Evolutionary Analysis Of RIPs Genes And Functional Study Of ZmRIP1 Gene In Maize

Ribosome inactivating proteins(RIPs)are toxic proteins mainly found in bacteria,fungi,and higher plants,which can inactivate ribosomes by damaging their structure.RIPs in plants have various biological resistance functions against viruses,bacteria,fungi,and insects,and have great potential for utilization.Currently,the evolution of RIPs genes and the types and functions of ZmRIPs genes in maize are relatively less studied.In this study,we mainly collected high-quality genomic and transcriptomic data from various species and used bioinformatics methods to accurately identify RIPs genes,analyze the characteristics of RIPs,phylogenetic features,genes expression patterns,and evolutionary driving factors to comprehensively explore the evolution of RIPs.We selected potentially biologically resistant ZmRIPs genes by molecular dynamics characteristics of adenine(ADE)binding to ZmRIPs,studied the functions of ZmRIP1 using transgenic Arabidopsis,transgenic maize,and maize mutants,and analyzed the resistance to Fusarium graminearum function of ZmRIP1 using RNA sequencing.We deeply explored the ZmRIPs with great potential for utilization.The main results were as follows:1.RIPs are distributed in bacteria,fungi,and plants,but their types vary.In this study,a total of 582 RIPs genes were identified in 764 bacterial species,46 RIPs genes in 39 fungal species,and 782 RIPs genes in 51 plant species.They can be roughly divided into proactive defense bacterial or fungal types and passive defense plant types based on their role in their respective organisms.2.The evolution of plant RIPs might be related to the developmental structure of fruits or seeds.We did not find any RIPs in non-seed plants with many reproductive offspring,but found RIPs in some seed plants including gymnosperms and angiosperms.Plants with seed or fruit development structures that are not conducive to seed dispersal might be more likely to have RIPs.The evolutionary characteristics of RIPs in monocotyledonous and dicotyledonous plants are different.Compared to dicotyledonous plants,monocotyledonous plants have more RIPs,higher codon usage bias,and more new types of RIPs with other domains.The proportion of RIP2 genes is lower.In addition,we also found that RIP2 might only exist in plants,and the RIP2 genes might originate from the common ancestor of angiosperms and gymnosperms.The origin of RIP2 in plants might be earlier than RIP1,and some RIP1 genes may be formed by tandem duplication of the RIP2 genes.3.ZmRIP1 might possess biological resistance.Through phylogenetic and gene expression analysis of ZmRIPs in maize,we found the type and number of secondary motif structures of ZmRIP1 and the toxic protein b-32 were completely consistent.Only the ZmRIP1 in ZmRIPs is up-regulated by infection with Fusarium graminearum and Colletotrichum graminicola.Through molecular docking and molecular dynamics simulation of maize ZmRIPs and ADE,we found that ZmRIP1 had a strong binding affiny with ADE(-4.8 kcal/mol),second only to b-32(-5.3 kcal/mol),and the active sites were same with b-32.4.Overexpression of the ZmRIP1 gene enhances the resistance of Arabidopsis thaliana and maize to Fusarium graminearum,while the maize mutation of ZmRIP1 gene reduces the resistance of to Fusarium graminearum.Through resistance identification experiments on Arabidopsis thaliana and maize infected with Fusarium graminearum,we found that overexpression of the ZmRIP1 gene significantly enhanced the resistance of Arabidopsis thaliana leaves and maize roots to Fusarium graminearum,while EMS mutagenesis of the ZmRIP1 gene significantly reduced the resistance of maize stems and ears to Fusarium graminearum,and CRISPR/Cas9 knockout of the ZmRIP1 gene significantly reduced the resistance of maize roots to Fusarium graminearum.5.Transcriptome data analysis revealed the important role of ZmRIP1 gene in maize resistance to Fusarium graminearum.By inoculating the wild-type,over-expressed and knockout strains of ZmRIP1 gene in the young roots of maize with Fusarium graminearum,and comparing the transcriptome data at 0h,24 h and 48 h after inoculation,we found that over-expressing ZmRIP1 gene before inoculation had a significant impact on the synthesis of chlorophyll or xanthophyll,energy metabolism,and stress response in maize.After inoculation,a large number of differentially expressed genes were enriched in pathways such as plant-pathogen interaction pathways,MAPK signaling pathways in plants,and plant hormone signaling transduction pathways,indicating that ZmRIP1 gene may play an important role in maize disease resistance,stress response,hormone signaling transduction,and growth and development.