Probing the Sinorhizobium meliloti-alfalfa symbiosis using temperature-sensitive and impaired-function citrate synthase mutants

To study the role of the decarboxylating leg of the bacterial TCA cycle in symbiotic nitrogen fixation, we used DNA shuffling and localized random polymerase chain reaction mutagenesis to construct a series of temperature-sensitive and impaired-function mutants in the Sinorhizobium meliloti Rm104A14 citrate synthase (gltA) gene. Reducing citrate synthase (CS) activity by mutation led to a corresponding decrease in the free-living growth rate; however, alfalfa plants formed fully effective nodules when infected with mutants having CS activities as low as 7% of the wild-type strain. Mutants with approximately 3% of normal CS activity formed nodules with lower nitrogenase activity and a mutant with less than 0.5% of normal CS activity formed Fix- nodules. Two temperature-sensitive (ts) mutants grew at a permissive temperature (25 degrees C) with 3% of wild-type CS activities but were unable to grow on minimal medium at 30 degrees C. Alfalfa plants that were inoculated with the ts mutants and grown with a root temperature of 20 degrees C formed functional nodules with nitrogenase activities approximately 20% of the wild type. When the roots of plants infected with the ts mutants were transferred to 30 degrees C, the nodules lost the ability to fix nitrogen over several days. Microscopic examination of these nodules revealed the loss of bacteroids and senescence, indicating that CS activity was essential for nodule maintenance.     

华癸根瘤菌外膜孔蛋白编码基因MCHK_1326突变株的构建与共生固氮表型分析

【目的】Mesorhizobium huakuii 7653R的MCHK_1326基因编码一种外膜孔蛋白,可能参与根瘤菌侵染宿主植物以及结瘤固氮过程,本研究旨在探索该基因在共生固氮中的功能。【方法】生物信息学分析MCHK_1326蛋白的结构特征及生物学功能,启动子原位表达技术检测MCHK_1326共生时空表达特征,利用Cre-loxp系统构建MCHK_1326缺失突变株,考察其共生固氮表型及早期侵染事件,通过植物盆栽并额外添加无机氮源,检测突变株接种紫云英后的共生固氮表型变化。【结果】MCHK_1326基因在侵染早期如侵染线的延伸等过程中表达,在成熟根瘤的侵染区表达,与野生型相比,突变体△1326侵染线和根瘤原基数量显著减少;植株地上部分鲜重与固氮酶活性极显著降低,根瘤数量和根瘤重量显著降低;额外添加无机氮源能恢复其共生缺陷表型。【结论】MCHK_1326基因参与根瘤菌早期侵染和结瘤,在根瘤发育与共生固氮过程中发挥作用。     

Photorespiratory metabolism and nodule function: behavior of Lotus japonicus mutants deficient in plastid glutamine synthetase

Two photorespiratory mutants of Lotus japonicus deficient in plastid glutamine synthetase (GS(2)) were examined for their capacity to establish symbiotic association with Mesorhizobium loti bacteria. Biosynthetic glutamine synthetase (GS) activity was reduced by around 40% in crude nodule extracts from mutant plants as compared with the wild type (WT). Western blot analysis further confirmed the lack of GS(2) polypeptide in mutant nodules. The decrease in GS activity affected the nodular carbon metabolism under high CO(2) (suppressed photorespiration) conditions, although mutant plants were able to form nodules and fix atmospheric nitrogen. However, when WT and mutant plants were transferred to an ordinary air atmosphere (photorespiratory active conditions) the nodulation process and nitrogen fixation were substantially affected, particularly in mutant plants. The number and fresh weight of mutant nodules as well as acetylene reduction activity showed a strong inhibition compared with WT plants. Optical microscopy studies from mutant plant nodules revealed the anticipated senescence phenotype linked to an important reduction in starch and sucrose levels. These results show that, in Lotus japonicus, photorespiration and, particularly, GS(2) deficiency result in profound limitations in carbon metabolism that affect the nodulation process and nitrogen fixation.     

Enhanced Nodulation and Nitrogen Fixation by a Revertant of a Nodulation-Defective Bradyrhizobium japonicum Tryptophan Auxotroph

In greenhouse studies, the symbiotic properties of a prototrophic revertant (TA11 NOD⁺) of a nodulation defective tryptophan auxotroph of Bradyrhizobium japonicum were compared with those of the normally nodulating wild-type strain, B. japonicum I-110 ARS. Strain I-110 ARS was the parent of auxotrophic mutant TA11. Plants inoculated with TA11 NOD⁺ contained significantly more nitrogen per plant than did plants inoculated with wild-type bacteria (275.9 ± 35 versus 184 ± 18 mg). Also, plants that received the revertant were larger, averaging 8.4 ± 0.9 g (dry weight) versus 6.4 ± 0.6 g for those that received the wild-type bacterial strain. Additionally, plants that received the NOD⁺ strain had 56% more nodules and 41% more nodule mass than did control plants. With both inocula, average nodule size and amount of nitrogen fixed per gram of nodule were about the same. These data indicated that the improvement in nitrogen fixation observed with the TA11 NOD⁺ resulted from an increase in the overall nodule number. The physiological basis for this increase in nodulation is not known, but enhanced tryptophan catabolism does not appear to be involved.     

GuaB activity is required in Rhizobium tropici during the early stages of nodulation of determinate nodules but is dispensable for the Sinorhizobium meliloti-alfalfa symbiotic interaction

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.     

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.     

Suppression of the Fix- phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K polysaccharide.

The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range. Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix- phenotype and results in a modified expression of the K polysaccharide. Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ+ differed from that of Rm6903 in molecular weight range. Importantly, the effect of LpsZ is not structurally specific, as the introduction lpsZ+ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain. This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.     

The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis.

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules. We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.     

The influence of lipopolysaccharides and glucans from two Rhizobium leguminosarum bv. viciae strains on the formation and efficiency of their symbioses with pea plants

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation experiments. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix+, efficient, moderately virulent, moderately competitive), and strain 248b (fix-, inefficient, highly virulent, highly competitive). Preparations of LPS-glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10-26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS-glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18-23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36-56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation-proficient but less virulent strain 31.     

Effect of nodules on dehydration response in alfalfa (Medicago sativa L.)

Drought is a key abiotic stress that negatively affects growth and development as well as symbiotic nitrogen fixation in alfalfa (Medicago sativa L.). To understand whether nodulation would affect drought stress response in alfalfa, we analyzed the lipid peroxidation, activities of antioxidant enzymes including superoxide dismutase (SOD), and catalase (CAT), contents of superoxide anion radical, non-enzymatic antioxidants including reduced glutathione (GSH) and proline, total protein, and soluble sugar in dehydration-stressed alfalfa. Three-month-old alfalfa plants without nodule, with active nodules, or with inactive nodules were dehydrated for 0, 1, 2, 4, 6, 8, and 10 h. We found that roots and leaves from plants with nodules, especially with active nodules, showed less lipid peroxidation which was associated with higher CAT activities and higher levels of GSH. Roots and leaves with active nodules also accumulated less free proline and soluble sugar compared to plants without nodules, suggesting that proline and soluble sugar may have a limited role in osmotic adjustment in these plants. The results suggested that active nodules may have a positive effect on drought stress tolerance in alfalfa.     

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.     

Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.     

豆科植物共生结瘤的分子基础和调控研究进展

豆科植物与根瘤菌共生互作的结果导致了一个新的植物器官――根瘤的形成, 根瘤菌生活在根瘤中, 它们具有将氮气转化为能被植物同化的氨的能力。该文阐述了根瘤的形成过程和类型, 并主要以模式豆科植物蒺藜苜蓿(Medicago truncatula)和日本百脉根(Lotus japonicus)为例, 对近年来共生结瘤过程中宿主植物对根瘤菌结瘤因子的识别和信号传递、侵入线形成和固氮的分子基础, 以及宿主植物对根瘤形成的自主调控机制、环境中氮素营养对结瘤的影响研究进行了综述, 指出当前豆科植物与根瘤菌共生互作研究存在的问题, 并对今后的研究方向作了分析与展望。     

Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.)

Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.     

Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress

Common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 formed a more active symbiosis than did its decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12 and HB13). The mutants formed partially effective (HB10, HB12) or almost ineffective (HB8, HB13) nodules (Fix(d)) under non-saline conditions. The DST mutant formed nodules that accumulated more proline than did the wild-type nodules, while soluble sugars were accumulated mainly in ineffective nodules. Under salt stress, plant growth, nitrogen fixation, and the activities of the antioxidant defense enzymes of nodules were affected in all symbioses tested. Overall, mutant nodules showed lower antioxidant enzyme activities than wild-type nodules. Levels of nodule catalase appeared to correlate with symbiotic nitrogen-fixing efficiency. Superoxide dismutase and dehydroascorbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules to salinity.