Screening And Validation Of Stalk Rot Resistance Genes In Maize

Maize stalk rot is one of the most devastating diseases,causing significant yield losses around the world and also posing a potential food safety problem.Fusarium verticillioides(F.verticillioides)has emerged as a dominant pathogenic bacterium of maize stalk rot,however,the underlying mechanisms of our defence against Fusarium stalk rot remain poorly understood.In this study,a combination of transcriptomic RNA-seq analysis and proteomic iTRAQ labeling approaches was developed by comparing inoculated F.verticillioides Differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)in maize stalks before and after F.verticillioides to identify pathogen related disease resistance genes.Parallel reaction monitoring(PRM)and real-time PCR(RT-q PCR)techniques were used for quantitative verification with F.verticillioides Expression levels of candidate genes associated with disease resistance in F.verticillioides.On the basis of multi omics analysis,the defense related genes ZmCTA1,ZmWIP1 and ZmLOX2 identified in the multi omics analysis were further evaluated by virus induced gene silencing(VIGS)in Potential role during F.verticillioides infestation.It was further analyzed that ZmLOX2 regulates the salicylic acid pathway dependent hypersensitive response(HR)in maize plants to resist the pathogen Infection by F.verticillioides.The results provide insight into the mechanism of action of the molecular mechanism of F.verticillioides interaction with host crops lays a certain theoretical foundation and provides a basis for the discovery of new key pathogenic genes of pathogenic bacteria.The main research results are as follows:(1)In this study,using transcriptomic and proteomic analyses,2814 DEGs and 387 DEPs were identified,respectively.Combined analysis of transcriptome and proteome revealed 74 DEPs matched with their DEGs,among which 57 DEPs showed the same trend as DEGs(49 up-regulated,8 down regulated),and finally 14 DEGs were identified to be associated with F.verticillioides response related candidate genes.RT-q PCR validation was performed on 14 candidate genes,and the expression levels of 11 candidate genes were consistent with the RNA-seq data of DEGs,showing an upward trend.The expression levels of 3 candidate genes were consistent with the corresponding RNA-seq data,showing a downward trend.In addition,PRM validation of the corresponding DEPs of the 14 candidate genes revealed that the DEPs corresponding to the candidate genes showed high concordance with the expression trends of the iTRAQ data,indicating that the transcriptomic and proteomic analysis results were accurate and reliable for further studies.(2)Select ZmLOX2,ZmCTA1,and ZmWIP1 genes closely related to the disease resistance pathway from the above 14 genes for further verification.To understand the potential roles of ZmLOX2,ZmCTA1 and ZmWIP1 in regulating defense responses in maize,a modified brome mosaic virus(BMV)silencing vector was used to induce ZmCTA1,ZmWIP1 and ZmLOX2 gene silencing.F.verticillioides were inoculated into the roots and culms of maize plants on days 7 and 10 after viral silencing,and RT-q PCR was performed on the leaves and roots of silenced plants at both time points.The results showed that the silencing efficiency of genes ZmCTA1 and ZmWIP1 reached 39% to 50% and were successfully silenced,and the plants in which the ZmCTA1 and ZmWIP1 genes were silenced showed higher disease grade and disease index after verticillioides inoculation compared with the controls.ZmLOX2 expression in BMV-ZmLOX2-inoculated plants was only 20.5% to 27% of that in control plants,and the expression of ZmLOX1,ZmLOX4,and ZmLOX5,which have high homology to ZmLOX2,was not affected,indicating that ZmLOX2 was successfully silenced.Disease grade and disease index according to the roots and stalks of silenced plants showed that ZmLOX2 silenced plants had heavier disease onset than controls.ZmLOX2,ZmCTA1 and ZmWIP1 are thus involved in maize resistance to resistance in F.verticillioides.(3)To explore the pathway involved in the regulation of maize defense responses by ZmLOX2.This study used the phytohormones methyl jasmonate(Me JA)and salicylic acid(SA)for external spray treatments while ZmLOX2 silenced plants were inoculated with F.verticillioides.SA and JA expression,as well as expression of disease resistance related PR genes,Lipoxygenase(LOX)activity,and contents of disease resistance related physiological indexes were examined after 24 h and 48 h,respectively.The results showed that exogenous application of SA enhanced maize resistance to F.verticillioides infestation.Taken together,ZmLOX2 may regulate maize defense responses to F.verticillioides through the SA pathway.