Investigating The Mechanisms Underlying Enhanced Nodulation And N₂-fixation Of Peanut By Phomopsis Liquidambari Under In Vivo And In Vitro

In natural environment,plant can establish symbiotic relationship with many different beneficial microbes.However,our knowledge on the interactions of different symbioses which occur in same plant is rare.Our group previously isolated a broad-spectrum endophytic fungus Phomopsis liquidambari B3 from the inner bark of Bischofia polycarpa.P.liquidambari could establish mutualistic relationship with many crops,which enhances nutrients uptake,increases resistance of plant against pathogens and promotes plant growth,development and yield.Most interestingly,symbiotic association of P.liquidambari with peanut enhances the association of host plant with another soil microsymbiont,rhizobia under controlled and field-realistic condition.Studies have reported that P.liquidambari enhances peanut nodulation and N2-fixation through stimulation of root flavonoids exudation.Considering the complexity of legume-rhizobium interaction,the mechanisms behind the P.liquidambari-mediated peanut nodulation and N2-fixation enhancement are largely unknown.Except as a plant endophyte,P.liquidambari also have strong saprophytic ability,which could degrade plant litterfall and soil pollutants.P.liquidambari would undergo a lifestyle transition from saprophyte to endophyte when it encounters host plant.Similarly,rhizobium also possesses these two different lifestyles.Therefore,the aim of this study is to reveal the underlying mechanisms that P.liquidambari enhances peanut-bradyrhizobium nodulation and N2-fixation under saprophytic and endophytic conditions.To study the interaction of P.liquidambari and bradyrhizobium in the non-rhizosphere soil,we performed a plate experiment and found that P.liquidambari could establish a close interaction with bradyrhizobium and bradyrhizobium is able cross the physical barrier with the facilitation of P.liquidambari network.Then,the P.liquidambari exudation was collected at 7 days after inoculation.The results showed that the hyphal exudation significantly increased bradyrhizobial growth,biofilm formation and chemotaxis behavior.We next set up a soil microcosm to determine whether P.liquidambari network could facilitate the disperse of bradyrhizobium.We found that fungal hyphae increased the dispersal of bradyrhizobium in soil condition by determinations of bradyrhizobial CFU counting and nif D gene abundance.Additionally,P.liquidambari promoted the root invasion of bradyrhizobium and subsequently increased effective nodule formation.These results suggested that P.liquidambari could facilitate the disperse of bradyrhizobium in soil condition,shorten the distance of bradyrhizobium and host plant,and assist the infection of bradyrhizobial root infection.Previous studies reported that application of P.liquidambari could degrade phenolic acids,improve the continuous cropping soil environment and promote the nodulation.However,it is rarely unknown how phenolic acid affect peanut nodulation.As the root is the site for nodulation and N2-fixation,we speculate that phenolic acids negatively affect peanut nodulation through inhibiting root system growth.In this study,we found a model phenolic acid,benzoic acid inhibited root elongation of monocotyledonous(Oryza sativa and Zea mays)and dicotyledonous(Medicago sativa and Arachis hypogaea)plants.When employing a model plant Arabidopsis thaliana,we found that benzoic acid inhibited primary root elongation through reducing lengths of meristem and elongation zones,and number of meristematic cells.Benzoic acid-mediated inhibition of root elongation was due to the increased accumulation of auxin in root tips,which resulted from the enhanced auxin synthesis and AUX1/PIN2-mediated auxin transport.Next,we found that ethylene increased auxin accumulation via stimulation of auxin synthesis and transport under benzoic acid stress.Meanwhile,ROS burst was involved in benzoic acid-mediated root elongation inhibition through an auxin/ethylene independent manner.These results suggested that benzoic acid inhibited root growth through auxin,ethylene and ROS signls.This root growth inhibition may reduce the potential of peanut-rhizobium nodulation under continuous cropping system.However,when P.liquidambari and bradyrhizobium reach rhizosphere,root surface or root endosphere,the interactions of P.liquidambari and bradyrhizobial symbioses are still unknown.We employed RNA-seq to analyse the early response of host plant to P.liquidambari and bradyrhizobial inoculation.The experiment contained four treatments:control,P.liquidambari inoculation alone,bradyrhizobium inoculation alone and P.liquidambari and bradyrhizobium co-inoculation.Compared to control,P.liquidambari inoculation primarily altered expression of genes related to plant secondary metabolites,including phenylpropanoid and flavonoids.These secondary metabolites might participate in signals exchange between symbiotic partners,and facilitate the adaption of P.liquidambari in host plants.By contrast,differential expressed genes(DEGs)of bradyrhizobial inoculation treatment are associated with plant primary metabolism,including starch,sucrose,fructose and mannose metabolism.For instance,mannan endo-1,4-beta-mannosidase(EC:3.2.1.78)is induced by bradyrhizobium.The modification of plant primary metabolism alters content of specific carbon-containing compound,which might provide resource for bradyrhizobial growth and reproduction.The DEGs of P.liquidambari and bradyrhizobium co-inoculation regime are associated with purine and primary metabolism.We next compared the DEGs between bradyrhizobium inoculation alone and P.liquidambari and bradyrhizobium co-inoculation treatments to reveal the gene response behind the P.liquidambari-mediated peanut nodulation enhancement.Results showed that co-inoculation altered the gene expression that associated with plant-pathogen interactions,endocytosis and plant primary metabolism.In addition,some DEGs are involved in flavonoids biosynthesis and nodulation process.For instance,flavonol synthase(EC:1.14.11.23)is induced by co-inoculation in relative to bradyrhizobium inoculation alone.Meanwhile,P.liquidambari and bradyrhizobium inoculation decreased ethylene biosynthetic and response genes.In addition,P.liquidambari and bradyrhizobium inoculation altered gene expression of auxin transport,metabolism and response.These results suggested that metabolism and plant hormones may play a role in associations of plant with P.liquidambari and bradyrhizobium.We next determined the role of auxin in P.liquidambari-mediated peanut-bradyrhizobium nodulation and N2-fixation.We first employed inhibitors of auxin biosynthesis and signaling.Results showed that auxin is required for peanut-bradyrhizobium nodulation.We demonstrated that P.liquidambari could activate plant auxin signaling and enhance peanut nodulation in a local and systemic mode when using peanut and transgenic Arabidopsis thaliana(DR5:GUS).The activated auxin was recruited by peanut for nodulation and N2-fixation enhancement through transcriptional activation of symbiotic signaling and promotion of nodule vascular bundle development.The promotion of vascular bundle development enhanced nodule carbon metabolism.In addition,the activation of auxin signaling was required to maintain the fungal symbiotic behaviours and the beneficial effects on plants.Taken together,these results suggested that P.liquidambari enhances peanut-bradyrhizobium nodulation and N2-fixation through auxin signaling,which highlight the importance of auxin in interactions between symbioses from different members of root microbiome.The establishment and maintance of peanut-P.liquidambari-bradyrhizobium tripartite system require a large amount of carbon from the host plant.The root colonization of P.liquidambari was increased rapidly and then decreased in peanut after co-inoculation of P.liquidambari and bradyrhizobium,indicating that peanut maximizes reproductive potential and ensures the success of nodulation and N2-fixation through restricting P.liquidambari root colonization.However,it is unknown how plant modulates root colonization of P.liquidambari.In this study,we used A.thaliana-P.liquidambari system and found that flowering restricted colozniation of P.liquidambari.Next,we determined root soluble sugars concentrations and jasmonate signaling at flowering.We found that jasmonate signaling was activated at flowering and thus reduced root glucose and fructose concentrations.When emplying jasmonate signaling mutants,our results found that the reductions of P.liquidambari colonization were due to the jasmonate-dependent root glucose and fructose depletions at flowering.Furthermore,the jasmonate-dependent root glucose and fructose depletions were probably resulted from inhibitions of root soluble invertase and soluble sugar transport.These results suggested that flowering restricts root colonization of P.liquidambari through jasmonate-dependent root soluble sugar depletion.Meanwhile,these results indirect indicated that restricted colonization of P.liquidambari at late stage of plant growth in tripartite system saves resource for plant growth and reproductive,and peanut-bradyrhizobium nodulation and N2-fixation.To summary,our study uses P.liquidambari-peanut-bradyrhizobium tripartite interaction as the experimental model to reveal the mechanisms underlying the enhanced nodulation and N2-fixation by P.liquidambari under in vivo and in vitro.Our study provides a theoretical basis for the practical use of P.liquidambari in field-grown leuguminous crops,and enhances our knowledge on the coordination and adaption of plant to multiply microbial infection.