Functional Study On Helianthus Annus Tetraspanin(TET) Family Genes In The Parasitic System Of Helianthus-orobanche Cumana

Orobanche cumana Wallr.is a fully parasitic plant,which is mainly parasitic on the roots of Helianthus annuus L.,and seriously affects the quality and yield of H.annuus.Therefore,the control of O.cumana is very important.Tetraspanin（TET）is widely present in mammals,insects,fungi and plants,and it regulates various physiological processes of cells and plays an important role in development and immune response.Plants carry out long-distance information exchange between plant cells through the phloem transport of exosomes specifically tagged with TET genes.Therefore,this paper studied the role of TET genes in the Helianthus-Orobanche cumana parasitism process,revealed the transcriptional regulation mechanism of TET genes,and provided a theoretical basis for the prevention and control of O.cumana.In this study,overexpression and virus-induced gene silencing（VIGS）techniques were used to construct a series of four exosomes specifically tagged with TET genes based on the Helianthus-Orobanche cumana parasitism process.Overexpression vectors of TET genes HaTETE3,HaTET6,HaTET8 and HaTET10and tobacco rattle virus（TRV）interference vectors.H.annuus seeds were infected by seed-soak agroinoculation（SSA）method,and then inoculated with O.cumana.The number of O.cumana.and growth phenotypes were counted and analyzed,and the expression levels of these related genes were detected by RT-qPCR.The main findings are as follows:（1）First,the extraction and identification of exosomes from the roots of H.annuus were carried out.The results showed that there were exosomes with a diameter of about 30-150 nm in the roots of H.annuus.（2）RT-qPCR technology was performed to detect the expression of the above HaTET genes before and after the parasitism of O.cumana.The results showed that the expression of the above HaTET genes were significantly upregulated after the parasitism of O.cumana.（3）In the overexpression experiments,the well-studied AtTET8 and AtTET9genes in Arabidopsis were used as controls,the results showed that the number of O.cumana increased after HaTET genes overexpression,with the highest increase of43.3%in HaTET8 overexpression.In the silencing experiment,the number of O.cumana decreased significantly after the silencing of the HaTET genes,with the highest decrease of 58.68%in HaTET8 silenced group.（4）HaTET3,HaTET6,HaTET8 and HaTET10 were located in the cytoplasmic membrane by subcellular localization.（5）This paper cloned the promoter sequence of HaTET genes and analyzed its expression characteristics,by recombining them into an expression vector containing a luciferase reporter gene（LUC）,and using Agrobacterium-mediated tobacco transient expression system to detects the activity of HaTET promoter.The results showed that different infection concentrations(OD₆₀₀=0.2,OD₆₀₀=0.4,OD₆₀₀=0.6,OD₆₀₀=0.8)of ProTET:LUC Agrobacterium infected tobacco leaves could drive the expression of LUC reporter gene.（6）H.annuus plants were treated with 1 mM SA,1 mM GR24 and crude extract of O.cumana respectively.The results showed that SA could significantly induce the promoter activity of HaTET genes,while GR24 and crude extract of O.cumana inhibited its activity.