Fine Mapping Of Rice Brown Planthopper Resistance Gene Bph34(t) And Mechanism Dissection Of OsphyB In Dim Light Modulated Insect Resistance In Rice

Rice is one of the most important food crops in the world,as the population grows,the demand for food is increasing.However,rice production is seriously threatened by various pests and diseases all the time.Brown planthopper（BPH）is one of the most serious pests to rice.The outbreak of BPH caused a large area of rice plants withered and lodged or even completely yiled loss.In addition,BPH is also a vector for the transmission of Rice Grassy Stunt Virus Disease（RGSVD）and Ragged Stunt Virus Disease（RSVD）.At present,chemical pesticides are still the main measures to control BPH.The long-term overuse of pesticides not only increases the production cost,pollutes the environment,and leads to pesticide residues,but also promotes the emergence of drug resistance of BPH.Which will cause the BPH to become rampant and more difficultly to control.Breeding BPH resistant rice varieties is considered to be the most cost-effective way to control BPH.And discovering resistance genes is the precursor and basis for breeding resistant varieties.Although a number of BPH resistance genes have been discovered and cloned,it has also been found that BPH have strong adaptability to resistant varieties and overcome the variety resistance in a relatively short period of time.Therefore,it is necessary to continuously discover new resistance genes to cope with the variability of BPH population.In addition,previous studies have found that the rice resistance against BPH is often affected by environmental factors,such as light.Dim light significantly reduce the BPH resistance,but the molecular mechanism of how light regulates BPH resistance in rice is poorly understood.In this study,we screened a rice cultivar W41126 with high resistance to BPH.The BPH resistance gene was fine-mapped and named Bph34（t）.In addition,we also found that the rice phytochrome Phytochrome B（OsPHYB）plays an important role in light regulating rice resistance to BPH,and investigated the molecular mechanism of OsphyB regulating BPH resistance under normal light and dim light conditions.The main researches as follows:1.Fine mapping of rice BPH resistance gene Bph34（t）（1）Comparative analysis of BPH resistance between W41126 and C418The indica variety W41126 is from Vietnam.We found that the variety was highly resistant to BPH by Seedling Bulk Test（SBT）.Consistent with the identification at the seedling stage,W41126 also showed strong resistance to BPH at the adult stage.So,W41126was a rice cultivar resistant to BPH at both seedling and adult stages.The feeding preference experiment found that the number of BPH on W41126 plants was significantly less than C418,indicating that W41126 had a rejection to BPH.However,after forced feeding,the excretion of BPH on W41126 and C418 plants has no significant difference.It indicating that there may be no difference in the feeding amount of BPH between the two cultivars.（2）Genetic analysis and gene mapping of BPH resistance in W41126In order to analyze the genetic basis of W41126 resistance to BPH,a F2:3 population with 120 individuals derived from a cross of W41126/C418 was used.Then evaluated the BPH resistance at seedling stage and the construction of molecular marker genetic map were completed.Finally,we detected a LOD value of 5.0 in the range of about 1.1 Mb between RM190 and RM3414 on the short arm of rice chromosome 6,and the contribution rate was36.9%.Which was the BPH resistance QTL locus q Bph6 in W41126（3）Fine mapping of the BPH resistance gene Bph34（t）In order to fine-map the gene q Bph6,a total of 21084 individual plants in backcross populations BC₁F₂,BC₂F₂ and BC₃F₂ were used.Recombinant individuals were screened using marker RM190 and RM3414 based on initial mapping information.We evaluated the BPH resistance of recombinant plants and its offspring plants.Then several SSR markers and In Del markers were newly developed according to the publicy available rice genomic sequences.Finally,the gene was finely mapped in the interval of about 203 kb between the molecular markers XH1 and NWD11.The comparative analysis with the previously reported BPH gene indicated that q Bph6 was a new BPH resistance gene,which was named Bph34（t）.The identification and cloning of Bph34（t）is not only helpful for elucidating the molecular mechanism,but also can speed up the application in breeding program.2.The role of OsphyB in light-modulated insect resistance in rice（1）Dim light reduces BPH resistance in riceIn order to study the effect of light intensity on rice BPH resistance,two-week-old seedlings of Nipponbare（NIP）were pretreated with dim light(DL,50μmol m^-2 s^-1),normal light(NL,200μmol m^-2 s^-1)and strong light(SL,650μmol m^-2 s^-1)for 24 hours,then infested with BPH and continuously exposed to SL,NL or DL treatment,respectively.The results showed that after 8 d of infestation,the seedling mortality rate of NIP under DL was 100%,while under NL was about 50%and under SL was only about 2%.To determine whether the effect of light on the BPH resistance varies among rice cultivars,three other rice varieties were used.Including Japonica rice Zhonghua 11（ZH11）,indica rice 93-11 and IR64（carrying BPH resistance gene Bph1）.Similarly,DL could significantly reduce BPH resistance in both japonica and indica rice or BPH susceptible and resistant varieties.（2）The inhibition of DL on rice BPH resistance depends on OsPHYBIn order to analyze the molecular mechanism of DL inhibiting rice BPH resistance.We evaluated the BPH resistance in three rice phytochrome mutants osphya,osphyb-1 and osphyc under NL and DL conditions,respectively.The results showed that the BPH resistance of osphya was significantly lower than wild tipe（WT）under NL,but there was no significant difference between WT and osphyb-1 or osphyc under NL;DL significantly reduced the BPH resistance of osphya,osphyc and WT,but there was no significant difference in the BPH resistance of osphyb-1 between NL and DL.It indicated that the inhibition of DL on rice BPH resistance might depend on OsPHYB.In order to further verify the role of OsPHYB in light regulating rice BPH resistance,two other allele mutants of OsPHYB（osphyb-2 and osphyb-N4）were evaluated.The results showed that DL effectively alleviated the BPH resistance in osphyb-2 and osphyb-N4 similar to osphyb-1.In addition,overexpression of OsPHYB significantly reduced the resistance of rice BPH under DL.These results showed that OsPHYB plays a key role in light regulating rice BPH resistance.（3）OsphyB plays a dual role in ethylene synthesis and signaling pathwayThe data of RNA-seq proved that DL significantly increased the expression of ethylene synthesis and signal pathway genes,indicating that the ethylene pathway was activated under DL.Further q RT-PCR experiments found that the expression of OsACO1 in the osphyb-1mutant was significantly up-regulated whether under normal light（NL）or dim light（DL）conditions,but the response gene downstream of the transcription factor OsEIL2 was significantly suppressed.These results indicated that OsphyB may affect the ethylene pathway by regulating the expression of OsACO1 and ethylene-responsive genes regulated by OsEIL2,but the effects of these two regulation on the ethylene pathway may be opposite.（4）ET negatively regulates the resistance to BPH in riceThe transcriptome data proved that DL activates the ethylene pathway,so we supposed that ethylene negatively regulates BPH resistance in rice.Firstly,we found that it can significantly reduce rice resistance against BPH through exogenous ethylene treatment,and the exogenous PZA（ethylene synthesis inhibitor）treatment can enhance BPH resistance in rice.In addition,we identified BPH resistance in a series of genetic materials,which including genes overexpressed or knocked out in ethylene synthesis and signaling pathway.These results showed that ethylene negatively regulates BPH resistance.（5）OsphyB inhibits the expression of OsACO1 by promoting the degradation of OsPIL14In order to investigate how OsphyB regulates the ethylene pathway,we noticed that OsphyB may interact with OsPIL14（Phytochrome-Interacting Factor-Like14）by yeast screen.The interaction between OsphyB and OsPIL14 was verified through pull-down in vitro and LUC experiments in vivo.Further research found that OsphyB can affect the stability of OsPIL14 protein,more OsPIL14 protein accumulates in osphyb-1 mutant compared to WT.As little OsphyB enters the nucleus under DL,the accumulation of OsPIL14protein also increases under DL.In addition,the yeast monohybrid and LUC experiments proved that OsPIL14 can bind to the promoter of OsACO1 and promote its expression.Therefore,it promotes the accumulation of OsPIL14 in the nucleus by reducing the entry of OsphyB into the nucleus under DL,which enhances the expression of OsACO1 and promotes ethylene synthesis.（6）OsphyB stabilizes OsEIL2 by competitively interacting with OsEBF1We also obtained the interaction between OsphyB and OsEBF1（a negative regulator in the ethylene signal pathway,EIN3 Binding F-Box Protein）by yeast screen,and the interaction was proved by pull-down in vitro and Bi FC experiments in vivo.Interestingly,we found that the interaction between OsphyB and OsEBF1 did not affect the stability of OsEBF1 protein.Next,we found that less OsEIL2 accumulated in the osphyb-1 mutant.OsEBF1 as an E3 ubiquitin ligase,which interacted with OsEIL2.Furthermore,we found that the interaction between OsphyB and OsEBF1 can inhibit the interaction between OsEBF1 and OsEIL2,then prevented the degradation of OsEIL2.In conclusion,we found that DL promoted ethylene synthesis and OsEIL2 expression by reducing OsphyB protein in nucleus,while OsphyB retained in nucleus inhibited BPH resistance by stabilizing OsEIL2 and ethylene signaling by competing for binding to OsEBF1.This study not only revealed the molecular mechanism of light-regulated ethylene pathway and the interaction between rice and brown planthopper,but also provided a new strategy for controlling BPH.