Mechanism Of Molecular Communication Between Endophytic Fungi Of Bupleurum Scorzonerifolium Willd.and Host Plant

Objective:Study on the communication between endophytic fungi and host plant to elucidate the molecular regulation mechanism of endophytic fungi on upstream pathway and dowmstream gene expression.Methods:(1)Fungi mycelium of endophytic fungi(CHS2,CHS3,CHS4,CHS5,CHL2 and CHR4)was separated from culture medium by using vacuum filtration.The protoplast was prepared by using enzymatic dissociation method from callus.The interactions of fungal supernatant and mycelium with protoplasts were detected by flow cytometry.(2)Suspension cells of Bupleurum scorzonerifolium Willd.were co-cultured with fungi supernatant and fungi mycelium for 2 days,respectively.Then,cAMP,cGMP and CaM of suspension cells were extracted for analysis of endophytic fungi regulation on upstream pathway.Moreover,extending co-cultivation upto 7 days,the communication began to stabilize.Total RNA was extracted from endophytic fungi and Real-Time PCR was used for determination of gene expression.The changes of genes in saikosaponin synthesis pathway were analyzed.Results:Compared with control group,fungi supernatant and fungi mycelium of endophytic fungi(CHS2,CHS3,CHS4,CHS5,CHL2)could promote ROS level of protoplast and the effect of fungi supernatant was greater than fungi mycelium.The results from cAMP showed that fungi supernatant and fungi mycelium of all endophytic fungi could decrease the content of cAMP,but the CHR4 had a comparatively faint inhibition.The results from cGMP showed that fungal mycelium of CHR4 and supernatant of CHS4 and CHR4 could raise the cGMP level,fungal supernatant of CHS3 and mycelium of CHS2,CHS3,CHS4,CHS5 and CHL2 decreased the cGMP level,the supernatant of CHS2,CHS5 and CHL2 had no significant effects.The results from CaM showed that fungal supernatant of CHS4 and CHR4 could enhance the level of CaM,fungal mycelium of CHS2,CHS3,CHS4,CHS5,CHL2 and CHR4 and supernatant of CHS3 and CHL2 decreased the CaM level,the supernatant of CHS2 and CHS5 had no significant effects.Under the effect of the endophytic fungi,the genes expression of saikosaponin synthesis pathway was investigated by real-time quantitative PCR.The results demonstrated that endophytic fungi can regulate genes expression of saikosaponin synthesis pathway.Conclusion:The results indicated that endophytic fungi(CHS2,CHS3,CHS4,CHS5,CHL2,CHR4)isolated from B.scorzonerifolium Willd.have regulating effects on upstream pathway and genes expression.The mechanisms of action of most endophytes(CHS2,CHS3,CHS4,CHS5,CHL2)are to enhance the level of ROS and to decrease the content of cAMP,cGMP and CaM.Thus,cells were forced to environmental stress and the expression of intracellular stress-resistant genes were induced.Among them,CHS4 and CHR4 could promote the level of CaM,and this is most obvious in CHR4.CHR4 could not raise the level of ROS and the probable regulation mechanism on gene expression is calcium-mediated signal transduction.