Exploration Of Brown Planthopper Resistance Gene Bph27(t) And Resistance Mechanism Of Rice Against Brown Planthopper

Brown planthopper(Nilaparvata lugens Stal)is one of most destructive insect in rice cultivation area.As a monophagous insect pest of rice,brown planthopper continuously feeds on rice phelom and xylem.Brown planthopper reproduces quickly.Once breakout,it can cause serious damage or even completly yiled loss.Besides,it could also transmit rice grassy stunt and rugged stunt vius,which damage rice production indirectly.At present,brown planthopper has rosed up as the "number one" pest in Aisa,and its control and management has attracted people's widely attention.Traditionally,we depend on chemicals sprying to deal with this insect.However,the expensive chemicals not only poison environment but also stimulate the adaptation of brown planthopper to chemicals,leading to new biotypes appearance.Plant resistance utilization is perceived as the most effective strategy to control this insect.Therefore,new resistant gene mining and application promises us an effective,economic and environment-friendly way to combat brown planthopper.Meanwhile,exploration of resistance mechanism that rice adopts to defend brown planthopper would benefit us to appropriately use resistance genes in breeding,especially in gene pyramiding program.In our present study,we started research with following aspects:1-Exploration and application of Bph27(t)(1)Fine mapping and candidate validation of Bph27(t)In our present study,we fine mapped Bph27(t)from Balamawee to 46 kb region on rice chromosome 4,in which contains 8 annotated genes and 6 of them are reported relative to insect or pathogen resistance.Then,we transformed each of them from resistant Balamawee into susceptible 02428 to valid which is Bph27(t).However,none of transgenic family could improve resistance of 02428.Given these results,we hypothesize that:1)none of the 6 genes is Bph27(t),which maybe a new resistance gene coded by the other genes in the region.2)Bp27(t),like Bph3,is not contributed by single gene,but a resistance gene cluser.Besides,we can not exclude the possibility that Bph27(t)functions as a non-coding gene.3)Bph27(t)mapping region maybe incredible.Bph27(t),a major QTL,its function would be influenced by environment change and different genetical background.In order to isolate Bph27(t)in future,another population now is used to assure and further narrow Bph27(t)region.Although Bph27(t)is yet to be determined,we have already got the closed linked makers to Bph27(t),which facilitates us to introduce it into rice cultivar.(2)Pyramiding breeding of Bph27(t)and Bph3 into Ningjing3With availability of closed linked markers to Bph27(t)and Bph3,We first introduced them into elite japonica rice cultivar Ningjing3,respectively.The introgression lines significantly improved NJ3's resistance,and reduced yield loss(-17.9%)caused by BPH.Among these introgression lines,R2256(carrying Bph27(t))and R2381(carrying Bph3)recovered the most genetic background of NJ3,99.52%and 95.75%,respectively.We pyramided Bph27(t)and Bph3 into NJ3 by crossing R2381 with R2256.The hyterozygous and homozygous pyramided line showed similar BPH resistance level with R2256and R2381.We perceive that pyramided line Bph27(t))/Bph3 could confer more durable resistance against BPH.We simultaneously backcrossed R2381 and R2256 with NJ3,respectively,and found that both genes are dominate resistant genes.This result implicates that only one allele could confer high resistance against BPH,which is especially important in hybrid cultivar breeding.2.Exploration of BPH resistance mechanism(1)Phenylalanine ammonia-lyase and diterpenoids biosynthesis metabolic pathways might participate in BPH resistanceBased on microarray experiments,we found that 24 hours post BPH infestation,RH up-regulated two secondary metabolic pathways compared with 02428 and RH non-infestation,which are diterpenoids biosynthesis and phenylalanine ammonia-lyase metabolic pathways.These two pathways also showed up-regulated in resistant plants compared with susceptible plants,which were selected from RH/02428 F2,implicating that the two pathways respond to BPH infestation and maybe regulated by resistant gene from RH,independent of genetic background.(2)Function of PAL and SA in BPH resistanceInorder to valid whether the PAL metabolic pathway up-regulated upon BPH feeding functions in BPH resistance,we interfered with PAL expression(based on RNAi method)in resistant variety IR64.Compared with IR64,the transgenic plants PAL(RNAi)showed significant reduced resistance.This result indicted that PALs play an important role in BPH resistance.Next,we measured the downstream production content of PAL,and find that total flavonoids,lignin content showed no differences,but SA content showed significant lower than IR64.Then,we exogenously sprayed SA or its analogs benzothiadiazola(BTH),survival rate of transgenic plants could rise up to 90%of IR64.In addition,we got SA free Nipponbare(NIP)transgenic plant(NIP(NahG)),which showed severer susceptibility compared to NIP.While,supplied with exogenous SA or BTH,NIP(NahG)showed comparable resistance to NIP against BPH infestation.These results conferred that PAL play a key role in SA biosynthesis facing BPH infestation,and SA,as an important signal molecular,play an important role in BPH resistance.(3)SA up-regulates diterpenoids phytoalexins biosynthesisPAL metabolic final production salicylic acid(SA),when exogenously sprayed on RH and 02428,could independently up-regulate diterpenoids biosynthesis pathway in both plants without BPH feeding.This suggested that diterpenoids phytoalexins response to and regulated by SA signal.Combined with our results from microarry experiments,real time RT-PCR,trangenic plant BPH resistance behavior,SA content measurement and its role in BPH resistance,we propose that BPH feeding up-regulates PAL pathway,leading to higher SA accumulation,SA may function as siganal molecular further up-regulates downstream molecular(eg:diterpenoids phytoalexin)as defensive materials to BPH.