Transport And Signaling Through The AM Symbiotic Phosphate Transceptor

The majority vascular plants are able to form symbiotic associations with arbuscular mycorrhizal fungi. The symbiosis, termed arbuscular mycorrhiza is a reciprocal symbiosis, improving plant uptake of phosphate. The more than80%of land plants form AM interactions, in which plants supply associatedAM fungi with carbohydrates, essential for fungalsurvival and growth. In exchange, AMfungi provide their host plants with mineral nutrients, phosphorus (P), nitrogen(N)and other benefits. The first step of the fungus-mediated uptake is carried out by fungal membrane Pi transporters (PT) that transfer Pi from the soil into the extraradical hyphae, then the AM-specific Pi transporters transport the phosphate from the apoplastic space to plant cell.,So far, however, the Pi metabolic pathway of AM fungi are still not fully understood. Pi as a signal to permit continued development of theAM symbiosis, however, the mechanisms involved in signaling are poorly understood.To characterize the detailfunctions of Pi transporters in AM symbiosis, we combined cellular localization, heterologous functional expression in yeast with expression/subcellular localization studies and reverse genetics approaches in planta.l.Here we report the cloning and the functional analysis of a gene encoding a phosphate transporter (GigmPT) from the arbuscular mycorrhizal fungus Gigaspora margarita during mycorrhizal association with Astragalua sinicus roots. The kinetic analysis of GigmPT reveals that it belongs to high affinity phosphate transporter family. The GigmPT polypeptide belongs to the major facilitator superfamily (MFS). Homology modeling reveals that GigmPT exhibits twelve transmembrane helices divided into two halves connected by a large hydrophilic loop in the middle.2.GigmPT, a fungal Pi transporter highlyinduced during AM symbiosis. GigmPT is expressed in arbuscules and intercellular hyphae. These analyses show that GigmPT expression is regulated in response to external Pi concentrations. Phosphate concentrations, typical of those found in the soil solution, result in expression of GigmPT. Carbon availability triggers fungal phosphate uptake andtransport in AM symbiosis.3.We show that GigmPTis essential for the acquisition of Pidelivered by the AM fungus. However, more significantly, GigmPT function is critical for AM symbiosis. Loss of GigmPT function leadsto premature death of the arbuscules; the fungus is unable toproliferate within the root, and symbiosis is terminated. Thus, Pitransport is also a requirementfor the AM symbiosis. 4. The GigmPTphosphate transceptor transports phosphate and mediatesrapid phosphate activation of the protein kinase A (PKA) pathway.Using Substituted CysteineAccessibility Method (SCAM) we identified A146in TMDIV and V357in TMD VIII as residues exposed with their side chaininto the phosphate-binding site of GigmPT. Our results provide to the best of our knowledge the first insightinto the molecular mechanism of a phosphate transceptor.5. There also exists a second AM-specific Pi-transporter AsPT1indispensable for the development of AM symbiosis indicots. Knockdown of AsPTl by RNA interferenceled to degenerating or dead arbuscule phenotypesidentical to that of;AsPT4silencing lines. Nonredundant regulation of A. sinicusAM. symbiosis byAsPT1and AsPT4. AsPT4but not AsPTl is necessary and sufficient to mediatesymbiotic Pi transfer.6.These results substantiatethe hypothesis that phosphate transporteracts as the main provider of phosphate to the cell, but alsomediates rapid activation of thePKApathway. The nutrientsensers haveevolved from nutrient transporters andthus provide further support for the proposed evolutionaryscheme from nutrient transporters to chemicalsignal receptors.