Functional Characterization Of A Rice Transcription Factor OsWRKY72 In Plant Growth And Development And Blast Resistance

Rice(Oryza sativa L.)is one of the important food crops,the harvestable parts of which,namely rice grains,serve as the staple food for nearly half of the global population.The root development,shoot architecture,and resistance to various biotic and abiotic stresses of rice are all crucial determinants for its firmly high yield.Root is an organ for plant anchoring and the uptake of water and nutrients,and agronomic traits determining rice shoot architecture such as plant height and the angles of tillers and leaves,are vital for lodging resistance and photosynthetic efficiency,respectively.In addition,rice blast caused by the rice blast fungus(Magnaporthe oryzae)is one of the major diseases confronted by rice plants which may lead to severe loss in productivity.The growth and development of crops is closely related to their resistance to pathogens.It has been documented that the improvement of development/productivity-related trait and enhancement of pathogen resistance can be coordinately achieved through manipulating the expression of a single gene.Nevertheless,the crosstalk between the pathogen resistance of crops and their growth and development as well as the underlying mechanism are widely unveiled.A large number of WRKY transcription factor(TF)-encoding genes are present in plants.In rice genome,more than 120 members have been annotated.WRKY TFs are involved in plant growth and development,and diverse responses to biotic and abiotic stresses.A single WRKY TF can be responsible for the regulation of two or more seemingly disparate biological processes.In our previous work,we provided evidence that one rice WRKY TF,OsWRKY72,is involved in maintaining P homeostasis.Here,we generated the mutant plants of OsWRKY72.By using its mutants and overexpression lines,we investigated the role of OsWRKY72 in rice growth and development.Furthermore,its involvement in rice blast resistance was also analyzed.The main results acquired are listed as follows.1.Our previous results showed that OsWRKY72 was transcriptionally induced by exogenous auxin and abscisic acid(ABA),and overexpression of OsWRKY72 led to a significant increase in primary root.Given that exogenous auxin and ABA both suppress rice root elongation,the mutants and overexpressors of OsWRKY72 were subjected to 1naphthaleneacetic acid(NAA)and ABA treatments,in an attempt to investigate the role of OsWRKY72 in rice root elongation and responses to exogenous auxin and ABA.Under control condition,the primary root length was significantly decreased and increased,respectively,in the mutants and overexpression lines compared with the wild type(WT)plants.The adventitious root(AR)number of OsWRKY72 overexpression lines was higher than that of WT plants,whereas no difference was found in the mutant plants.Furthermore,in the basal node where AR emerge,the expression of two genes encoding positive regulators of AR emergence was not altered upon OsWRKY72 mutation or overexpression,suggesting that the regulation of OsWRKY72 on AR emergence occurs independent or downstream of these two genes.When exposed to NAA or ABA,the alteration trend in the primary root length of the mutants and overexpressors was similar to that found under control condition.Notably,the primary root elongation of OsWRKY72 overexpression plants was insensitive to ABA,as evidenced by increased fold change of relative root elongation rate.These results suggest that OsWRKY72 is involved in both root elongation and abiotic stress resistance.2.Since stem-elongation stage,OsWRKY72 overexpressors but not mutants showed a dwarf phenotype.This results from decreased length of each internode,consistent with our previous findings.Unexpectedly,the cell length of the peduncle of OsWRKY72 overexpressors was larger than that of WT plants.By contrast,the cell number of the peduncle of OsWRKY72 overexpressors was significantly decreased compared with WT plants,suggesting that the dwarf phenotype observed in OsWRKY72 overexpressors is attributed to impaired cell proliferation.A reverse transcription-polymerase chain reaction(RT-qPCR)analysis showed that the gene encoding the DELLA(a repressor protein functioning in the GA signaling cascade)homolog in rice,OsSLRl(Slender Rice 1),was upregulated in the culm of OsWRKY72 overexpressors,and an electrophoretic mobility shift assay(EMSA)demonstrated that OsWRKY72 physically interacts with the promoter of OsSLR1.These results indicate that OsWRKY72 is a negative regulator of GA signaling through activating OsSLRl expression.In addition,the expression of three GA biosynthetic genes(OsKS1,OsKAO and OsGA20ox3)and one GA inactivation gene(OsGA2ox4)was enhanced and suppressed,respectively,upon OsWRKY72 overexpression.This might be a feedback regulation of GA biosynthesis due to impaired GA signaling(increased OsSLR1 transcript and thus DELLA protein).Furthermore,the diameter of the cross section of the culm and that of the medullary cavity of OsWRKY72 overexpressors were markedly reduced compared with WT plants,whereas the alteration in wall thickness showed an opposite trend.Whether these changes are also caused by the up-regulation of OsSLR1 remains to be studied.3.Grown in paddy field,the OsWRKY72 overexpressors at the reproductive stage was hypersensitive to rice blast as evidenced by visible brown spots in leaves.To verify whether OsWRKY72 is indeed involved in rice blast resistance,we inoculated the leaves of the mutants and overexpressors of OsWRKY72 as well as WT plants at seedling stage with rice blast fungi(Magnaporthe oryzae,strain Guy11).The expression of OsWRKY72 was induced by inoculated rice blast fungi and also by exogenous salicylic acid and jasmonic acid(JA),two phytohormones involved in plant resistance to pathogens.Unlike that found under field condition,OsWRKY72 overexpressors showed no alteration in rice blast resistance.Interestingly,oswrky72 mutants were more resistant to rice blast.These results suggest that OsWRKY72 is a negative regulator of rice blast resistance and that its regulation may be dependent on the developmental stage of rice plants and/or the strain of rice blast fungi.Furthermore,RT-qPCR analysis showed that one of the pathogen resistance-related genes,OsPAL9,was significantly up-regulated upon OsWRKY72 mutation.In addition,it has been established that OsWRKY72 functions as a negative regulator of rice resistance to blight disease via suppressing the expression of a JA biosynthetic gene,OsAOS1.It is of interest and significance to investigate whether OsWRKY72-regulated rice resistance to blast disease is also achieved through modulating JA biosynthesis.In summary,this study demonstrates that in addition to maintaining P homeostasis,OsWRKY72 is involved in several other seemingly disparate biological processes,namely root elongation,response to abiotic stress,stem elongation and rice blast resistance.Thus OsWRKY72 is probably a central regulator integrating the signaling events in the aforementioned processes.It represents a novel entry point to dissect the crosstalk between GA and JA signaling pathways,and provides new ideas for breeding of crops with multiple eminent traits.