Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.     

KINETIN-INDUCED PSEUDONODULES ON TOBACCO ROOTS

Arora , Nirmal , F. Skoog , and O. N. Allen . (U. Wisconsin, Madison.) Kinetin-induced pseudonodules on tobacco roots. Amer. Jour. Bot. 46(8): 610–613. Illus. 1959.—Tobacco stem segments were cultured on modified White's medium containing 0.02–0.1 mg./l. of kinetin, 2 mg./l. of IAA and 2 g./l. of casein hydrolysate. Roots grew profusely from these stem pieces. In the presence of kinetin, especially when a second supply (0.2 mg./l.) was introduced after the roots had developed, large numbers of pseudonodules were formed along the root surfaces. The protuberances were spherical in shape and were invariably associated with the axial area of lateral rootlets derived as root primordia from parent root pericycle. Development of the pseudonodules started with the division and enlargement of cortical cells. The center of each pseudonodule contained either a simple provascular strand constituting a modified rootlet with a poorly defined apex and layers of tracheids at the base, or a normal lateral root primordium capable of continued growth and eventual emergence as a rootlet. In later stages, the swellings became lobed and the mature cells showed a high starch content. Although these kinetin-induced pseudonodules arose from cortical tissue, their anatomical structure remained undifferentiated and unspecialized and bore no resemblance to that of typical nodules caused by rhizobia.     

Cytokinin-induced Pseudonodules on Alnus glutinosa

Alder root nodules are capable of atmospheric nitrogen fixation. A differential treatment with kinetin and 2IP (2-isopentenyl adenine) was applied to the rooting medium of alder plants growing in test tubes containing Crone solution. The experiment led to the formation of a fair number of pseudonodules scattered on the root system of the plants treated with cytokinin levels higher than 0.6 μg/ml. Kinetin had a lowering effect on the overall growth of the plant, whereas the plants treated with 2IP showed no differences in growth as compared to the non-treated control plants. Histological sections of the pseudonodules show an unorganized tissue originating from the parental root cortex, and the presence of groups of tracheids giving the aspect of unorganized vascular bundles.     

根瘤菌在大麦和水稻根上形成拟瘤的细胞结构

用3种方法使紫云英根瘤菌(Rhizobium astragali Huikui)、田菁根瘤菌(R.sesbania sp.)分别入侵大麦(hordeum vulgare L.)和水稻(Oryza sativa L.),形成拟瘤状组织。一是用一定磁场强度处理根瘤菌和植物,并接种培养。二是用含有水稻幼苗根提取物的培养基培养根瘤菌,接种水稻。三是重复别人用2,4-D外源激素处理植物,接种根瘤菌。镜检观察,用紫云英根瘤菌接种形成的大麦根拟瘤细胞结构非常精细,保持各种细胞器。有侵入线结构和根瘤菌从侵入线释放。根瘤菌被宿主细胞来源的膜包围,成为拟菌体。这些形态结构与豆科根瘤细胞相似,有共生状态特征,但拟菌体有泡状化现象。田菁根瘤菌入侵水稻根形成的拟瘤,在细胞间隙和细胞内都有细菌分布。受侵染的细胞结构粗糙,根瘤菌裸露,无胞膜包围。用2,4-D处理接种根瘤菌的拟瘤细胞结构也如此,但在维管系统内有大量密集的细菌存在。这种结构完全不同于豆科根瘤细胞结构,细菌与植物细胞的形态学相互关系是一种非共生联合作用。     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Dissection of Nodule Development by Supplementation of Rhizobium leguminosarum biovar phaseoli Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 10⁵ to 10⁶ bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule.     

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.     

Transposon Mutants of Bradyrhizobium japonicum Altered in Attachment to Host Roots

Transposon mutants of Bradyrhizobium japonicum 110 ARS were produced and screened for changes in attachment ability. Mutant CFK4 produced twice as many piliated cells, attached in 2.5-fold-higher numbers to soybean root segments, and colonized roots in about 2-fold-higher numbers than did the parental strain, 110 ARS. Mutants CFK35 and CFK38 were reduced in their attachment about 2-fold and 3.5-fold, respectively. This corresponded to reductions in piliated cells in their populations, reduced reaction with anti-pilus antiserum, and reduced hydrophobic attachment. Mutants CFK4 and CFK38 nodulated soybeans at about the same level as the parent strain, but CFK35 induced only pseudonodules. Two-dimensional gel analyses of the proteins from the mutants showed relatively few changes in proteins.     

Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.     

SOME EFFECTS OF KINETIN ON THE GROWTH AND FLOWERING OF INTACT GREEN PLANTS

Wittwer , S. H., and R. R. Dedolph . (Michigan State U., E. Lansing.) Some effects of kinotin on the growth and flowering of intact green plants. Amer. Jour. Bot. 50(4): 330–336. Illus. 1963.—Dry matter accumulation of aerial parts, and heights of tomato, cucumber, and pea plants were markedly reduced when kinetin was incorporated into the culture solution root medium in concentrations ranging from 10^–5 to 10^–7 M. Concentrations which suppressed top growth (height, dry weight) generally had lesser effects on root growth and, in some instances, enhanced it. Thus top/root ratios were greatly reduced and approached unity in kinetin-treated peas and tomatoes. Flowering was inhibited in tomatoes and accelerated in peas. There were marked changes in root morphology, including the formation of pseudonodules. Kinetin had an effect which was opposite to that of gibberellin on internode elongation, root extension, top/root ratios and flowering of dwarf peas. N⁶-benzyladenine was more active than kinetin in suppressing the growth of intact green plants. The data show that kinetin can markedly alter the behavior of intact plants when absorbed by the roots from culture solutions in which the concentrations are comparable to those which are biologically active on explants.     

Analysis of Rhizobium meliloti Sym Mutants Obtained by Heat Treatment

Deletions in the pSym megaplasmid of Rhizobium meliloti were produced at a high frequency, and their lengths varied according to incubation temperature. Morphological differentiation into large and small colonies occurred after heat treatment. Small colonies elicited pseudonodules on alfalfa roots.     

Cloning and Identification of <Emphasis Type="Italic">lpsH</Emphasis>, a Novel Gene Playing a Fundamental Role in Symbiotic Nitrogen Fixation of <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis>

Mesorhizobium huakuii 7653R forms a symbiotic relationship with Astragalus sinicus to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. By screening its transposon-inserted mutant library, a mutation in the lpsH gene was found to form pseudonodules on A. sinicus. Its effect was further confirmed by double-crossover mutant HK242. DNA sequence analysis revealed that the lpsH gene encodes a deduced protein of 397 amino acids with a predicted molecular mass of 43.6 kD. LpsH contains a putative signal peptide, 11 transmembrane helices, and an O-antigen polymerase domain, which locates on the periplasmic membrane and is necessary to lipopolysaccharide synthesis. Plant studies showed that the lpsH gene mutant formed ineffective nodules, and this symbiotic phenotype was linked to abnormal nodule development.     

Relationships Amongst the Fast-growing Rhizobia of Lablab purpureus, Leucaena leucocephala, Mimosa spp., Acacia farnesiana and Sesbania grandiflora and their Affinities with Other Rhizobial Groups

Colony characteristics, growth in litmus milk, precipitation in calcium glycerophosphate medium and utilization of carbon sources of the root-nodule bacteria isolated from the tropical legumes Leucaena, Mimosa, Acacia, Sesbania and Lablab were similar to fast-growing rhizobia of temperate legumes, particularly Rhizobium meliloti. In agglutination tests, isolates from each host shared antigens with one or more of five Rhizobium strains from Leucaena. Infective characteristics of the fast-growing rhizobia were studied in modified Leonard jars and in agar culture. Cross-infections by rhizobia between these plants were common and the association often effective. Lablab was effectively nodulated by its own fast-growing isolate but only formed root swellings, possibly ineffective pseudonodules, with the other isolates. Slow-growing rhizobia which were able to nodulate Macroptilium atropurpureus were unable to form nodules on these legumes except Lablab which was considered more akin to the cowpea group. All fast-growing isolates nodulated, often effectively, Vigna unguiculata and V. unguiculata ssp. sesquipedalis. The isolate from Lablab also effectively nodulated a number of other tropical legumes which have previously only been reported to nodulate with slow-growing nodule bacteria and it also produced ineffective nodulation on Medicago sativa. This is the first record of an effective fast-growing isolate from Lablab.     

Varying the abundance of O antigen in Rhizobium etli and its effect on symbiosis with Phaseolus vulgaris

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region alpha, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region alpha elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region alpha and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.     

2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudate and is eliminated in a symbiotically defective mutant

When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395 alpha 395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395 alpha 395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps(+) strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.     

Nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis

The role of Mesorhizobium loti surface polysaccharides on the nodulation process is not yet fully understood. In this article, we describe the nodulation phenotype of mutants affected in the synthesis of lipopolysaccharide (LPS) and beta(1,2) cyclic glucan. M. loti lpsbeta2 mutant produces LPS with reduced amount of O-antigen, whereas M. loti lpsbeta1 mutant produces LPS totally devoid of O-antigen. Both genes are clustered in the chromosome. Based on amino acid sequence homology, LPS sugar composition, and enzymatic activity, we concluded that lpsbeta2 codes for an enzyme involved in the transformation of dTDP-glucose into dTDP-rhamnose, the sugar donor of rhamnose for the synthesis of O-antigen. On the other hand, lpsbeta1 codes for a glucosyltransferase involved in the biosynthesis of the O-antigen. Although LPS mutants elicited normal nodules, both show reduced competitiveness compared with the wild type. M. loti beta(1-2) cyclic glucan synthase (cgs) mutant induces white, empty, ineffective pseudonodules in Lotus tenuis. Cgs mutant induces normal root hair curling but is unable to induce the formation of infection threads. M. loti cgs mutant was more sensitive to deoxycholate and displayed motility impairment compared with the wild-type strain. This pleiotropic effect depends on calcium concentration and temperature.     

2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps⁺ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.     

A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes.

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.