Effect Of Transcription Factor NtPHR Gene On Phosphorus Uptake And Arbuscular Mycorrhizal Symbiosis In Tobacco

Arbuscular mycorrhizal(AM)symbiosis that is formed by soil fungi and most terrestrial plants is the most widespread mutualistic associations in the nature.In the root cortex,the fungus develop highly differentiated hyphae called arbuscules,which are the major sites for the exchange of nutrients such as nitrogen(N),phosphorus(P)and potassium(K)between the two symbionts.Multiple genes involved in Pi transport across the symbiotic interface have been ioslated in various plant species and characterized to be strongly induced in response to AM symbiosis.Since it has been well established that PHR transcription factors play central roles in controling Pi signaling regulatory pathway in diverse plant species,it is tempting to speculate that the AM symbiotic P uptake pathway might also be regulated by the PHR-mediated Pi signaling pathway.In plants,the PHR transcription factors are encoded by a multi-gene family with members having different physiological roles.In the model plants,Arabidopsis and rice,AtPHR1 and OsPHR2 were evidenced to be the key regulators in P signaling pathways.AtPHR1 and OsPHR2 belong to MYB-CC family,as both of them contain MYB domain and CC domain.In this study,two PHR genes,NtPHR1 and NtPHR2,with substantial homology to AtPHRl were isolated from the tobacco(Nicotiana tabacum).To explore the potential roles of the PHR genes in regulating Pi uptake and AM symbiosis,detailed investigations,such as expression patterns,in vitro binding activties,and physiological responses of the transgenic plants with reduced or enhanced expression abundance were further carried out.The main results were as follows:1.Bioinformatic analysis showed that both NtPHRl and NtPHR2 are transcription factors with an MYB domain and a predicted coiled-coil(CC)domain defined as members of the MYB-CC family.In comparison with NtPHR1,NtPHR2 has a more closer phylogenetic relationship with the AtPHRl.Subcellular localization analysis revealed that the proteins encoded by both the two PHR genes are nuclear-localized proteins.2.Expression analysis revealed that NtPHRl and NtPHR2 were almost constitutively expressed in roots and leaves,and their transcripts were significantly induced in response to Pi starvation.The transcripts of NtPHR2 in roots were much higher than those in the tobacco leaves.The proteins encoded by both the two PHR genes can bind to the promoter of NtPht1:4 in a heterologous yeast system.3.Transgenic tobacco plants expressing an RNA interference(RNAi)construct targeting both NtPHRl and NtPHR2 were generated.Knock down of NtPHR1 and NtPHR2 impaired tobacco plant growth and lateral root development.The P concentration in RNAi transgenic plants was significantly decreased compared with that in the wild-type(WT)plants under low-Pi supply condition.Additionally,repressing the expression of NtPHR1 and NtPHR2 by RNAi caused signifincant decline of the expression level of Pht1;4,an AM-specific Pi transporter gene,and the P concentration,as well as the mycorrhization intensity and arbuscule abundance in mycorrhizal tobacco plants.4.Overepression of NtPHR2 in tobacco plants had no significant effect on plant biomass,but significantly increased the Pi accumulation in leaves under either high-Pi or low-Pi supply condition.The AM colonization rate and arbuscule incidence in the mycorrhizal roots of NtPHR2-overexpression plants were significantly decreased as compared to those in the mycorrhizal roots of WT plants.In concluion,there exist at least two PHR paraloguous genes belonging to the MYB-CC family in the tobacco genome.The two PHR genes might play an important role in regulating AM symbiotic Pi uptake and mycorrhization intensity in solanaceous species by interacting with the P1BS motif in the promoters of AM-induced Pi transporters.