Fine Mapping Of Brown Planthopper Resistance Gene Bph33(t) And Exploration Of Resistance Mechanism Of Rice Against Brown Planthopper

Browm planthopper(Nilaparvata lugens Stal)is the most destructive insects in rice-growing area.It always attaches to rice stem and penetrates the phloem to suck sap,resulting in consuming the plant nutrients.When a large number of BPHs outbreak,causing a "hopper-burn" phenotype,which seriously affects the rice yield and quality.In addition,BPH is also a carrier of two viruses,rice grassy stunt and rugged stunt vius.For a long time,chemical control is the common control strategy of BPH,but excessive use of chemical pesticides,not only leads to time-consuming,environmental pollution,but also increases the adaptation of BPH on chemical pesticides.Exploration the host resistance is considered as an effective and economical strategy to control BPH.Thus,it needs to screen the resistance germplasm resources and explore new resistance genes constantly.Identification and cloning of insect-resistance genes is helpful for elucidating the molecular mechanism of BPH resistance,speed up the resistance varieties breeding process.In this study,we obtain a Vietnamese landrace W41123 from a total of 295 local cultivars coming from Southeast Asia and South Asia which exhibited high resistance to BPH at both rice seedling and maturity stage.the evaluationg of preference feeding showed that W41123 have centain antixenosis against BPH compared to the susceptible cultivar C418,When BPHs were forced to feed on W41123 and C418,the nymph survival rate and honeydew extretion of BPHs feeding on W41123 were significantly lower than that on C418.These findings implied the strong antibiosis of W41123 on BPH.Then,genetic analysis of the F2 population including 105 individuals derived from the cross between W41123 and C418 showed that the resistance trait in W41123 was controlled by muti-genes.Genome linkage map was constructed using 118 molecular markers which showed polymorphism among the two parents and combined with its BPH resistance scores,we detected two quantitative trait loci(QTL)of BPH resistance on chromosomes 4 and 6,Qbph4 and Qbph6,which accounted for 30%and 15.5%of the phenotypic variance,and the major quantative QTL,Qbph4,was renamed as Bph33(t).Further analysis showed the two locis have cumulative effects on the resistantce against BPH in W41123.To finely map Bph33(t),we developed a near isogenic lines(NIL)with C418 background by backcrossed three timely.Genetic background detection of NIL showed that the background recovery rate was 91.8%.In addtion,resistance evaluation showed the NIL-Bph33(t)displayed high resistance against BPH at seedling and maturity stage.Then a total of 10600 individuals from F2?BC1F2?BC2F2 and BC2F3 population were used to screen for recombinants.Combined with the BPH resistance score of these recombinants,Bph33(t)was finally mapped to a region with 49 kb physical distance.There are two ORFs predicated in this region.ORF1 was predicated as a functionally uncharacterized expression protein which contains a zing-finger domain;and ORF2 is predicted to be a carboxypeptidase 10.Furtherly we sequenced the two genes and found that ORF1 had multiple amino acid differences,whereas ORF2 had only one substitution.Real-time PCR analysis showed that the expression level of ORF1 gene in BPH-resistantce parent was higher than that of BPH-susceptibility parent 12 h later after BPH infestation.The expression level of ORF2 was induced after BPH infestation in both resistant and susceptible parent but which was significantly higher in BPH-resistant parent than that in susceptible parent at 3,6 and 24 h after BPH infestation.Fine mapping of Bph33(t)can establish the foundation of its map-base clonning as well as the utilization in molecular breeding.Although at least seven BPH-resistant genes have been cloned,the underlying interaction mechanisim between rice and BPH was largely unkown in rice.Brassinolides(BRs)is widely reported to be involved in the regulation of plant growth and defense response to pathogen infection,but the role in the response to herbivores feeding in plant is less reported.Two hundred and fifty-six activation-tagging T-DNA insertion rice lines with background of Dongjin were screened for BPH resistance at rice seedlings stage and a mutant,slg-D,reported as a BR overproduction mutant previously showed more susceptibility to BPH.Further tests showed that the BPH nymphs fed on mutant had higher survival rate than those fed on wildtype(WT)plants.Preference feeding indicated that BPH prefered to feed on mutant plants rather the WT plants.Like slg-D,another BR overdose mutant,m107,also showed more sensitive to BPH.Contrary to BR overproduction mutant,BR deficient mutant,lhdd10,exhibited more resistant to BPH.In addition,exogenous BL application can also promote susceptibility to BPH.Collectively,these results indicated BR played a negatively role in rice response to BPH infestation.Real-time PCR analysis showed BR pathway was suppressed while the salicylic acid(SA)and jasmonic acid(JA)pathways were induced by BPH infestation sucessively.Meanwhile,BPH infestation inhibited the accumulation of BRs(CS and 6-deoxoCS)contents.Moreover,foliar spaying with brassinolide(BL)or endougenous BR overproduction can suppress SA-synthesis genes,OsICS1 and OsPAL,and reduce the SA content.Conversly,the expression levels of OsICS1 and OsPAL and SA content were increased in lhdd10.Additionally,transcript levels of JA-related genes OsAOS2,OsLOX1 and OsMYC2 as well as JA content were increased in BL-treated plants or endougenous BR overproduction plants while decreased in BR-deficient plants.These findings indicate BR can induce JA pathway while suppress SA pathway during BPH infestation.Furtherly,studies showed that the inhibition of SA pathway mediated by BR was abolished in JA-related mutants,ogl and coil-18.Collectively,these results indicated that BR could suppress SA pathway by positively regulating JA pathway in rice.This study firstly reports that BR participates in the interaction between rice and BPH,and its mechanism is expounded,which broadens our understanding of BR function,deepens our understanding of the interaction between rice and BPH and provides theoretical basis of use of host resistance to prevent brown planthopper.