Responses Of Robinia Pseudoacacia To Rhizobial Nod Factor And The Mechanism Of Heme Degrading Enzyme HmuS In Symbiosis

The establishment of symbiotic association between legumes and soil bacteria,generally referred to as rhizobia,results in specialized plant organs known as root or stem nodules in nitrogen-deprived soils.Elaborate molecular dialogues and complex regulations between legumes and rhizobia result in successful symbiotic associations.In recent decades,genes of legume plants involved in establishment or regulation symbiosis have been greatly studied,and most of functional research focused on model legume plants,such as Medicago truncatula and Glycine max.However,the symbiotic mechanism of woody legumes with their rhizobial partners is still poorly understood.Here,the symbiotic association of Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia,a woody legume species,was studied.The aim of the present study is to elucidate the responses of R.pseudoacacia to nod factor of M.amorphae CCNWGS0123,and to identify the difference in symbiotic properties between NF signaling and T3SS signaling.In addition,M.amorphae CCNWGS0123 encodes two hemin degrading factors:one(hmu S_pSym)is located in the symbiotic plasmid(Sym-plasmid),and the other(hmu S_ch)is located in chromosome.Whether both of the two genes are involved in symbiosis with R.pseudoacacia remains unclear,and their biological functions in symbiosis are still unknown.The main findings in this study are listed as follows:1.Symbiosis of R.pseudoacacia is nod factor dependent;the NF-and T3SS-mediated symbiotic signals are different,and NF affects host compatibility.The majority of the nod factor(NF)genes,which could be activated by plant flavonoids,were identified in the symbiotic plasmid of M.amorphae CCNWGS0123.Two nod-boxes were identified upstream of nod L and nod A,and they were considered to be the conserved sequence to which Nod D binds.Compared to the plants inoculated with WT strain,the plants infected by the NF-deficient strain were significantly shorter and weaker,with few etiolated leaves and shoot dry weight.No nitrogenase activity was detected in the white bumps from the plants inoculated with the NF-deficient strain.Despite a large amount of root hair tip swellings formed in the plants inoculated with the NF-deficient strain,the rhizobia did not enter the root hair cells but were attached to the surface of roots,and no normal infection threads were detected.The NF-deficient strain did not enter root hair cells,but the T3SS strain could be entrapped into curled root hair cells.However,the infection thread development from the plants infected by T3SS-deficient strain was aborted at the elongation stage.At the early stage of nodulation,the most obvious difference of plant hormones between the roots inoculated with the NF-and T3SS-deficient strains were the content of jasmonic acid(JA)and gibberellic acid(GA).Furthermore,the expression patterns of the plant defense and symbiotic genes were quite different between the plants inoculated with the NF-and T3SS-deficient strains.Additionally,the host compatibility assay indicated that the symbiosis of R.hispida was similar to R.pseudoacacia.A large number of root tips induced by the NF-deficient strain displayed a long-cylindrical nodule-like structure that finally developed into an abnormal lateral root.Interestingly,the symbiosis of Amorpha fruticosa was NF-dependent and T3SS-independent,which formed determinate-like nodules when inoculated with the WT strain.M.amorphae CCNWGS0123 was incompatibility with either Sophora japonica or Sophora xanthantha.2.hmu S_pSym located in symbiotic plasmid of the strain,rather than hmu S_ch on chromosome,plays a vital role in symbiosis with R.pseudoacacia.Phylogenetic analysis indicated that strains harbor two hemin degrading factors are exclusive to Mesorhizobium.hmu S_pSym of M.amorphae CCNWGS0123 is highly homologous to hmu S_ch with 79%similarity at the amino acid level.The number of infection threads and primordia were significantly decreased in?hmu S_pSym strain-infected plants compared with WT strain-infected plants.The phenotypes of the plants inoculated with the?hmu S_pSym strain displayed retarded growth with chlorotic leaves,and the height of shoots and the dry weight of aerial parts of plants were significantly shorter and lower than those of plants inoculated with the WT strain.Moreover,nodules of plants inoculated with the?hmu S_pSym strain remained small,and most were spherical in shape,whereas nodules from plants inoculated with the WT strain were elongated and displayed a cylindrical shape.The number of pink nodules and the nodule dry weight of plants inoculated with the?hmu S_pSym strain were significantly decreased.Inactivation of hmu S_pSym suppressed the nodule nitrogenase activity.However,the growth phenotypes of plants inoculated with the?hmu S_ch strain were not different to those of plants inoculated with the WT strain.3.Inactivation of hmu S_pSym disrupts plant immune homeostasis in symbiosis,and impairs the viability of differentiated bacteroids lived in plant cells.Plant defense responses were analyzed during the different stages of symbiosis.In contrast to plants inoculated with the WT strain,levels of both H₂O₂ and O₂^-were generally higher in plant roots inoculated with the?hmu S_pSym strain before nodule maturation.However,in matured nodules(4 wpi),the quantification results indicated a remarkable decrease in plants infected by the?hmu S_pSym strain,consisting with the results of ROS-staining of nodules.Moreover,the expression of NADPH oxidase genes,Rboh A and Rboh D,were significantly higher in nodules from plants infected by the WT strain than in plants infected by the?hmu S_pSym strain.These results imply an unbalanced cellular immune homeostasis in plants inoculated with the?hmu S_pSym strain.Furthermore,compared to nodules of plants infected by the WT strain,the majority of infected cells of plants inoculated with the?hmu S_pSym strain displayed collapsed peripheries,and bacteroids diffusely distributed in cells.The symbiosomes of?hmu S_pSyminoculated plants presented ruptured membranes with shrunken cytoplasm,and several symbiosomes were packaged into one membrane-like structure.In addition,Hmu S_pSym was strongly expressed in nitrogen-fixation zone of mature nodules.Unlike free-living state,rhizobia in nodules from plants infected with the?hmu S_pSym strain were differentiated into elongated bacteroids(3-5 fold),similar to those of nodules from WT strain-inoculated plants in terms of shape and size.Likewise,there was no difference in the DNA content of bacteroids extracted from those two types of nodules,indicating comparable endoreduplication of both strains.However,the surface of bacteroids extracted from plants infected by?hmu S_pSym strain displayed a collapsed and compressed morphology with shrunken cytoplasm,which was remarkably different from bacteroids isolated from plants infected by the WT strain.These results confirm that bacteroids extracted from plants infected by the?hmu S_pSym strain were necrotic.4.Hmu S_pSym is not an effector protein of type three secretion systems.Transient expression of hmu S_pSym in Nicotiana benthamiana leaves partially suppressed Bax-triggered cell death,and a predicted tts-box in the promoter region of hmu S_pSym was found,indicating Hmu S_pSym may be an effector protein.The result of flavonoids induction showed that the expression level of hmu S_pSym was significantly decreased in tts I deletion(?tts I)strain,whereas the expression of rhc J,rhc N(two structural genes of T3SS1)and nop P(a secretory effector gene of T3SS1)that served as the control was not detected.However,the secretion assay revealed that Hmu S_pSym was not detected in flavonoid-induced culture supernatants from the parent M.amorphae CCNWGS0123 strain or the three T3SS mutant strains,but largely existed in the cell pellets.Accordingly,Hmu S_pSym can not be secreted extracellularly by T3SS in vitro.5.Hmu S_pSym binds to but does not degrade heme,inactivation of Hmu S_pSym impairs the expression of genes related to oxygen sensitivity in nitrogen fixation.The expression levels of hmu S_pSym and hmu S_ch were not affected by the presence of hemin as a sole iron source,whereas hmu S_ch expression was significantly decreased in iron-replete(Fe Cl₃)conditions compared with hmu S_pSym expression.The results suggested that hmu S_ch,but not hmu S_pSym,was negatively regulated by iron concentration.The relative hemin level in nodules of plants infected by the?hmu S_pSym strain was significantly lower than in plants infected by the WT strain.Furthermore,heme binding and degradation assays were carried out for three recombinant proteins Hmu S_pSym,N-Hmu S_pSym,and Hmu S_pSym-C.Absorption spectral profiles strongly suggested that all proteins were able to bind hemin,based on typical heme-protein complex spectrographic curves with a prominent Soret peak at 412 nm,and the peak height increased with increasing hemin concentration.However,using ascorbic acid as the reductant,Soret peaks were basically unchanged,indicating that Hmu S_pSym was unable to degrade heme in vitro.In addition,subcellular localization of Hmu S_pSym revealed that the protein was present both in membranes and cytoplasm of N.benthamiana,and was strongly aggregated in plasma membranes.More importantly,hmu S_pSym mutation resulted in significantly down-regulated the expression of genes involved in oxygen sensing systems(fix L/fix J)and its downstream transcription factors(nif A,fix K)in nodule.Taken together,Hmu S_pSym of M.amorphae CCNWGS0123 may plays an essential role in symbiosis,by storing or regulating oxygen via its heme binding ability.This study enriches our understanding of the nodulation and symbiosis mechanisms of legumes,which provides theoretical reference for the establishment of the optimal symbiotic associations.