Analysis Of The Symbiotic Interaction Between Bread Wheat Roots And Arbuscular Mycorrhizal And The Function Of Phosphate Transporter TaPT29-6A

Phosphorus(P)deficiency is one of the main growth-limiting factors for plants.As the most ancient,common and widespread beneficial symbionts,arbuscular mycorrhizals(AM)have been found in more than 90 % of vascular plant species,and they can significantly promote the mineral elements uptake,especially phosphorus.In order to analyze the symbiotic relations of wheat and AM fungus and excavate phosphate transporter genes involved in AM symbiosis,the symbiotic symstems between AM fungus and wheat under different phosphorus were constructed in this paper,which was analyzed by RNA-sequencing.Then the expression profiles of all the PHT1 gene members were detected,and four novel mycorrhizal-induced PHT1 genes of wheat were obtained.The virus induced gene silencing(VIGS)and yeast two-hybrid(Y2H)techniques were used to verify the functions of TaPT29-6A gene in P transport and immunity.The major results were as follows:1.The symbiotic system between wheat roots and AM fungus was established in the paper.The results of RNA-seq transcriptome analysis of 12 samples(symbiosis under high P condition: Ta_HPFM;symbiosis under low P condition: Ta_LPFM;un-symbiosis under high P condition: Ta_HPCK;un-symbiosis under low P condition: Ta_LPCK,three replicates for each treatment)obtained an average of 11.33 Gb data.And the average GC content was about 56 %,the standard Q20 and Q30 for evaluating reads quality were all higher than 97 % and 93 %,respectively.There were 960(959 up-regulated genes and one down-regulated gene)and 744(all up-regulated)differential expression genes existed in the comparisons between Ta_HPFM vs.Ta_HPCK and Ta_LPFM vs.Ta_LPCK samples.All the above genes were involved in pathways of basal metabolism,secondary metabolite metabolism and absorption of mineral nutrients,and nine mycorrhizal-induced PHT1 genes of wheat were obtained,indicated that PHT1 genes were involved in the symbiotic of wheat and AM fungus.2.Based on the latest high-quality bread wheat genome,35 memberss of PHT1 family in wheat were identified in the paper,which were named as TaPT1-2B to TaPT34-Un,according to their location on 13 wheat chromosomes.Phylogenetic analysis revealed that 35 PHT1 genes clustered on five of eight branches,and several novel PHT1 genes clustered on the same branch with the known mycorrhizal-specific PHT1 gene of other plant species.Sequence analysis indicated that there were 11 types of motif architectures and five types of exon-intron structures in the PHT1 family,and all PHT1 genes shared the characteristic motif of PHT1 protein.Chromosomal location and gene duplication mode analysis indicated that the PHT1 family has expanded mainly through segmental and tandem duplication events,and that all duplicated gene pairs have been under purifying selection.3.The symbiosis of Zhoumai 26 and Funneliformis mosseae,Glomus versiforme belonged to the negative responses,which suppressed the P direct uptake pathway and decreased the concentration of AM-symbiotic plants.Transcription analysis of the 35 PHT1 genes revealed that except for the known mycorrhizal-inducible genes Ta Pht-myc,TaPT15-4B(TaPT11)and TaPT19-4D(TaPT10),four novel mycorrhizal-inducible genes(TaPT3-2D,TaPT11-4A,TaPT29-6A,and TaPT31-7A)were highly up-regulated in AM-infected wheat roots.Furthermore,there are differences in the expression of mycorrhizal-inducible genes in wheat roots at different stages of infection by colonizing fungi.Transient Agrobacterium tumefaciens-mediated transformation expression in onion epidermal cells showed that TaPT29-6A was a membrane-localized protein.Tabacco rattle virus induced TaPT29-6A-silencing plants increased P content of the wheat plants,which is apparently required for the symbiotic and direct P pathway.And the silencing of TaPT29-6A also exhibited reduced levels of AM fungal colonization and arbuscules,but increased susceptibility to biotrophic,hemi-biotrophic and necrotrophic pathogens.In conclusion,TaPT29-6A was an essential gene for AM symbiosis,phosphorus absorption and immunity responses.4.The high quality yeast c DNA library of wheat roots colonized by AM under low P condition was constructed in the paper.The bait recombinant plasmid p GBKT7-TaPT29-6A was constructed on the GAL4-based two hybrid assay,and TaPT29-6A had no autoactivation and toxicity in yeast.As the bait protein,we obtained four potential interacting proteins of TaPT29-6A,which were the other protein of PHT1 family and proteins of plastid synthesis,respectively.It indicated that PHT1 proteins might play roles as protein complexes.The research provides a theoretical basis for the symbiosis between wheat root and arbuscular mycorrhizal,which influence the absorption and utilization of phosphorus,and enriched the functions of PHT1 genes involved in AM symbiosis.It also provides a theoretical basis for improving the absorption and utilization efficiency of phosphorus in wheat.