Research On Germination Specificity Of Orobanche Cumana And Regulation In Its Interaction With Host Sunflower

Seeds of the root parasitic plants of the genus Orobanche germinate specifically in response to host-derived germination signals,which enables parasites to detect and attack preferred hosts.Orobanche grows in harmony with its host plants in the natural environment,while in agricultural environment,it absorbs the nutrients of host crops and causes serious damages to them.Most Orobanche plants germinate in response to strigolactone(SLs),such as Orobanche cernua parasitizes tomato and other Solanaceous crops and responds to orobanchol,the major SLs from tomato roots,whereas Orobanche cumana specifically parasitizes sunflower(Helianthus annuus)and responds to DCL.When O.cumana recognizes the germination stimuli secreted by sunflowers,it germinates and then developes haustorium attached to the roots of sunflowers,severely affecting plant growth and development.Due to the connection between O.cumana and the roots of sunflowers,the effect of herbicides is minimal.Therefore,in order to effectively prevent and control Orobanche,we mainly conduct researches in three aspects: the germination specificity of Orobanche,the interaction between sunflowers and O.cumana,and the relieving effect of brassinolide(BR)on O.cumana infection.The main research results are as follows:1.We used genetic and genomic approaches for two closely related species,Orobanche cumana and Orobanche cernua,to investigate the role for KAI2 d in regulating germination specificity in parasitic plants.For each species we developed a set of comprehensive transcriptomes spanning pre-and post-germination stages to identify genes involved in stimulant perception.O.cernua contains six KAI2 d genes(assigned Orce KAI2d1-6),while O.cumana contains eight genes(Orcu KAI2d1-8).Then DNA from 94 F2 hybrids was genotyped to identify the KAI2 d gene composition and these were correlated with germination phenotype.The segregation pattern indicated that the KAI2 d genes are linked,but pointed to Orce KAI2d2 as a likely orobanchol receptor,while response to DCL was associated with inheritance of all O.cumana KAI2 d genes together.Each KAI2 d gene was expressed in the Arabidopsis thaliana kai2 mutant background and tested for the ability to recover the mutant phenotype when exposed to SLs(including orobanchol,5-deoxystrigol and GR24)or DCL.One O.cernua gene,Orce KAI2d2,responded to all SLs,but not DCL in this system.Interestingly,an O.cumana gene,Orcu KAI2d5(the ortholog to Orce KAI2d2)also responded to SLs,but not DCL.No DCL-specific KAI2 genes were identified.In summary,we have identified the likely SL receptor in O.cernua,and show evidence that the DCL receptor is either not a KAI2 d protein,or uses KAI2 d in combination with other signaling pathway components.2.We used the susceptible cultivar TK0409 and the resistant cultivar JY207 to study the interaction process between sunflowers(Helianthus annuus)and O.cumana.Analysis of the germination stimuli secreted by the two sunflower cultivars showed no difference,indicating that the resistance of sunflower may originate from its own defense response.During the interaction,O.cumana established a complete connection with the sunflower vascular system in TK0409,resulting in plant growth retardation,which was not observed in JY207.The successful infection of sunflower involves a series of interactions,including disrupting the cell wall,transforming the host root from a reservoir to a source of nutrients for the parasitic plant by using transporters of parasitic plants and inhibiting nutrient assimilation and / or transport in sunflower,as well as interfering with the defense system of host by disease-related proteins.In contrast,sunflower resistant cultivar successfully recognized the effectors of O.cumana.Moreover,we successfully cloned 28 candidate effector genes from O.cumana through transcriptome sequencing and secretome prediction.Then,with the help of the gene transient expression technology,we further selected 5effector genes that could inhibit the plant defense response.Among them,3 effector genes were found localized in the nucleus and plasma membrane using subcellular localization.Altogether we conclude that the activation of defense system during parasitic interactions may be related to sunflower resistance.This study will help to explore the molecular mechanisms of parasitic interactions between O.cumana and sunflower,and provide important genetic resources for sunflower resistance breeding and O.cumana control.3.We studied the effect of BR pretreatment on the growth and physiological regulation of sunflower plants under O.cumana stress.After sunflower was infected by O.cumana,plant height and biomass decreased significantly,ROS and MDA accumulation increased,enzyme activities also changed accordingly,mesophyll and root tip cell structure were seriously damaged,and plant growth was severely damaged.After pretreatment with0,1,10,and 100 n M of BR,the growth of sunflowers underwent significant changes.Compared with infected plants,the biomass of sunflowers significantly increased when BR was applied,while the number,fresh weight and dry weight of O.cumana significantly decreased by 77.8%,90.8% and 74.2%,respectively;GSH content,GR activity and GSH/GSSG ratio in sunflowers also significantly increased,while ROS levels and MDA content significantly decreased,the activities of POD and SOD increased significantly,and APX and CAT activities decreased.Overall,this effect was significantly enhanced as the BR concentration increased from 1 n M to 10 n M,and was best at 10 n M.We explored the effects of different BR concentration pretreatment on the ultrastructure of sunflower mesophyll and root tip cells through transmission electron microscopy(TEM).The chloroplasts of sunflower mesophyll cells were distorted and deformed,while the root tip cell membrane system was severely damaged and the nucleus was even disintegrated after being attacked by O.cumana alone;After BR pretreatment,the ultrastructure of sunflower cells under stress was improved in varying degrees,with the most significant relief effect observed under 10 n M BR treatment.At the same time,qRT-PCR analysis indicated that the expression levels of brassinolide responsive genes such as BRI1,BR6OX2,BAK1,BSK1,and BSK2 were significantly upregulated after 10 n M BR treatment compared to infection alone.These results indicate that BR alleviates the damage of sunflowers caused by O.cumana infection and enhances the resistance of sunflowers to parasitic weeds by regulating the antioxidant enzyme system and reactive oxygen species(ROS)levels,ameliorating cell ultrastructure and regulating the expression of related genes.This study provides new insights into the prevention and control of parasitic weeds.