Functional Analysis Of MtPPI1 In Medicago Mycorrhizal Symbiosis

The symbiosis between land plants and arbuscular mycorrhizal fungi is the most common symbiosis,which impact on the carbon cycle in nature.The symbiosis between plants and arbuscular mycorrhizal fungi is crucial for plants to obtain phosphorus nutrients,and plants also provide carbon elements necessary for the growth and development of arbuscular mycorrhizal fungi.The arbuscule is the main site of active nutrient exchange between plant and arbuscular mycorrhizal fungi.It is a tree-like structure formed by the hyphae of arbuscular mycorrhizal fungi entering the inner cortical cells,which are highly bifurcated and eventually formed.In the arbuscule,mycelium branches are surrounded by periarbuscular membranes derived from plant cell membranes,on which some mycorrhizal symbiosis specific ion transporters and proton pumps are located to participate in bidirectional nutrient exchange.Among them,H~+-ATPase plays an important role in maintaining the potential difference between the two sides of the membrane and regulating the nutrient exchange of mycorrhizal symbiosis.However,how H~+-ATPase activity is regulated during mycorrhizal symbiosis is still unknown.Therefore,in-depth analysis of the molecular mechanism of H~+-ATPase operation will help to increase the understanding of nutrient exchange in mycorrhizal symbiosis.We found that Proton Pump Interactor 1(MtPPI1)gene expression in Medicago truncatula is specifically induced by AM symbiosis.By constructing RNA interference vectors,we found that reducing MtPPI1 expression in Medicago truncatula did not affect the normal growth of the plant,but significantly reduced the rate of mycorrhizal infection.Meanwhile,the development of arbuscule was severely affected in MtPPI1-RNAi plants.q RT-PCR analysis showed that the expression of symbiotic marker genes,including Mt PT4,Mt Fat M and Mt RAM2 genes involved in nutrient exchange in MtPPI1-RNAi plants,was significantly lower than that in the wild type,suggesting that MtPPI1 plays an important role in mycorrhizal symbiosis.We further analyzed the interaction proteins of MtPPI1 in mycorrhizal symbiosis by Co IP-MS,and found that the proteins interacting with MtPPI1 included H~+ATPase,suggesting that MtPPI1 plays an important role in mycorrhizal symbiosis through Mt HA1.The results of subcellular localization in Nicotiana Benthamiana leaves indicated that MtPPI1 and Mt HA1 co-located on the cell membrane,further suggesting that MtPPI1 may affect nutrient absorption in mycorrhizal symbiosis through regulating H~+-ATPase.Therefore,this study revealed the role of MtPPI1 in mycorrhizal symbiosis and its possible role in the process of mycorrhizal symbiosis by regulating plant H~+-ATPase activity,which laid a foundation for further understanding of plasma membrane proton gradient balance and nutrient exchange in arbuscular structures.