Study On The Changes Of Gibberellin Signaling Transduction Passway In Resistance To Aphids Mediated By RMES1 Gene In Sorghum

Sorghum bicolor is one of the five major cereal crops in the world.It plays an important role in grain reserved,modern industry,wine making and fermentation.Sorghum aphid is one of the main pests threatening the survival of sorghum,which seriously affects the yield and quality of sorghum.Gibberellin(GA)is a tetracyclic diterpenoid plant hormone.At present,research on GA mainly focuses on the regulation of plant growth and development,while the research on plant disease/insect resistance is less.It is not clear whether the GA signaling pathway is activated in the defense process of sorghum against aphids.In this study,HN16(wild type,with RMES1 gene)and asm1(RMES1 gene mutation)were used as research materials.Through transcription analysis,hormone content determination and exogenous hormone experiment,the hormone signal pathway activated in sorghum aphid resistance response was comprehensively analyzed to provide a theoretical basis for sorghum aphids resistance breeding.The main results are as follows:1.Transcriptome data analysis showed that hormone related differentially expressed genes mainly included GA,auxin,cytokinin and phytosulfophenyl signaling pathway.There were 8 genes in the GA signaling pathway,and Sb CIGR,Sb GA20ox-2,Sb GA20ox-1,Sb GSA genes were verified selected for expression verification.The results were consistent with transcriptome data.2.Analysis of GA signaling pathway under external hormone application:(1)The external hormone application experiment revealed that after spraying GA with aphid ligation treatment,resistance was enhanced in WT compared to the control(Con),while susceptibility was enhanced in asm1,presumably involving GA in the hormonal signaling pathway of sorghum aphids resistance response.(2)Analysis of the gradient of external GA concentration revealed that external application of 0.05 m M and 0.5 m M GA with aphid ligation treatment resulted in increased resistance in WT compared to Con and increased susceptibility in asm1.After topical application of 5 m M GA with aphid induction,there was no significant change in resistance compared to Con.It was hypothesized that GA was involved in the aphid resistance response of sorghum in a concentration-dependent pattern.(3)Analysis of the expression pattern of GA signaling pathway marker genes after topical GA application revealed that the expression of GA signaling pathway marker genes after topical GA application revealed that the expression of GA synthesis genes Sb GA20ox-1 Sb GA20ox-2 and Sb GA3ox-1 was reduced in both WT and asm1 after GA spray compared to Con,presumably not affecting GA synthesis in RMES1-mediated aphid resistance response;compared to Con the expression of Sb GA2ox-1 and Sb GA2ox-2 increased in WT and decreased in asm1 after GA spraying,presumably the RMES1-mediated aphid resistance response affected the GA metabolism process;compared with Con,the expression of Sb DELLA-1 and Sb DELLA-2,the genes of DELLA protein(inhibiting GA synthesis),increased in WT and asm1 after GA spraying.The expression of both Sb DELLA-1 and Sb DELLA-2 in WT showed an increasing trend after GA spraying compared with Con,while there was no significant change in asm1.It was speculated that the mutation of RMES1 gene reduced the synthesis of DELLA protein.Thus affecting the GA signaling pathway.3.GA/JA,GA/SA interaction studies under external hormone application:(1)Compared with Con,aphid resistance was enhanced in WT after spraying either GA or SA alone,and further enhanced by spraying both GA and SA,indicating a synergistic effect between GA and SA in WT;while in asm1,aphid susceptibility was enhanced after spraying both GA and SA alone,and further enhanced by spraying both in asm1,the susceptibility of aphids was increased by spraying GA and SA alone,and decreased by spraying GA and SA together.It can be seen that the mutation of RMES1 gene shifted the mode of action between GA and SA from synergistic to antagonistic.(2)Compared with Con,the aphid resistance was enhanced by spraying GA or JA alone in WT,but not changed by spraying both GA and JA,indicating that there was no reciprocal relationship between GA/JA;while in asm1,the aphid susceptibility was enhanced by spraying either GA or JA alone,and reduced by spraying both GA and JA.It can be seen that the mutation of RMES1 gene made GA antagonistic to JA.(3)Expression analysis of SA and JA signaling pathway marker genes under external GA application revealed that the expression of SA and JA pathway genes were increased in WT,while the expression of SA pathway genes was decreased in asm1(except for ICS1),and the expression of JA pathway genes positively regulated genes(AOS2,EIN2)was unchanged or decreased under external GA application compared with that of Con.The expression of the negative regulatory gene JAR1 increased,indicating that the mutation of RMES1 gene suppressed the expression of positive regulatory genes of SA and JA pathways and promoted the expression of negative regulatory genes,which is consistent with the previous conclusion that the mutation of RMES1 gene made the antagonistic effect between GA and SA and GA and JA.4.Analysis of GA signaling pathway during aphid feeding:(1)GA content measurement during aphid feeding revealed that GA1 and GA4 were not detected,GA3,and GA7 underwent larger changes and accumulated more in the late stage of aphid feeding,indicating that GA3 and GA7 play more important roles in RMES1-mediated aphid resistance response.(2)Analysis of the expression pattern of GA signaling pathway marker genes during aphid feeding revealed that all GA synthesis-related genes were highly expressed in WT in the early stage of aphid feeding and in asm1 in the late stage of aphid feeding,speculating that GA synthesis-related genes may be involved in the RMES1gene-mediated aphids resistance response in the early stage of aphid feeding.Analysis of the expression pattern of GA signaling pathway marker genes during aphid feeding revealed that the expression of genes related to GA metabolism did not change significantly in WT,while it continued to increase in asm1,indicating that mutations in the RMES1 gene caused an increase in the expression of genes related to GA metabolism,which reduced the GA content in the plant and was detrimental to sorghum resistance to aphids infestation.The expression of the DELLA protein gene decreased in WT and did not change significantly in asm1,indicating that the RMES1-mediated aphid resistance response promotes the accumulation of GA by reducing the synthesis of DELLA protein to achieve aphids resistance.5.Construction of Fo MV-mediated gene silencing system in sorghum variety HN16:successful construction of VIGS system by phenotypic observation and RT-PCR results indicating PDS and RMES1 gene were silenced.