Mechanism Of Regulation Of Rice Resistance Against Brown Planthopper By OsEBF1/2

Brown planthopper（BPH,Nilaparvata lugens St?l）is one of the most destructive rice pests,which mainly feeds on the phloem sap from rice plants through their stylet,causing severe yield loss in rice.Plant hormone signaling is involved in regulating stress responses and tolerance in rice.Among them,ethylene signaling pathway plays an important role in rice resistance to biotic stresses.However,the role of OsEBF1/2,two negative regulators of ethylene signaling,in rice resistance to BPH is still unknown.In this study,the molecular mechanism of OsEBF1/2 in the regulation of rice resistance against BPH was investigated.The main findings are as follows:1.Firstly,OsEBF1/2 homologous proteins in different species were analyzed,and phylogenetic analysis showed that OsEBF1/2 of rice had the highest homology with EBF1 of Sorghum bicolor and brachypodium distachyon.The tissue-specific expression of OsEBF1 and OsEBF2 genes was detected using real-time fluorescence quantitative PCR,and the results showed that OsEBF1 and OsEBF2 were expressed at the highest level in rice leaf sheaths,the main feeding site of BPH.Both OsEBF1 and OsEBF2were significantly induced by BPH infestation,and OsEBF2 expression was higher than that of OsEBF1.To further understand the biochemical properties of OsEBF1/2,we conducted tobacco transient expression assay,and found that both OsEBF1 and OsEBF2 were localized in the nucleus.Compared with wild type,both ebf1 and ebf2 exhibited significantly shorter roots upon ACC treatment.However,the root growth of EBF1/2-overexpressing rice OE-EBF1 and OE-EBF2 was barely inhibited,indicating that EBF1 and EBF2 are negative regulators of ethylene signaling.2.BPH resistance of ebf1 and ebf2 mutants were determined,ebf1 and ebf2 were found to be more sensitive to BPH than Nip.The survival rate of Nip,ebf1 and ebf2 rice after BPH infestation was 83.33%,33.33%and16.67%,respectively.The survival rate of Nip,ebf1 and ebf2 rice after BPH infestation was 83.33%,33.33%and 16.67%,respectively.The BPH body weight increments in Nip,ebf1 and ebf2 were 0.91,1.39,and 1.44 mg.In the honeydew excretion test,BPHs fed on ebf2 plants excreted a higher volume of excretion than those fed on Nip and ebf1.These results indicate that OsEBF1/2 positively regulated the resistance of rice to BPH.3.The cotents of lignin and cellulose contents in ebf2 mutant were analyzed,we found that their cotents in ebf2 were significantly lower than those in other materials,with a reduction of 31.21%and 15.32%,respectively,compared to Nip.q RT-PCR showed that ebf2 exhibited lower expression of lignin and cellulose synthesis-related genes than those of Nip before and after BPH infestation.Most of these genes were significantly lower in ebf2 than those in Nip after BPH infestation for 12 h,indicating that OsEBF2 regulated the expression of lignin and cellulose synthesis related genes.We also found that OsEBF2 interacts with the gibberellin signaling suppressor Os SLR1 by a yeast two-hybrid experiment,suggesting that OsEBF2 may regulate the BPH resistance of rice by interacting with Os SLR1.Bioassay showed that exogenous application of GA₃ couldenhance the resistance of rice to BPH,with the best effect at 10μM concentration of GA₃.To further determine whether Os SLR1 is involved in regulating rice resistance to BPH,the BPH resistace phenotype of slr1-gain of function mutant（slr1-gain）was analyzed.slr1-gain was found to be more sensitive to BPH compared with Nip.Bioassay showed that the survival rate of slr1-gain was significantly lower than that of Nip.However,the honeydew,weight gain,and survival of BPH on ebf2 were significantly higher than those fed on Nip.These result suggests that Os SLR1 is a negative regulator of rice resistance to BPH.Taken together,OsEBF1/2 are negative regulators of ethylene signaling in rice and plays a positive role in the regulation of rice BPH resistance.OsEBF2 may integrate ethylene and gibberellin signaling in to regulate rice defense against BPH.Our study reveals the molecular mechanism of EBF1/2 in regulating rice BPH resistance,and can be beneficial for genetic improvement for BPH resistance in rice.