Zhi-Ping Xie

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10–9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.

Symbiosis-Promoting and Deleterious Effects of NopT, a Novel Type 3 Effector of Rhizobium sp. Strain NGR234

Establishment of symbiosis between certain host plants and nitrogen-fixing bacteria (rhizobia) depends on type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS). Here, we report that the open reading frame y4zC of strain NGR234 encodes a novel rhizobial type 3 effector, termed NopT (for nodulation outer protein T). Analysis of secreted proteins from NGR234 and T3SS mutants revealed that NopT is secreted via the T3SS. NopT possessed autoproteolytic activity when expressed in Escherichia coli or human HEK 293T cells. The processed NopT exposed a glycine (G50) to the N terminus, which is predicted to be myristoylated in eukaryotic cells. NopT with a point mutation at position C93, H205, or D220 (catalytic triad) showed strongly reduced autoproteolytic activity, indicating that NopT is a functional protease of the YopT-AvrPphB effector family. When transiently expressed in tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. Arabidopsis plants transformed with nopT showed chlorotic and necrotic symptoms, indicating a cytotoxic effect. Inoculation experiments with mutant derivatives of NGR234 indicated that NopT affected nodulation either positively (Phaseolus vulgaris cv. Yudou No. 1; Tephrosia vogelii) or negatively (Crotalaria juncea). We suggest that NopT-related polymorphism may be involved in evolutionary adaptation of NGR234 to particular host legumes.

Exo-Oligosaccharides of Rhizobium sp. Strain NGR234 Are Required for Symbiosis with Various Legumes

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

A gene encoding a receptor-like protein kinase in the roots of common bean is differentially regulated in response to pathogens, symbionts and nodulation factors

Abstract Using mRNA differential display for healthy or Fusarium -infected roots of common bean ( Phaseolus vulgaris L. cv. Saxa), we have isolated a bean cDNA encoding a putative receptor-like protein kinase (RLK), designated PvRK20-1 , that is induced during pathogen attack. The catalytic domain of the deduced polypeptide is highly homologous to protein kinases whereas the extracellular domain shares no similarity to any known RLK, suggesting that PvRK20-1 might represent a new class of RLK. PvRK20-1 mRNA accumulated rapidly and transiently in the roots for 3–6 h upon handling and treatment, probably in response to wounding. Subsequently, transcript levels returned to background levels in control roots but started to accumulate again to high levels between 9 and 24 h in roots infected with the virulent pathogen Fusarium solani f. sp. phaseoli . The transcript remained at control levels or was suppressed upon infection with the mutualistic symbionts tested, the arbuscular mycorrhizal fungus Glomus mosseae and the nodule-forming bacterium Rhizobium tropici . A similar expression pattern was observed when the roots were challenged with a nodB mutant strain of R. tropici, unable to nodulate common bean. Surprisingly, PvRK20-1 mRNA accumulation increased in roots treated with purified Nod factors from R. tropici, whereas Nod factors from the non-host strain Rhizobium sp. NGR234 had only little effect. The expression of PVRK20-1 at early stages of plant–microbe interactions is consistent with PvRK20-1 playing a role in the differentiation between mutualistic and antagonistic symbionts.

Ethylene Responsiveness of Soybean Cultivars Characterized by Leaf Senescence, Chitinase Induction and Nodulation

Summary A total of 161 cultivars of soybean (Glycine max L. Mere.) were tested for their responsiveness to ethylxad ene treatments, using senescence of the primary leaves and the induction of chitinase activity in the roots as response markers. Cultivar «Gong jiao 6308-1» showed rapid chlorosis and the highest chitinase inducxad tion upon treatments with ethylene. The inducibility of chitinase by ethylene increased with increasing age of the plant. In addition, it was found that upon repeated ethylene treatments nodule formation of cultixad var «Gong jiao 6308-1» was completely blocked in the lower part of the root system when inoculated with Bradyrhizobium japonicum. In several other cultivars, e.g. in «Bai tie jia qing», ethylene treatments did not induce leaf senescence or induction of chitinase activity. Cultivar «Bai tie jia qing» also showed completely normal nodulation even after repeated ethylene treatments. These results demonstrate that different soyxad bean cultivars show a wide variation and strongly differ in their ethylene responsiveness.

Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234 SYMBIOTIC EFFECTS, PHOSPHORYLATION, AND INTERFERENCE WITH MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING

Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) directly into eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation in the symbiosis with host legumes. Nodulation outer protein L (NopL) of Rhizobium sp. strain NGR234 is a Rhizobium-specific type 3 effector. Nodulation tests and microscopic analysis showed that distinct necrotic areas were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by strain NGRΩnopL (NGR234 mutated in nopL), indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of nopL in yeast disrupted the mating pheromone (α-factor) response pathway, whereas nopL expression in N. tabacum suppressed cell death induced either by overexpression of the MAPK gene SIPK (salicylic acid-induced protein kinase) or by SIPKDD (mutation in the TXY motif resulting in constitutive MAPK activity). These data indicate that NopL impaired function of MAPK proteins or MAPK substrates. Furthermore, we demonstrate that NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro pattern, a typical motif in MAPK substrates. Taken together, data suggest that NopL mimics a MAPK substrate and that NopL suppresses premature nodule senescence by impairing MAPK signaling in host cells.

Effects of Nitrate on Accumulation of Trehalose and other Carbohydrates and on Trehalase Activity in Soybean Root Nodules

Summary Soybean ( Glycine max cv. Maple Arrow) plants were infected with Bradyrhizobium japonicum (strain 61-A-101), grown in sterilized Leonard jars, exposed to various amounts of nitrate either from the beginning or after completion of nodulation. The presence of 5 mM and more nitrate during nodulation caused a considerable reduction of the number and biomass of nodules per plant, of nitrogenase activity per nodule fresh weight. The carbohydrate content of nodules was determined on a dry weight basis. The level of the disaccharide trehalose, produced by the microsymbiont, was 50% lower in nodules formed in the presence of 20 mM nitrate than in control nodules formed in its absence. With regard to the non-structural carbohydrates produced by the plant, nodules formed in the presence of high amounts of nitrate contained about 75 % less starch but three- to fourfold higher levels of sucrose and pinitol than control nodules. Sucrose was the most abundant non-structural carbohydrate in nodules formed in the presence of 20 mM nitrate, accounting for 4–5 % of the dry weight. When plants with fully established nodules, grown in the absence of nitrate, were shifted to 20 mM nitrate, the levels of trehalose and starch decreased over a period of 3 weeks while the level of sucrose increased, until the carbohydrate levels attained similar values as found in nodules established in the presence of nitrate. The activity of trehalase, an enzyme known to be induced in nodules, was about 75% lower in nodules formed in the presence of nitrate than in control nodules. However, trehalase activity did not change in established nodules during a 3-week exposure to 20 mM nitrate. Similarly, the number of colony- forming bacteria recovered from the nodules and the activities of endochitinase and endoglucanase, two plant defense hydrolases, were not affected during a 3-week exposure to nitrate.

Accumulation of soluble carbohydrates, trehalase and sucrose synthase in effective (Fix+) and ineffective (Fix–) nodules of soybean cultivars that differentially nodulate with Bradyrhizobium japonicum

Roots of soybeans have the ability to form symbioses with nitrogen-fixing rhizobial bacteria to form nitrogen-fixing (Fix+) nodules, thus allowing the plant to grow in the absence of mineral nitrogen. Several soybean cultivars from China nodulated normally with Bradyrhizobium japonicum USDA110 spc4, but developed only a few nodules with 61-A-101, another B. japonicum strain. When soybeans were infected with Rhizobium sp. NGR234, ineffective (Fix-) nodules that do not fix nitrogen were formed. Plants infected with NGRΩnodD2, a mutant strain overproducing lipo-chitooligosaccharidic nodulation signals (Nod factors), showed significantly higher numbers of ineffective nodules. Nodules from the different plant-microsymbiont combinations were characterized with respect to their accumulation of soluble carbohydrates and their induction of trehalase and sucrose synthase. These two plant enzymes are known to be nodule-stimulated proteins. Pool sizes of soluble carbohydrates in nodules showed strain-specific alterations in sucrose and trehalose, whereas myo-inositol and pinitol were affected in a more cultivar-specific way. Immunoblots with nodulin-specific antiserum indicated that sucrose synthase is induced in Fix+ nodules, but undetectable in Fix- nodules, indicating a strain-specific induction profile. Trehalase activity in nodules showed a similar strain-specific induction profile. High enzyme activity was measured for nodules harboring the Bradyrhizobium strains, whereas ineffective nodules containing NGR234 exhibited activities in the range of uninfected roots. Nodules induced by NGRΩnodD2 showed increased trehalase activity. A similar induction of trehalase was observed when uninfected roots were treated with Nod factors purified from NGR234. The data obtained are discussed in the context of carbohydrate allocation in nodules and the question of how rhizobial bacteria influence the carbohydrate metabolism of their host plant is addressed.