Genome-wide Identification Of The R2R3-MYB Transcription Factor Family And Functional Dissection Of OsMYB18 And OsMYB98 In Rice

Rice,as the most important food crop in China,accounting for nearly 70%of the total grain output and providing food for more than half of the population.As a result,China is the world’s largest producer and consumer of rice,the production and stability of rice are essential for maintaining food security in both China and the global context.However,rice growth and development are susceptible to water,temperature,and pests and diseases.Therefore,identifying and studying stress-related genes in rice are important for breeding resistant rice varieties and improving yield.The MYB family consists of a group of transcription factors（TFs）with various functions.Among which,the R2R3-MYB type of proteins are the most abundant group in plants,controlling various biological processes such as plant growth and development,physiological metabolism,defense,and responses to biotic and abiotic stresses.In this study,a genome-wide identification of the MYB TF family in rice was conducted,and potential stress-related genes（OsMYB18 and OsMYB98）were identified and analyzed.Which were found to be involved in rice development,salt,drought,cold,and other abiotic stresses as well as biotic stress responses.The specific experimental results were as follows:1.Through bioinformatics analyses,a total of 190 MYB TFs including 99 R2R3-MYBs were identified in rice,with an uneven distribution across the 12 rice chromosomes.Analysis of cis-acting elements within the 2000 bp upstream regions of Os2R＿MYBs demonstrated that all Os2R＿MYBs feature hormone-related or stress-responsive elements,with respective occurrences of 91.9%,79.8%,79.8%,and 58.6%for ABRE,TGACG,CGTCA,and MBS sequences.Protein network analysis indicated that Os2R＿MYBs were involved in metabolic and biosynthetic processes as well as tissue development.Furthermore,certain genes displayed tissue-specific or developmental stage-specific expression patterns.These findings suggest that Os2R＿MYBs may play some significant roles in both rice development and stress response mechanisms.Two unreported TFs were choosed for further studied.2.Two previously unreported transcription factors were selected for further study.OsMYB18 and OsMYB98 were identified as TFs with potential resistance capabilities.Quantitative real-time PCR（q RT-PCR）analysis demonstrated that OsMYB18 and OsMYB98were expressed in various rice tissues with varying expression intensities.The relative expression level of the OsMYB18 was higher in the roots,leaves and roots at seedling stage,the OsMYB98 was higher in the leaves at seedling stage,in the roots and stems at heading stage of rice.Transient expression experiments in both tobacco and rice protoplast transformation studies confirmed the nuclear localization of OsMYB18 and OsMYB98proteins.3.Stable overexpression transgenic plants（OsMYB18-OX and OsMYB98-OX）and OsMYB18 and OsMYB98 knockout mutants（osmyb18 and osmyb98）were produced using CRISPR technology.In comparison to the wild-type Nipponbare,osmyb18 and osmyb98plants demonstrated a decreased stature,shorter root length,and reduced grain size.Conversely,OsMYB18-OX and OsMYB98-OX plants exhibited enhanced shoot height,elongated root length,and increased grain dimensions in terms of both length and width.4.There were multiple stress response pathways in plants which interact to form a complex regulatory network.Under abiotic stress,differential expression and functional analysis of OsMYB18 and OsMYB98 showed that the phenotype of the gene knockout plant osmyb18 was more tolerant to high salt environments than wild-type Nipponbare while the overexpression plant OsMYB18-OX was more sensitive to high salt environments.Additionally,after cold treatment,osmyb18 showed enhanced resistance to cold environments,while OsMYB18-OX and OsMYB98-OX showed increased sensitivity to cold environments.Abiotic stress-related gene expression detection showed that OsMYB18may negatively regulate the salt tolerance of rice by regulating the Na⁺/K⁺distribution ratio and ABA signal transduction pathway.OsMYB98 negatively regulates the drought resistance of rice by reducing the transcription levels of OsDT11 and OsCCD1,which depend on the ABA signaling pathway.Under cold conditions,OsMYB98-OX negatively regulates the cold resistance of rice by down-regulating the expression levels of kinase activity or Ca²⁺channel-related genes OsCOLD1,OsSAPK8,and OsCPK24.The experimental results showed that under salt stress,OsMYB98 showed the accumulation of reactive oxygen species and oxidative damage to cell membrane lipids in different expression modes of knockout and overexpression,resulting in a decrease in salt tolerance of rice plants.This indicated that OsMYB98 may participate in the complex regulation network of stress resistance,which was affected by other pathways,resulting in a decrease in salt stress tolerance.Further experiments are needed to study this mechanism.Similarly,OsMYB18 knockout mutants and overexpression plants were more sensitive to cold and drought stress,and H₂O₂ and superoxide anions accumulate under stress,leading to damage to the plant leaf membrane system and severe plant damage.The mechanism of OsMYB18 and OsMYB98 in regulating salt or cold stress in rice needs further study.5.In vitro leaf inoculation assays using rice blast fungus revealed that osmyb18 and osmyb98 plants exhibited enhanced resistance to rice blast in comparison to the wild-type Nipponbare,as evidenced by shorter lesion lengths and decreased fungal relative biomass.In contrast,OsMYB18-OX and OsMYB98-OX plants displayed heightened susceptibility to rice blast,characterized by extended lesion lengths and elevated fungal relative biomass.6.Through yeast library screening and yeast two-hybrid validation methods,that OsMYB18 and OsMYB98 interact with OsVDAC6 were determined.The interaction was further substantiated using bimolecular fluorescence complementation（Bi FC）and co-immunoprecipitation（Co-IP）techniques.