Mechanisms Of AM Symbiosis: Recognition Between AM Fungi And Plant Roots And Characterization Of AM Functional Related Genes

By means of headspace solid phase microextraction, gas chromatography-mass spectrometry,quantitative Realtime-Polymerase Chain Reaction (qRT-PCR), protein in vitro expression system,Electrophoretic Mobility Shift Assay (EMSA), RNA interference and Laser Scanning ConfocalMicroscope (LSCM), the recognition between arbuscular mycorrhizal (AM) fungi and plant rootsduring pre-symbiotic stages were verified and two AM-induced genes were functional characterized.AM fungi Gigaspora margarita, Glomus mosseae, and Glomus intraradices (currently Rhizophagusirregularis) and plants Lotus japonicus, Daucus carota, Cichorium intybus, Arabidopsis thaliana,Sorghum bicolor and Trifolium repens were used during the research. The main results andconclusions are as follow:1. Investigation of the viability of AM fungal spores in the rhizosphereAll the five tree species (Caragana korshinskii, Hippophae rhamnoides, Robinia pseudoacacia,Populus simonii and Populus euramericana Moench.108) investigated were well colonized by AMfungi, and the colonization rates were79.2%,76.0%,63.8%,59.9%and71.0%separately. Byintroducing two new indicators, density of viable spores (DVS) and density of spores that couldgerminate (DSG), we found that in each species, both the DVS and the DSG were lower than thespore density (SD); for DVS, the upper and lower limits were835(C. korshinskii) and35per100gdry soil (R. pseudoacacia) separately; and for DSG, the lowest value was9per100g dry soil with P.simonii and the highest is428per100g dry soil with R. pseudoacacia. There was a contradictionbetween the high AM colonization rates and the low DVS and DSG in the rhizosphere, whichindicates the presence of high efficient perception mechanisms between AM fungi and plant roots.The universal presence of AM in different plants and the high AM colonization rates indicatefunctional importance of AM to host plants.2. In vitro dual-culture between AM fungi and root organsBased on the fact that the spores of AM fungi are relatively small in size and are prone toattach to the substratum, a reusable tool for transferring AM fungal spores, which is easy to handleand sterilize, was invented; we also introduced a novel surface sterilization method of AM fungalspores which is easy to operate and with least hazard to spores during the sterilization procedures. The isolated-root of T. repens was created by cuting root tips from the aseptic seedlings; the callusof D. carota was induced by exotic plant hormones; and the hairy root of D. carota was obtained byagrobacterium-transformation; the dual-culture systems between G. mosseae and Ri T-DNAtransformed transformed root of carrot, G. intraradices and Ri T-DNA transformed transformed rootof cicoria were constructed.3. Sensing and responses of AM fungi to host plantSix different kinds of crude root exudates or plant aqueous extracts were collected or extracted,and their effects on G. mosseae development were evaluated via a germination and branching assayin vitro. Crude root exudates had no effects on spore germination and hyphal branches, while plantaqueous extracts could significantly promote spore germination, hyphal elongation and branching.The highest stimulation effects on spores germination, hyphal elongation and branching were foundwith leaf and stem extracts (LSET), isolated root extracts (IRET) and IRET respectively, and thevalues were2.3times,2.5times and5.2times of the control separately. Plant extracts could alsoinduce secondary spore formation.The recognition between AM fungi and plant roots through volatiles were verified throughco-culturing AM fungi and transformed carrot root in separated compartments of two-compartmentpetri-dish. After receiving the volatiles emitted from AM fungi, root branches were significantlyincreased, while the number of root hairs was significantly decreased. The numbers of root branchesat the presence of AM fungi were1.9times (G. mosseae) and1.7times (G. intraradices) of thecontrol separately, and the incidences of root hair were decreased to76.8%(G. mosseae) and67.6%(G. intraradices) of the control respectively. The hyphal growth of AM fungi was reoriented andtowards root after sensing the volatiles emitted from roots.4. Responses of plant roots to AM fungiSpores of the AM fungus Gi. margarita and the plants were inoculated in the same plateseparated by cellophane membranes (GSEs experiment) or in isolated different mediums (GVCsexposure). The root development was monitored with a time series. Both GSEs and GVCs couldpromote lateral root formation in wild-type plants of L. japonicus, and there were significantdifferences between the co-cultured plants and the control plants from day6(GSEs) and day7(GVCs) onwards separately. The effect was diminished with GSEs treatment in LjCASTOR mutant,while enhanced under the presence of GVCs. LjCCD7, an important component of strigolactonessynthesis pathway, was differentially expressed following GVCs exposure. GVCs could alsostimulate LRF in A. thaliana, while by contrast, GSEs show an inhibitory effect.5. Effects of AM fungi inoculation on root volatile organic compounds emissionSorghum plants were inoculated with/without G. mosseae and G. intraradices respectively. By means of headspace solid-phase microextraction (HS-SPME) and gas chromatography-massspectrometry (GC-MS), altogether44VOCs emitted from sorghum roots were identified, with21compounds from roots inoculated with G. mosseae,17from roots inoculated with G. intraradicesand18from the control. Among all the three treatments, alkanes were always the dominantcompounds and accounted for73.6%(control),56.8%(G. mosseae) and34.9%(G. intraradices) ofall VOCs contents separately. Compared with non-mycorrhizal plants, those inoculated with AMfungi emitted more alcohols and acids; there were no branched alkanes or alkenes detected from thenon-mycorrhizal plants, and no aldehydes were identified from mycorrhizal roots. Both thecomponents and contents of volatiles were dramatically altered by the presence of AM fungi, andthese changes have species specificity.6. Functional analysis of an-AM induced transcript factor LjMAMIThe LjMAMI in vitro expression system was constructed, and LjMAMI protein was extractedand purified with the Denaturing method. By applying the EMSA technique, LjMAMI was provedbinding to the cis-acting elements (MYCS and P1BS) of LjPT4gene, thus verifying the possibletranscriptional regulation of LjPT4by LjMAMI protein.Morphological analysis of LjMAMI TILLING mutants revealed that there are more shootbranches with the2084mutant seedlings (1.3times of the control); however, along with the growthof seedlings, this difference diminished. This result indicates that the divergent is only presentedwhen the plants are relatively young. The growth of the mutants was always retarded comparedwith the wildtype plants, implying the possible involvement of LjMAMI in plant growth regulation.7. Functional characterization of AM-induced phosphate transporter LjPT4By means of the RNA interference technique, we characterized the function of LjPT4. Theexpression of LjPT4is well downregulated, and the expression level is only14.6%of the control.By using fluorescence staining and LSCM, we found that when LjPT4was partially silenced, thedevelopment of arbuscules was hampered and up to74.6%arbuscules were collapsed. This resultindicates that LjPT4is indispensible for maintaining the morphology of arbuscules. Polyphosphateswere largely accumulated in the arbuscules of the LjPT4RNAi mutants as revealed by the TBOstaining, which gives hint that LjPT4is an important phosphate transporter in AM symbiosis.