Cloning And Functional Analysis Of Main Effect QTL In Resistance To Tobacco Brown Spot

Tobacco brown spot(BS)is a foliar disease caused by Alternaria alternata(fries)Keissler and has caused devastating harm to tobacco production.The most fundamental and effective control method is to discover and utilize resistance genes.However,the molecular breeding of BS-resistant tobacco is still in the preliminary mapping stage,which restricts the utilization of resistant resources.Previously,we have identified a novel major BS resistant QTL-qBSR12-1 by using a RIL population.In this study,we cloned the major gene and carried out the functional verification and investigation.The main results are as follows:1.Preliminary analysis of molecular mechanism in plant-pathogen interaction.In the study,BH-1 and XHJ were innoculated with A.alternata spore suspension and sampled at 0 hpi,6hpi,12 hpi,24 hpi,48 hpi and 72 hpi for RNA-seq.Transcriptome analysis showed that a large number of genes enriched in photosynthesis-associated pathways were down-regulated in response to A.alternata infection.DEGs between infected BH-1 and XHJ also mainly enriched in pathways associated with cell wall and photosynthesis.2.Identification of the qBSR12-1 candidate gene.In the study,qBSR12-1was mapped to the interval of bin12-96 and bin12-96 b,which is about 2 c M.The target region was further narrowed down to 84.0 Mb ～ 84.8 Mb of chromosome 7 by combining the GWAS result.Resequencing data analysis showed that a 936 Kb fragment covering the target region was deleted in BH-1.Among the 6 encoded genes within the 0.8 Mb,NtERF170 was the only one induced by pathogen infection.Therefore,NtERF170 was identified as the candidate gene of qBSR12-1.3.Functional verification of NtERF170.The loss-of-function mutants of NtERF170 was developed using CRISPR/Cas9 technology.Phenotypic assay showed that loss-of-function mutants exhibited increased resistance compared to the wildtype control.Moreover,the transient expression of NtERF170 in XHJ contribute to its susceptibility to A.alternata.Therefore,NtERF170 was identified as the dominant gene of qBSR12-1.4.Functional investigation of NtERF170.Transient expression of NtERF170 in Nicotiana benthamiana could induce cell death.Therefore,N.benthamiana transiently expressed NtERF170 was sampled for RNA-seq.Transcriptome analysis showed that a large amount of genes enriched in photosynthesis-associated pathways were down-regulated in NtERF170-overexpressed tissue,suggesting NtERF170 might facilitate A.alternata infection by inhibiting photosynthesis.Proteins interact with NtERF170 was screened by using yeast two-hybrid system and verified by gyration validation.