Effects Of Brown Planthopper Nilaparvata Lugens(St(?)l) Saliva On Rice Resistance,and Characteristic Analysis Of Important Resistance-adapted Genes

In the process of co-evolution of plant and insect,plants continue strengthening resistance agaist insects,on the other hand,insects keep trying to adapt to these anti-insect properties.Plants activate their defense against insect by detecting herbivore-associated molecular patterns(HAMP).Usually directly contact is needed in the interaction between insect and plant,the saliva of insect therefore plays an important role.The saliva is also a hot spot in the research of interaction between rice and brown planthopper(BPH)Nilaparvata lugens(St?l).But previous studies favored the rice related analysis when challenged by BPH as a whole,ignoring the endosymbionts in the saliva,which plays an important role in plant-insect interaction.In order to detect the effector or elicitor existed in the BPH's saliva,several related studies have been done.Firstly,the activities of three defense related enzymes were detected when treated with endosymbiont-containing/endosymbiont-free BPH saliva.Then,based on the effect of saliva on rice,different expression transcriptsfrom BPH's saliva gland were analysed,and two important genes were choosed for further cloneing and analysing the characteristic.The main results are as follows:1.BPH saliva was collected to simulate BPH feeding by sting on rice seedlings.The enzyme activities of three defense related enzymes including peroxidase(POD),catalase(CAT)and polyphenoloxidase(PPO)were measured within 24 h.The results showed that both saliva group and endosymbiont-free saliva(EF-saliva)group caused significant increase(about 50% higher than control)of POD activity at 6 h after treatment,and no significant difference was observed between this two treating with BPH feeding.This means the change of POD activity is caused by saliva.While the CAT activity in EF-saliva group is about 25~30% higher than in EF-saliva group,this proofed the endosymbionts in the saliva lead to this changes.An more interesting result is in the PPO activity,comparing the enzyme activities in saliva group,EF saliva group and control,we know,both the componts in and the endosymbionts in saliva contribute the change of PPO activity,To sum up,both the componts and endosymbionts in BPH saliva can act as effectors or elicitor to suppressed or trigured the rice resistance.2.According to results above,both the components of BPH saliva and its endosymbionts may act as elicitor or effector to elicite or suppress rice defense,hence,to isolated the candicate elicitor or effector is very important.By analyzing the suppression subtractive hybridization library of BPH saliva gland,16 distinctively expressed ESTs were found,which were predicted to be related to carbohydrate metabolism,cellular signal transduction,plant defence and detoxiation et al.To further study characteristic of these genes,two up-regulated ESTs were chosen to clone their full length,and named BPHef-hand and BPHsec1 respectfully.BPHef-hand contained an ORF coding a polypeptide of 181 amino acids(AA).First 20 AA was an obvious signal peptide,indicating it may be an secreted protein.There was an EF-hand motif in the area of 58-135 AA.The vector of protein expression was contructed and induced,after purification,the target protein was obtained.Relative expression analysis by fluorescent real-time PCR showed that BPHef-hand was generally expressed higher in larvae than in adults.Expression level was highest in second instar,about 3 times of the adult BPH.No significant expression differences were observed in BPHs of different gender or different wing type.The other gene BPHsec1 contained an ORF coding a polypeptide of 312 AA.No signal peptide was observed.Among the area of 103-254 AA,a CRAL-TRIO domain was observed,which is a general domain in SEC14 protein superfamily.CRAL-TRIO domain is mainly responsible for lipid binding,transporting and metabolism and it is pretty conservative.Some proteins containing CRAL-TRIO domain were aligned by MEGA6.0,and then phylogenetic tree was constructed by Neighbour-Joining method.Phylogenetic result indicated that BPHsec1 shared a cluster with CRAL-TRIO domain proteins of Aphididae,Psyllidae and Pentatomidae.Relative expression analysis by fluorescent real-time PCR showed that BPHef-hand was generally expressed higher in larvae than in adults.First instar larvae expression was twice as much of adults.No significant difference was observed between male/female short-wing BPHs,but both were about 10% lower than long-wing BPHs.A silencing vector promoted by T3 was constructed,and ds RNA was synthesised in vitro.Efficiencies of silencing BPHsec1 were compared by injecting and feeding ds RNA.Results showed that injecting ds RNA caused about 60% decrease in expression and had a faster and more durable gene silence effect.By feeding ds RNA,1 day after treatment saw a minor rise of target gene and then went down steadily to 50% of control.In this study,BPH saliva expecially the endosymbionts in saliva were preliminary confirmed to relate to rice resistance;and two saliva gland genes,BPHef-hand and BPHsec1,were cloned and and analyzed.Results of this study are the foundation of functional study,which may also offer a new point in studying the BPH-rice interaction.