Physiological Response Mechanism Of Phaseolus Vulgaris To Rhizobiun Etli CFN42 Nitrogen-deficient Strain

Legume-rhizobia symbiotic nitrogen fixation system is the most efficient biological nitrogen fixation system in nature.However,rhizobial genotypes vary substantially in the amount of nitrogen they fix for legume hosts and often include ineffective genotypes that instigate nodule formation but fail to fix any nitrogen.Can host plants respond to the difference of fix nitrogen ability of rhizobia,so as to establish and maintain a stable symbiotic relationship?It has been found in soybean,mimosa and other plants that the host restricts the proliferation of non-fixing rhizobia and bacteroid differentiation.but its mechanisms remain unclear.Therefore,in this experiment,we choose Phaseolus vulgaris and Rhizobium etli CFN42 as models to study response mechanism of host plants to different fix nitrogen ability of rhizobia.Previous works found that Rhizobium etli CFN42(wild type-fixing strain)and nifHa/bDK mutant strain(ΔnifHa/bDK,nitrogen-deficient strain)were co-inoculated at 1:1 ratio,host had a certain restriction on the formation of non-fixing strain ΔnifHa/bDK,and the proportion of ΔnifHa/bDK strain at a low level,while the proportion of WT strain gradually increased to 80%.There was no difference in the number of bacteria between the two genotypes rhizobia.In this paper,we study the response of Phaseolus vulgaris to nitrogendeficient strain ΔnifHa/bDK by single inoculating WT and ΔnifHa/bDK strains separately.The main results are as following.(1)To test whether nifHa/bDK effect nodulation and nitrogen fixation of plants.Firstly,we detected the nitrogenase activities of WT and ΔnifHa/bDK in nodules following single inoculation and co-inoculation of Phaseolus vulgaris for 2-5 weeks.The results showed that the ΔnifHa/bDK single inoculated plants had almost no nitrogenase activity.The plants single with WT and had highest nitrogenase at the 3rd week and shows downward trend with the growth of plants.After that,nodulation kinetics of plants inoculated with WT ΔnifHa/bDK separately and 1:1 mixture of them were measured.The number of nodules in the plants inoculated with WT single and mixed with two strains 1:1 was about 120,but the number of nodules of the plants inoculated with ΔnifHa/bDK single reached about 220,which resulted in the phenomenon of autoregulation of nodulation.The above results showed that the nodule formed by the nitrogen-deficient strain ΔnifHa/bDK lost the ability of nitrogen fixation and could not provide enough nitrogen source for the host plant,so the plant would produce more nodule to obtain nitrogen source.(2)To study the physiology response of Phaseolus vulgaris to ΔnifHa/bDK.We observed the color and morphology of nodules by using the super deep scene microscope.TheΔnifHa/bDK formed small white nodules in the 2nd week and brown nodules in the later stage,while WT formed larger pink nodules.This showed that the nodules ΔnifHa/bDK strain were senescent in advance.Compare the fitness of the two rhizobia with different nitrogen fixation ability,we detected the number of the two strains in nodules following single inoculation for 2-5 weeks.The number of WT strain was higher than that of ΔnifHa/bDK strain,and there was a significant difference between them.The average number of WT reached highest in the 4th week.ΔnifHa/bDK reached highest in the 2nd week,and then decreased gradually.At the 5th week,the ΔnifHa/bDK was not detected in nearly 1/6 of nodules,which indicated that the fitness of WT better than ΔnifHa/bDK in nodules.The results of toluidine blue staining showed that there was no difference between the two strains in the infection of nodules.In the 2nd week,the infected cells of WT developed well and had full shape with vacuoles,while vacuole rupture in some infected cells of ΔnifHa/bDK.In the 4th week,the infected cells of ΔnifHa/bDK showed irregular deformation and shrinkage compared with WT,and vacuole rupture in most infected cells of ΔnifHa/bDK.The results showed that the infected cells of ΔnifHa/bDK were senescent in advance.Comparative bacteroid morphology of WT and ΔnifHa/bDK by TEM.It was found that there were signs of symbiosome membrane degradation in the nodules formed by ΔnifHa/bDK in the 2nd week.By the 4th week,most of the symbiosome membrane had been degraded,and the proportion and size of the bacteria after differentiation were lower than those of WT,and the cells broke down and the bacteroid were released to the cell gap.The above results show that Phaseolus vulgaris can sanction the nitrogen-deficient strain ΔnifHa/bDK to maintain the stability of symbiotic relationship by reducing the number of nitrogen-deficient strain in the nodule or forcing the nodule to senescence in advance.(3)To explore the physiology response mechanism of Phaseolus vulgaris against nitrogen-deficient strain ΔnifHa/bDK,RNA-Seq technology was used to sequence and analyze the nodule following single inoculation.It was found that compared with WT.the expression level of 5977 genes changed significantly under treatment of ΔnifHa/bDK inoculation,of which 3264 genes were up-regulated genes and 2713 genes were downregulated.Through KEGG pathway analysis,the differentially expressed genes were mainly enriched in plant hormone signal transduction,starch and sucrose metabolism,phenylalanine metabolism,plant pathogen interaction,carbon metabolism,glycolysis/gluconeogenesis,cell apoptosis,nitrogen metabolism and other pathways.In the nodule inoculated withΔnifHa/bDK,Defense-related gene PR1 Phvul.006G196900,PR4 Phvul.006G102300,PAL Phvul.008G289500,R Phvul.004G115600,WRKY29 Phvul.002G293200 and WRKY70 Phvul.005G080400 were up-regulated(log2 ratio was 2.35,2.14,2.48,13.66,2.36 and 16.18 respectively).The results showed that the host had obvious immunosuppressive effect on theΔnifHa/bDK.senescence-associated protein SAG29 Phvul.008G001100,Caspases3(the key gene of plant apoptosis)were up-regulated(log2 ratio was 2.33 and 15.41 respectively).The genes encoding WRKY,bZIP5 MYB and bHLH transcription factors,Phvul.005G080400,Phvul.009G073800,Phvul.007G069400 and Phvul.009G189200 were up-regulated(log2 ratio was 16.18、14.38、14.08 and 15.51 respectively),indicating that transcription factors play an important role in the physiological response of host to ΔnifHa/bDK.Phvul.008G289500,Phvul.007G135600 and Phvul.010G135300,which encode phenylalanine ammonia lyase(the key enzyme of salicylic acid synthesis).1-aminocyclopropane-1-carboxy late synthase(the key enzyme of ethylene synthesis),lipoxygenase(the key enzyme of jasmonic acid synthesis),up-regulated expression(log2 ratio was 2.48、6.43 and 4.02 respectively).The differential expression of these genes suggests that plant hormone pathway may be involved in the response of host to ΔnifHa/bDK.The genes encoding calmodulin-like(the key protein of Ca2+signal transduction pathway)and glutathione peroxidase(remove the excess reactive oxygen species),Phvul.007G169601and Phvul.007G175400 were up-regulated(log2 ratio was 17.06 and 14.92 respectively).The differential expression of these genes suggests that Ca2+ signal pathway and oxidative stress pathway may be involved in the response of host to nitrogen-deficient ΔnifHa/bDK.Ten differentially expressed genes were selected for q RT-PCR detection.The results showed that the results of qRT-PCR of 10 genes were consistent with the results of RNA-Seq,which confirmed the reliability of the results of RNA-Seq.According to the results of RNA-Seq,the content of salicylic acid in nodules was determined to verify whether the salicylic acid pathway of plants changed.We detected the salicylic acid content of nodules.The results showed that salicylic acid content in 4wpi nodules following single inoculated with WT was 18.40 pmol/g,while the content of salicylic acid in nodules following single inoculated withΔnifHa/bDK was 25.79 pmol/g.The results showed that the physiological response of Phaseolus vulgaris to nitrogen-deficient ΔnifHa/bDK may be a process of plant hormone signal transduction,Ca2+signal transduction,active oxygen signal molecules,and a variety of transcription factor families.All sum up,the sanction response of Phaseolus vulgaris to nitrogen-deficient strainΔnifHa/bDK mainly occurs after the formation of nodule for three weeks,which will strongly control the number of ΔnifHa/bDK,and produce certain sanction by reducing the fitness of nitrogen-deficient strain or forcing the nodules to senescence in advance.Phaseolus vulgaris to nitrogen-deficient-nitrogen strain ΔnifHa/bDK may be a process of plant hormone signal transduction,Ca2+ signal transduction,reactive oxygen signal molecules,and a variety of transcription factor families,but the complex regulatory network needs to be further study.