Molecular Mechanisms Of GH3.4 On The Formation Of Arbuscular Mycorrhizal Symbiosis In Tobacco And Tomato

Arbuscular mycorrhizal(AM)symbiosis that is formed by soil fungi belonging to Glomeromycota and most terrestrial plants is the most widespread mutualistic associations in the nature.The establishment of symbiosis occurs in the plant roots where the colonization process undergoes sequential steps that implicate both the epidermal and cortical cells.In the root cortex,the fungus develop highly differentiated hyphae called arbuscules,which are the major sites for nutrients and signal exchange between the two associates.The complex cellular interaction between AM fungi and host roots requires a continuous exchange of signals to ensure the development of AM symbiosis,and many genes have been proposed to be involved in such symbiotic interactions.Additionally,careful scrutiny of the released microarray dataset on the transcriptome of mycorrhizal roots revealed positive responses of some auxin-regulated genes,such as GH3(Gretchen Hagen)and Aux/IAA,to AM symbiosis in several mycorrhizal plant species,making a role for this phytohormone in AM formation plausible.In plants,GH3 gene family is widely considered to be involved in a broad range of plant physiological processes,through modulation of hormonal homeostasis.Based on the phylogenetic relationship,plant GH3 genes have been classified into three major groups(?,?,and?).Multiple GH3 genes from Group?have been functionally studied and biochemically evidenced to have IAA-amido synthetase activity.In this study,though gnome-wide hunting of the released tomato genomic sequences,a total of 15 GH3 paralogues(SIGH3.1-15)have been identified from tomato.Expression analysis of these tomato GH3 genes revealed that all the six paralogues from Group?(SlGH3.2,SIGH3.3,SlGH3.4,SIGH3.7,SIGH3.9)showed inducible responses to external IAA.Particularly,SlGH3.4(EC 6.3.2.8),of which the transcripts were Barely detectable under normal growth condition,was strongly activated in not only the IAA-treated roots,but also the AM fungal-colonized roots.We also isolated the orthologous gene of SIGH3.4 from tobacco which was accordingly named as NtGH3.4.Detailed investigations,such as promoter driving GUS gene expression,and functional analysis of the transgenic plants with reduced or enhanced expression activity,were then carried out in order to elucidate the regulatory profiles and the potential physiological roles of the auxin-and mycorrhiza-inducible GH3.4 genes in response to AM symbiosis in tomato/tobacco plants.The main results were as follows:1.The effect of polar auxin transport inhibitor NPA on mycorrhizal symbiosis was investigated.It was shown that external application of high concentration of NPA to tomato and potato roots resulted in significant repression of mycorrhizal infection rate and impaird the development of arbuscules in the mycorrhizal roots of the two solanaceous plants.2.DR5-GUS,which serves as an indicator of auxin response,was employed to investigate the auxin response in the AM fungal colonized cells.It was revealed that GUS expression was highly activated in arbuscule-containing cells of tobcaoo mycorrhizal roots,suggesting that arbuscule development or functioning is accompanied by an auxin response.3.The expression patterns of the tomato GH3 genes in response to different phytohormone applications and AM symbiosis was determined.All the six tomato GH3 genes(SlGH3.2,SlGH3.3,SIGH3.4,SlGH3.7,SlGH3.9 and SlGH3.15)from GH3 Group?were found to show inducible responses to external IAA,and three members were significantly up-regulated in response to AM symbiosis.In particular,SIGH3.4,the transcripts of which were Barely detectable under normal growth conditions,was strongly activated in the IAA-treated and AM fungal-colonized roots.A further time-course expression analysis was also performed,which provided further strong evidence of the high correlation between the transcript abundances of SlGH3.4 and colonization intensity.4.Histochemical staining demonstrated that a 2258-bp SlGH3.4 promoter fragment was sufficient to drive the GUS expression strongly in both IAA-treated and AM fungal colonized tobacco roots.It was observed that GUS staining was not detectable in untreated transgenic plant roots,but was strongly detected in the root tips,steles and cortical cells of the IAA-treated roots of transgenic plants.However,the GUS staining could be detected predominantly in distinct areas of the fungal-colonized roots.5.The orthologous gene(NtGH3.4)of the tomato SlGH3.4 was isolated from the tobacco mycorrhizal roots.Overexpression of the NtGH3.4 resulted in the remarkably reduction of IAA content,repression of mycorrhizal infection rate,and in particular imparement of the formation of normal arbuscules in transgenic tobacco roots colonized by AM fungi.Additionally,the expression of expansin genes NtEXPA1?NtEXPA14 and NtEXPB3,and auxin receptor gene NtTIR1 was significantly down-regulated in these transgenic plants with enhanced NtGH3.4 expression.In conclusion,in the present work we have characterized the transcriptional regulation profiles of tomato GH3 genes,which led to the identification of SIGHS.4 that was specifically and strongly induced in response to IAA supply and AM symbiosis.The specialized and strong expression of SlGH3.4 in fungal-colonized cells of AM roots provided a strong hint that this symbiosis-activated GH3 gene may perform a crucial regulatory role during the establishment of AM symbiosis.Such functional expectation was further verified by characterizing the transgenic plants with enhanced expression activity.The findings obtained from the current work offer new insights into the molecular mechanisms underlying the co-ordination between the auxin and mycorrhizal signaling pathways in the regulation of AM symbiosis in plants.