Evolutionary Analysis Of CBP60 Protein Family And Functional Validation Of RBOHB Related To Calcium-Induced Pathways In Plant Immunity

Plants are affected by various biotic and abiotic stresses,such as bacteria,fungi,drought and extreme temperature.During the long term of evolution,plants formed an elaborate immunity system to response to the infection of these pathogens to avoid their influence.Calcium which is an important second messager in plant immunity enhances plants’ability to resist to environmental stress by activating or depressing the activity of given enzymes in plant cells or by inducing the transcription and translation of stress tolerance genes.Evolutionary analysis and functional validation of genes involved in pathways mediated by calcium in plant immunity in Zea mays L.and Arabidopsis thaliana will provide useful information to molecular mechnisam in plant immunity and the genetic improvement of crops.Our previous results demonstrated that AtCBP60a,AtCBPg and At SARD1 in Arabidopsis were key regulation genes of plant immunity pathways mediated by calcium.Based upon this,this research used the genomic/transcriptomic data of 318 public available plant species to conduct evolutionary analysises in CBP60（Camodulin-Binding Protein 60）families which are important in plant immunity.Meanwhile,two maize DH lines under Fusarium graminearum stress with no stress as controls were used for Whole Genome Bisulfite Sequencing（WGBS）and data analysis found that RBOHB in RBOH（NADPH oxidase/respiratory burst oxidase homolog）protein family may be involved in the calcium-mediated pathways which are response to maize ear rot.Functional validation and evolutionary analysis were further conducted,finding that this gene was closely related to mazie resistance to ear rot.In this study,the evolutionary analysis and functional validation of two calcium-related gene families not only provide important gene source for maize disease-resistant breeding,but also lay a solid and theoretic foundation for molecular mechanisms in plant immunity.The major results obtained in this study were listed as follows.1.A protein blast database was built by ncbi-blast suite based on the public available data of318 land plant species.Based on this database,eight CBP60 proteins sequences from Arabidopsis were used as query for protein blast.As a result,1170 CBP60 orthologs were obtained and used to infer phylogenetic trees,and all CBP60 family was divided into five subfamilies of CBP60a,CBP60b-d,CBP60e-f,CBP60g and SARD1.2.Based on the analysis of phylogenetic trees of CBP60 protein family,two divergences occured during its evolution.The first divergence leads to immune-related clade neofunctionalized from its prototypical clade,and the second one occured in immune-related clade,leading to immune-related subfamily to evolve differently,among which CBP60a subfamily is negative regulator while CBP60g and SARD1 subfamilies are positive regulators.Sequences analysis of CBP60a,CBP60b-f,CBP60g and SARD1 by using PEAES（Protein Evolution Analysis of Euclidean Space）found that CBP60g subfamily has the highest diversity（0.312）and least conserved amino acid sites（51）and that CBP60b-f subfamily has the highest conservation（0.097）and most conserved amino acid sites（174）.Coevolutionary analysis among CBP60a,CBP60g and SARD1 subfamily found three interaction sites(the 238^th aa from CBP60g-SARD1 and the 66^th and 204^th aa from CBP60a-SARD1),implying that these sites may be key sites for CBP60a protecting CBP60g and SARD1.CBP60a,CBP60g and SARD1 among 12 plant species were used as common sequences for comparison.The results of significant test based on PESES found that when CBP60g was used as common sequence,10 out of 12 plant species shown significantly positive,however,when SARD1 was as common sequence,only 3 shown significantly positive,indicating that CBP60g is preferentially protected over SARD1 in many linages.The distribution of the colors of site bars does not show strong similarity between more closely related species,confirming that the lineage divergences were sufficiently old and that observed selections at the sites are consequence of the linage-specific selection after linage divergence.Based on the above,a conclusion was drawn that many sites in the CBP60-conserved domain of the three immune regulator subfamilies have likely been coevolving under the pressure from pathogen effectors in a plant linage-specific manner3.WGBS and data analysis in resistant line and susceptible line under Fusarium graminearum found that difference of genomic DNA methylation levels under three contexts（CG,CHG and CHH）between resistant line and susceptible line is not significant,but distributions of DNA methylation levels on chromosomes in resistant line and susceptible line are significantly different.DNA methylation levels under CG and CHH context vary a lot on the chromosome of susceptible line（IBM-85）while that stabilize on the chromosomes of resistant line（IBM-81）,inferring that the difference may be the reason that these two lines are response to ear rot differently.Differential analysis of DNA methylation levels in promotor regions showed that 14737,19300 and 17903 differential methylation genes were obtained under CG,CHG and CHH context,respectively.Genes in given datasets were used for KEGG enrichment,the results demonstrated that plant-pathogen interaction pathway has the highest significance,and the significance of the pathway under CG context is highest among three contexts.In addition,RBOH is highly enriched;Bioinformatical analysis found that RBOHB contains EF-Hand motifs,implying that RBOHB may be involved in maize restant to Fusarium graminearum by binding to calcium.These results demonstrated that this gene family plays an important role in plant immunity.4.Evolutionary analysis in RBOH family found that RBOHB can be divided into six subfamilies.During the evolution of RBOH family,gene duplication occurred several times,forming the modern RBOH family.And the first and second duplication occurred before basal angiosperm,leading to four RBOH subfamilies（RBOH III,IV,V and VI）.After that,duplication occurred several times in monocots and eudicots.Further analysis showed that RBOHB subfamily involved divergently before angiosperm,and diverged at least once in other species but twice in maize,suggesting that RBOHB were under strong selection so as to involve rapidly to respond to stress.Taking the evolutionary analysis of RBOH and CBP60,both of them shared the same evolutionary pattern,of which the first divergence via obtaing neofuntions from the prototypical clade occurred before basal angiosperm and the following divergences or gene duplication and selection occurred in high plants.5.In order to validate its functions,full length of c DNA sequence was cloned by PCR and sequenced,showing that the coding region has a length of 2697 bp which codes 899 aa.Combination of maize protoplast and subcellular localization,RBOHB was demonstrated to be localized in membrane.To validate its ability to resist disease,CRISPR/Cas9 construct was designed and built.Genetic transform was employed to validate its function and ability to resist maize ear rot,which provide important gene source for maize disease resistant breeding.Bioinformatics was used to predict the co-expression genes of ZmRBOHB.As a result,9directly related genes and 5 in-directly related genes were screened.q RT-PCR was used to test the related expression of these genes in resistant and susceptible lines after 2 days,3days and 4 days after inoculated by Fusarium graminearum spores.The results showed that Zm CPK5 and Zm WRKY48 were significantly differential expressed.Meanwhile,key genes in the biosynthesis pathways of jasmonic acid,salicylic acid and ethylene were used as marker genes to test how the production of JA,SA and ET between the two maize lines changed after inoculation.The results showed that sid2 in SA biosynthesis pathway was significantly differential expressed between resistant line and susceptible line.Taking all results above together,a model was proposed for maize in response to maize ear rot and the theoretical foundation was laied for studying its molecular mechanism.