Functional Verification Of The Key Gene OcEXPA6 For Orobanche Cumana Parasitism And Characterization Of SiRNASiganature

Orobanche cumana is a holoparasitic plant that attaches to host-plant roots and seriously reduces the yield of sunflower(Helianthus annuus L.).Effective control methods are lacking with only a few known sources of genetic resistance.In this study,we used host-induce gene silencing(HIGS)technology to interfere with the parasitic process of key genes on the early effects of O.cumana parasitism.We explored the effects of knockdown Oc QR1,Oc CKX5,Oc WRI1 and OcEXPA6 with TRV infectious clone in the stage of parasitism.Knockdown of target genes can restore sunflower plant heights to varying degrees(16-41%)and reduced the haustoria number(30-48%).In the control p TRV2-GFP group,the haustoria grew normally,whereas in p TRV2-Oc QR1,p TRV2-OcEXPA6,p TRV2-Oc CKX5,and p TRV2-Oc WRI1 groups the haustoria showed lagged development.Furthermore,incompatible attachment was observed in p TRV2-Oc QR1,p TRV2-OcEXPA6,p TRV2-Oc CKX5,and p TRV2-Oc WRI1 groups,where the haustoria showed failed connection to the host vascular tissues,whereas compatible attachment was seen in the control p TRV2-GFP group.The results demonstrate that these genes play an important role in the processes of O.cumana parasitism.After silencing the OcEXPA6 in the early stage of parasitism,the phenotype was significantly different,so the function and expression pattern of the OcEXPA6 were further analyzed.OcEXPA6 belongs to the EXPA subfamily of genes,and its expression level increases significantly during the germination and haustorium formation of O.cumana.It is found that the subcellular location is mainly located in the cell wall.Through heterologous overexpression,it was found that OcEXPA6 could cause the alfalfa(Medicago sativa)and sunflower roots growth retarded.High-throughput small RNA sequencing and bioinformatics analyses unveiled the distinct features of target gene-derived siRNAs in O.cumana such as siRNA transitivity,strand polarity,hotspot region,and 21/22 nt siRNA predominance,the latter of which was confirmed by Northern blot experiments.The possible RNAi mechanism is also discussed by analyzing RNAi machinery genes in O.cumana.In summary,this study provides theoretical guidance for exploring the parasitic mechanism of O.cumana.At the same time,it provides new control strategies for O.cumana.