Study On The Mechanism Of Nitrogen Regulation Of The Symbiotic Interaction Between The Endophytic Fungus P. Sylvestris And Plants

Nitrogen(N)is one of the main elements that limits plant growth and development.However,irrational fertilization patterns have resulted in the uneven distribution and excessive quantities of N resources in the soil,which have caused serious environmental issues.In recent years,researches have reported that endophytic fungi promote N absorption and metabolism of host plants.However,an increasing number of studies have shown that N signals regulate the plant-endophyte interaction and symbiotic benefits in the agricultural ecosystem.Given the ecological importance of plant-fungal symbioses in agricultural production,there is great interest in understanding how N signal regulates plant-fungal symbioses and the mechanisms by which this regulation occurs.Our results could be employed to manage plant-microbe interactions in N-affected environments.Our previous studies have reported a fungal endophyte Phomopsis liquidambaris B3,isolated from the inner bark of the stem of Bischofia polycarpa.This fungus could establish symbiotic associations with monocotyledonous rice(Oryza sativa)and dicotyledonous Arabidopsis thaliana,and promote plant N absorption and final yield.Interestingly,our previous studies showed that low-N application benefits plant-Ph.liquidambaris interaction,indicating that external N levels play an important role in mediating these symbioses.In addition,Ph.liquidambaris possesses strong saprophytic ability,and can regulate the nutritional dynamics in the mycosphere.This fungus could also establish an interactive relationship with soil bacteria,and affect the composition of inorganic N in the rhizosphere of plants,indicating that the N forms may also involve in regulating plant-Ph.liquidambaris interaction.Thus,in this study,the symbiotic rice-Ph.liquidambaris and Arabidopsis-Ph.liquidambaris interactions were established to explore the rules and mechanisms underlying N levels-and N forms-regulated plant-fungal interaction,respectively.First,we explored the impact of N levels on plant-microbe interactions.Rice,one of the most important crops in the world,relies on large amounts of N fertilizers for higher yields.This part of study was thus performed with the rice-Ph.liquidambaris system.Plant immune response is required for restricting fungal colonization,whereas N has been reported to contribute to multiple levels of plant resistance,such as the accumulation of plant defense proteins and signal transduction.Therefore,we hypothesized that N levels regulated the colonization dynamics of Ph.liquidambaris by triggering the basic defense response of rice.At 14 days post inoculation,low N application promoted root colonization,accompanied by the accumulation of hydrogen peroxide and nitric oxide,indicating that fungal inoculation could enhance the oxidative responses of rice under low N conditions.Moreover,low N promoted the accumulation of salicylic acid and increased the expression of salicylic acid-related genes in rice,which were independent of the colonization of fungal endophyte.In addition,we found that Ph.liquidambaris did not significantly activate activities of defense enzymes in response to external N levels,such as polyphenol oxidase,and phenylalnine ammonialyase.Next,we extracted the crude protein of the rice root and the crude cell wall of the fungus.The data showed that root colonization by Ph.liquidambaris impaired plant ability in degrading the fungal cell wall in response to N levels.In addition,by tracking the fungal response to root extracts of rice,we found that the fungus-derived antioxidant enzymes were not significantly affected by fungal inoculation or N levels.These results suggested that high N-restricted colonization by fungal endophyte might not primarily depend on the natural immunity of the host plants.Then,we hypothesized that host nutrient metabolism was involved in plant-microbe interactions in response to N levels.We monitored the dynamics of rice carbon metabolism over time under different N levels.The results showed that Ph.liquidambaris inoculation promoted plant photosynthesis,including the synthesis of leaf chlorophyll and ribulose bisphosphate carboxylase/oxygenase(Rubisco)under low N conditions.More importantly,fungal inoculation promoted the accumulation of water-soluble carbohydrate(WSCs)in rice roots and root exudates under a low N supply.In vivo and in vitro experiments have also proved that the accumulation of carbohydrate facilitated the growth and colonization of Ph.liquidambaris.In summary,these results indicated that Ph.liquidambaris promoted the primary photosynthesis of rice and induced the allocation of carbohydrates from source to sink,facilitating the establishment of symbiotic interaction under low N conditions.We further hypothesized that N regulated plant-fungal interactions by influencing the carbohydrate metabolism.The mutualistic fungus Ph.liquidambaris was found to prioritize hexose resources through in vitro culture assays.Rice-Ph.liquidambaris systems were exposed to N gradients ranging from N-deficient to N-abundant conditions to study whether and how the sugar composition was involved in the dynamics of N-mediated fungal colonization.We found that root soluble acid invertases were activated,resulting in increased hexose fluxes in inoculated roots.These fluxes positively influenced fungal colonization,especially under N-deficient conditions.Further experiments manipulating the carbohydrate composition and root invertase activity through sugar feeding,chemical treatments,and the use of different soil types revealed that the external disturbance of root invertase could reduce endophytic colonization and eliminate endophyte-induced host benefits,even under N-deficient conditions.Collectively,these results suggested that the activation of root invertase was related to N deficiency-enhanced endophytic colonization via increased hexose generation.Subsequently,we explored the effects of environmental N forms on plant-microbe interactions with the Arabidopsis-Ph.liquidambaris system.The uneven distribution of inorganic N forms in the soil is an important environmental cue,which has caused serious agricultural issues.However,the ways in which nitrate(NO₃^-)/ammonium(NH₄⁺)influence plant-fungal interactions and the underlying mechanisms governing this effect remain elucidated.Most fungal endophytes experience a saprophytic life cycle in the soil before establishing a symbiotic relationship with plants,but the effect of inorganic N forms on this process remains poorly understood.In vitro experiments showed that Ph.liquidambaris preferred to use NO₃^-as its N source,when compared with high NH₄⁺level.Later,we found that NO₃^-facilitated the migration of fungal hyphae in the soil and the utilization of plant litter.Moreover,NO₃^-facilitated the earlier establishment of a symbiotic relationship with Arabidopsis.Additionally,our results indicated that soil bacteria were involved in the adaptation of Ph.liquidambaris to the soil environment with high NH₄⁺levels.To further describe whether the bacterial behavior in the mycosphere was related to fungal capability in utilizing N resources,we conducted a nitrite reductase-deficient strain(?nir)by using CRISPR-Cas9 system.The?nir strain hardly utilized NO₃^-,but had better NH₄⁺uptake.Then,we collected the soil adjacent to the hyphae and performed 16S r RNA sequencing.This fungus established an unique bacterial community in its mycosphere and recruited several bacterial species related to the nitrogen cycle,such as N-fixing bacteria.BIOLOG analysis showed that the microorganisms in the mycosphere possessed enhanced metabolic capability of carbon sources,which was closely related to the changes in bacterial communities.These processes were deeply affected by the ability of fungi to utilize inorganic N sources.These results indicated that the form of inorganic N affected the saprophytic behavior of a fungal endophyte,depending on fungal habits in utilizing N sources;the bacterial community participated in the fungal adaption to the soil N environment.Next,we studied the influence of inorganic N forms on the Arabidopsis-Ph.liquidambaris interaction after the symbiotic association have been established.In this study,we used the wild-type strain,which had better NO₃^-uptake,and the?nir strain,which had better NH₄⁺uptake.The fungus-inoculated and noninoculated Arabidopsis were exposed to different N forms for measurements of plant biomass,fungal colonization,NO₃^-/NH₄⁺concentrations and ion fluxes on the root surface.We showed that NO₃^-facilitated beneficial Arabidopsis-Ph.liquidambaris interactions,whereas NH₄⁺suppressed this symbiosis.Moreover,we found that fungal inoculation regulated NO₃^-/NH₄⁺fluxes in the root tips,resulting from the modulation of the ion degrees in the plant-fungal interface.The transcriptomic analysis suggested that N form-regulated symbiosis might be caused by the altered transcription of energy metabolism and ion transport genes.Overall,our data indicated that root-associated fungi may influence host adaptation to different N forms by modulating the ion degrees in the plant-fungal interface.In conclusion,this study revealed the dynamics and mechanisms underlying environmental N-regulated plant-fungal symbiotic interaction with the rice-Ph.liquidambaris and the Arabidopsis-Ph.liquidambaris systems as the models,from the two perspectives of N level and N form.This study will provide a theoretical basis for the comprehensive management of N input and microbial application in agriculture,and also enhance our knowledge on the interaction between fungal endophytes and their host plants.