Analysis Of The Mechanism Of Brown Planthopper-induced Rice Resistance To Meloidogyne Graminicola

Rice plants in paddy fields are at risk of co-infestation by above-and below-ground pests.The ternary interactions between rice and above-and below-ground pests are more complex than the binary relationships between pathogen-hosts,and the exploration of the mechanisms of ternary interactions has received increasing attention in recent years.Brown planthopper（BPH,Nilaparvata lugens）and Meloidogyne graminicola are important above-ground pest and below-ground pathogen of rice,causing serious damage on rice.In this study,we investigated the mechanism of rice-mediated interaction between BPH and M.graminicola through physiological,biochemical and molecular biological technologies.This study enhances the understanding of the interactions between rice and above-and below-ground pests,and provides a theoretical basis for the scientific prevention and control of co-feeding between BPH and M.graminicola co-infestation in paddy.The major results are as follows:1.Infestation of BPH gravid adult females leads to a significant resistance to M.graminicola on rice.The difference in rice susceptibility to M.graminicola was systematically characterized among differentially infested rice plants（the rice cultivar Nipponbare）that are susceptible to both BPH and M.graminicola through a nematode infection assay.Results showed that the parasitism of M.graminicola on rice plants pre-inoculated with BPH（1 BPH per plant）for 1 day was significantly inhibited compared to rice plants only infected by M.graminicola.The number of galls and nematodes per gram of roots was significantly reduced from 399 to 254 and from 307 to13,respectively.There was no significant difference in the number of galls and nematodes compared with rice plants only inoculated by M.graminicola when 1 and 2BPH per plant were inoculated on rice plants at 3 dpi of M.graminicola,whereas only a significant decrease in the number of galls was found when 5 BPH per plant were inoculated.Meanwhile,data from chemotaxis analysis indicated that BPH gravid adult female infestation significantly reduced the attraction of rice root tips to M.graminicola compared to rice plants without BPH inoculation.Collectively,these results suggest that BPH gravid adult females could induce a remarkable rice resistance to M.graminicola that mainly involves in the resistance to M.graminicola invasion on roots.2.Reduction of sucrose supply to rice roots caused by BPH infestation is the key mechanism of BPH infestation-induced resistance to M.graminicola.Soluble sugar content of rice roots was quantified by GC-MS.Results showed that BPH gravid adult female infestation significantly reduced the contents of sucrose in rice root phloem and soluble sugars in the roots with the reduction from 11.60μg 400 cm^-1 roots to 8.17μg400 cm^-1 roots and from 742.54μg g^-1 FW to 583.54μg g^-1 FW,respectively,compared with rice not inoculated with BPH.RNA-seq data combined with q RT-PCR analysis elucidated that both glycolysis and tricarboxylic acid cycle in rice roots were inhibited at1 dpi of BPH gravid adult females.These data indicate that the infestation of BPH gravid adult females lead to the inhibition of sugar metabolism in rice roots.In addition,phloem loading of exogenous sucrose,glucose and fructose could significantly increase the soluble sugar content of rice roots infested with BPH gravid adult females,resulting in the inhibition of rice resistance to M.graminicola induced by BPH gravid adult females.These results suggest that BPH adult female-caused inhibition of sucrose supply to rice roots plays a pivotal role in BPH adult female-induced rice resistance to M.graminicola.3.Os SUT2 is induced at BPH oviposition sites and its functional deficiency inhibits rice resistance to M.graminicola induced by BPH gravid adult females but not by BPH nymphs.q RT-PCR analysis showed that Os SUT1 and Os SUT2,members of the Os SUTs family being responsible for sucrose efflux,were induced at BPH oviposition sites.Induced expression of Os SUT2 was dependent on the ABA signaling pathway in rice.In addition,the Os SUT1 silenced and Os SUT2 loss-of-function lines were used to conduct infection assay of M.graminicola.The results showed that the Os SUT2 mutant suppressed the resistance to M.graminicola induced by BPH gravid adult females,while it just had a slight effect on rice resistance to M.graminicola induced by BPH nymphs.Os SUT1 silencing had no significant effect on rice resistance to M.graminicola induced by BPH gravid adult females.4.Os SUT2 loss-of-function lines cause callose deposition and inhibits sucrose phloem loading in rice which leads to rice resistance to BPH,while BPH oviposition attenuates the negative regulation of Os SUT2 mutant-caused callose deposition by BPH feeding.Firstly,effects of Os SUT2 mutant on the honeydew excretion,oviposition and egg hatching rate of BPH were evaluated.Results indicated that compared with rice wild type,the amount of honeydew per BPH excreted by BPH gravid adult females and nymphs on two Os SUT2 mutant lines significantly decreased,and the number of eggs per BPH also decreased from 34 to 29 and 20.Meanwhile,the egg hatching rate was reduced from97.43%to 29.53%and 38.40%as compared to rice wild type.The effect of Os SUT2mutants on callose deposition in rice phloem and the effect of BPH feeding and oviposition on this callose deposition were then evaluated.Data showed that Os SUT2mutant led to callose deposition in phloem of rice sheaths compared with rice wild type.In addition,Os SUT2 mutant resulted in a significant increase in soluble sugar content in rice leaves and decrease in sucrose content in rice leaf phloem.These results suggest that Os SUT2 mutant could inhibit the phloem loading of sucrose that is presumed to be one of the mechanisms how Os SUT2 mutant induces rice resistance to BPH.