The Roles And Regulatory Mechanisms Of A Putative Tomato Indole-3-Acetic Acid Amido Synthetase Gene,Slgh3.4,in The Establishment Of Arbuscular Mycorrhizal Symbiosis

In nature ecosystems,plants as sessile organisms have evolved a series of ingenious skills to cope with diverse environmental stresses,such as pathogen infection,nutrient deficiencies,soil salination and drought.One of the most promising adaptations for plants reacting to these challenges is the formation of symbiotic interactions with arbuscular mycorrhizal(AM)fungi.The primary benefit for the both symbionts is the acquisition of nutrients:the fungus facilitates acquisition of water and nutrients,in particular P and N,from the soil to their host,whereas,in return,the host plant transfers its fixed carbohydrate to the fungus for their propagation.A number of genes showing strong inducibility in arbuscule-containing cells have been revealed to be essential for regulating AM symbiosis and arbuscule development.Auxin,the first phytohormone to be described,has been well recognized to be a key regulator acting in plant-pathogen interactions.It has recently become more evident that auxin concentrations could be elevated upon AM fungal colonization,in particular in the cells containing arbuscules,of various plant species,suggesting the involvement of auxin in regulating AM symbiosis.However,the mechanisms underlying the auxin-mediated AM symbiosis have not been well deciphered.We have previously reported a GH3 gene,SlGH3.4,encoding a putative IAA amido synthetase,that was strongly activated upon both external IAA induction and AM fungal colonization.SIGH3.4 belongs to the Group ? of GH3 family.Multiple homologues of this subgroup from diverse plants have been demonstrated to be involved in a number of physiological processes,including interactions between plants and pathogen,through modulation of IAA homeostasis in particular cells or tissues.The specialized expression of SlGH3.4 in fungal-colonized cells,in particular arbuscule-containing cells,provided a strong hint that this auxin-and AM-induced GH3 might be an important component of the auxin signaling pathway controlling AM fungal colonization in tomato.To testify this hypothesis,the regulatory profiles of the GH3 gene in response to AM fungal coloniation and its physiological roles during the establishment of the AM symbiosis were extensively investigated.The main results were as follows:1.Histochemical staining demonstrated that the tomato SIGH3.4 promoter could drive GUS expression strongly in both IAA-treated and AM fungal colonized roots.A 2258bp promoter fragment of SlGH3.4 was suficient to drive GUS expression strongly in mycorrhizal roots of tobacco,soybean and rice plants.2.A promoter deletion assay revealed three cis-acting motifs,the auxin-responsive element,AuxRE,and two newly identified motifs named MYCS1 and MYCS2,involved in the activation of auxin-and AM-mediated expression of SlGH3.4.Deletion of the AuxRE from the SIGH3.4 promoter caused almost complete abolition of GUS staining in response to external IAA induction.Seven repeats of AuxRE fused to the Cauliflower mosaic virus(CaMV)35S minimal promoter could direct GUS expression in both IAA-treated and AM fungal-colonized roots of tobacco plants.Four repeats of MYCS1 or MYCS2 fused to the CaMV35S minimal promoter was sufficient to drive GUS expression in arbuscule-containing cells.3.Transgenic tomato and rice plants constitutively expressing SlGH3.4 under the control of the strong maize ubiquitin promoter were generated.The free IAA concentration in the transgenic tomato and rice plants was significantly decreased,while the IAA-Ala conjugate level was increased,as compared to those in the wild-type control plants.Overexpression of SlGH3.4 in either tomato and rice plants resulted in a remarkably reduction in total mycorrhizal colonization rate and caused severe imparement in arbuscule development.4.To gain further insights into the potential role of SIGH3.4 during AM colonization,the knock-out and knock-down mutant lines were generated by using the CRISPR/Cas9 system or expressing a RNAi construct targeting SlGH3.4.The free IAA concentration in the mycorrhizal roots of the mutant lines was significantly higher than that in the mycorrhizal roots of control plants.The total fungal colonization level of the mutant lines showed no significant difference with that of the control plants.By contrast,the arbuscule morphology within the mycorrhizal roots of these mutant lines was not remarkably altered as compared to those in the control plant roots.5.Multiple genes associated with the auxin signaling pathways were investigated at the transcriptional level in both the wild-type and transgenic plants.Overexpression of the SIGH3.4 significantly repressed the expression of an auxin receptor gene,TIR1,and several expansin genes,such as SlEXPLB2,SlEXPLB4,OsEXP1,OsEXP5 and OsEXP10 in the transgenic tomato and rice plants.In contrast,the transcripts of SlEXLB4,SlEXLB2,SIEXPA9 and SIEXPA3 were significantly increased in the SlGH3.4-knock out or knock down mutant lines compared with that in the wild-type plants.In conclusion,in the present work we have identified three cis-acting motifs,the auxin-responsive element,AuxRE,and two newly identified motifs named MYCS1 and MYCS2,responsible for the AM-induced expression of SIGH3.4.Based on the physiological analysis of transgenic plants with enhanced or reduced expression activity revealed that SlGH3.4-mediated auxin homestasis in fungal-colonized cells is essential for modulating mycorrhizal colonization and arbuscule development in cortical cells.