ were significantly lower than RNAi-.The high expression of ZmAuxRPl can promote the growth of maize roots.Expression analysis and IAA content test showed that the expression of ZmAuxRPl was positively correlated with IAA content.The higher expression of ZmAuxRPl,the higher IAA content.7.Exogenous NAA can induce the expression of ZmAuxRPl,and the resistant allele is faster than susceptible allele.The resistant allele promoter is also more responsive to NAA than the susceptible allele promoter.8.Transcriptome analysis revealed that DEGs are mainly involved in defense and immune-related responses after elevated levels of ZmAuxRPl.KEGG enrichment found that DEGs are mainly enriched in secondary metabolite synthesis-related pathways.Metabolome analysis showed that after the expression of ZmAuxRPl increased,the content of IAA increased and the content of BXs decreased.ZmAuxRPl coordinates the balances between root growth and stalk rot resistance by regulating the distribution of IGP.