The Effect Of Nitrogenase Genes On Host Fitness In Rhizobium Etli CFN42

Rhizobial bacteria instigate the formation of root nodules on legumes which fix atmospheric nitrogen to convert into ammionia for the host.This process promotes the growth of plants and plays an important role in agricultural production.However,legumes often encounter ineffective rhizobia that trigger the formation of nodules but fix little nitrogen.Although these non-fixing rhizobia have an advantage in evolution,they can not replace fixing rhizobium,which has caused widespread concern.In this experiment,we choose Phaseolus vulgaris and Rhizobium etli CFN42 as models to study the fitness of rhizobia with different nitrogen fixing ability in nodule.In this experiment,two operons encoding full nitrogenase nifHaDK and nifHbDK were knocked out in Rhizobium etli CFN42 to construct ΔnifHa/bDK strain,which has no nitrogen fixation ability.These strains were used to study rhizobium of different nitrogen-fixing ability in response to Phaseolus vulgaris.Firstly,2-4 weeks after single inoculation of the host plants with the wild-type strain(Wild Type-WT)or the non-nitrogen-fixing mutant strain(ΔnifHa/bDK),the nitrogenase activity was determined.The results showed that the WT single inoculated plants had highest nitrogenase acitivity at the 2nd week anda downward trend with the growth of the plants.The plants single inoculated with ΔnifHa/bDK had almost no nitrogenase activity.Therefore,the constructed ΔnifHa/bDK strain can be used for subsequent experiments.Subsequently,the nodulation kinetics of WT and ΔnifHa/bDK were measured when they were separately inoculated and mixed inoculated at different ratios(1:1,1:9,and 9:1).The number of nodules under different treatments was determined.There was no significant difference in the number of nodules,which was around 120.And no autoregulation of nodulation was observed.In order to study the adaptability of the two strains in the host,the WT and ΔnifHa/bDK strains were labeled with fluorescence or antibiotic resistance as follows:WT(Cm),ΔnifHa/bDK(Tc),WT(GFP),ΔniHla/bDK(mCherry).Growth curves and nitrogenase activity assays were performed on different labled strains.The results showed that the resistance markers and fluorescent markers had no effect on the growth of the strains.There was no difference in the nitrogenase activity of differently labeled WT strains and differently labeledΔnifHa/bDK strains,which have no nitrogenase activity.Later,1:1 mixed culture and root hair adsorption experiments in vitro showed that WT(Cm)and ΔnifHa/bDK(Tc)strains did not have a competitive effect when co-cultured in vitro.There was no difference in root hair adsorption capacity.These experiments indicate that both rhizobia and early infection had no difference in the ability to grow.In the mixed inoculation experiment,the differences in host fitness between the two rhizobia with different fixing ability were compared by measuring the nodule occupation and the number of bacteria in the nodule of both strains.The results showed that there was no difference in the number of bacteria of WT(Cm)andΔnifHa/bDK(Tc)no matter in mixed infected root nodules or single infected nodules.TheΔnifHa/bDK(Tc)strain single infected nodules occupation rate was at relatively low level throughout the experimental period.While WT strain single occupation rate escalated and eventually reached 80%.The data showed that under the experimental conditions,the host plants have a certain limition on the number of nodule in the ΔnifHa/bDK(Tc)strains that can not fix nitrogen.There is a phenomenon of host sanctions.The WT(GFP)and ΔnifHa/bDK(mCherry)were mixed inoculated in the plants to observe the differentiation and distribution of the two strains.The results showed that the loss of nitrogen fixation did not affect the differentiation of ΔnifHa/bDK(mCherry).The morphological characteristics of its in-plant cells were consistent with that of WT(GFP)strains,and the cells exhibited a certain degree of prolonged differentiation state.Both strains were independently distributed in the centrally infected area of root nodules It is presumed that the host will perceive the difference in nitrogen-fixing ability of the strains,thereby affecting the distribution of the strains in the nodules.WT and ΔnifHa/bDK strains were studied in this experiment.First,the strains were subjected to fluorescence and resistance markers.Then the proportions and distributions of of the two different bacteria in the host were studied.The host have certain sanctions against non-fixing nitrogen-tolerant rhizobia,limiting the number of ineffective nodules,and affecting its distribution in root nodules,revealing that the host is dominant in the symbiotic nitrogen fixation process and capable of distinguishing the nitrogen fixation ability of rhizobia and influencing its symbiotic nitrogen fixation process.