Steven G. Pueppke

Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O-acetylated or sulphated

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non‐legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod‐factors which are N‐acylated chitin pentamers carrying a variety of substituents. The terminal non‐reducing glucosamine is N‐acylated with vaccenic or palmitic acids, is N‐methylated, and carries varying numbers of carbamoyl groups. The reducing N‐acetyl‐glucosamine residue is substituted on position 6 with 2‐O‐methyl‐L‐fucose which may be acetylated or sulphated or non‐substituted. All three internal residues are N‐acetylated. At pico‐ to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non‐sulphated lipo‐oligosaccharide signals.

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo‐oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis‐specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR‐factors to signal‐production‐deficient mutants of the broad‐host‐range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 −7 M and 10−6 M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non‐host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild‐type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257

Rhizobium fredii strain USDA257 produces nitrogen‐fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposonmutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain U5DA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolWand nolX are of the opposite polarity. The initiation codon for nolW lies 155bp upstream from that of nolB, and it is separated from nolXby 281 bp. The predicted NolT and NolW proteins have putative membrane‐spanning regions. The N‐terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site‐directed mutagenesis with mudll1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU anti nolX is induced as much as 30‐fold by flavonoid signal molecules, even though these genes lack nod‐box promoters. Histochemical staining of McCall roots inoculated with nolB–, nolU–, or nolX–lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU–ORF4–nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad‐host‐range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

The Genetic and Biochemical Basis for Nodulation of Legumes by Rhizobia

Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium are collectively termed rhizobia. They share the ability to penetrate legume roots and elicit morphological responses that lead to the appearance of nodules. Bacteria within these symbiotic structures fix atmosphere nitrogen and thus are of immense ecological and agricultural significance. Although modern genetic analysis of rhizobia began less than 20 years ago, dozens of nodulation genes have now been identified, some in multiple species of rhizobia. These genetic advances have led to the discovery of a host surveillance system encoded by nodD and to the identification of Nod factor signals. These derivatives of oligochitin are synthesized by the protein products of nodABC, nodFE, NodPQ, and other nodulation genes; they provoke symbiotic responses on the part of the host and have generated immense interest in recent years. The symbiotic functions of other nodulation genes are nonetheless uncertain, and there remain significant gaps in our knowledge of several large groups of rhizobia with interesting biological properties. This review focuses on the nodulation genes of rhizobia, with particular emphasis on the concept of biological specificity of symbiosis with legume host plants.

Soybean Lines Lacking the 120,000-Dalton Seed Lectin

Seeds of 102 lines of Glycine max (L.) Merr., the soybean, were screened quantitatively for the presence of the 120,000-dalton soybean lectin. Wide variation in the content of this lectin was noted, and five lines of soybean whose seed totally lacked the lectin were identified. Roots of all five lines were effectively nodulated by several strains of Rhizobium japonicum, thus indicating that the 120,000-dalton soybean seed lectin is probably not required for the initiation of soybean-Rhizobium symbiosis.

Characterization and localization of rice (Oryza sativa L.) seed globulins

Abstract Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of the globulin fraction of rice endosperm reveals two abundant proteins with apparent molecular weights of 16 kDa and 25 kDa. These globulins were purified from preparative SDS-PAGE gels, and polyclonal antibodies were raised in rabbits. Poly(A) + RNA isolated from developing rice grains, translated in vitro in a rabbit reticulocyte lysate, directed the synthesis of five major translation products of 53 kDa, 27 kDa, 18 kDa, 16 kDa and 12 kDa. Incubation of the total translation products with the 16 kDa globulin antiserum immunoprecipitated an 18 kDa protein, while the 25-kDa globulin antibodies specifically immunoprecipitated the 27-kDa polypeptide. Western blot analyses indicated that 16-kDa globulin antiserum cross-reacted with similarly sized proteins from several cereals, while the 25-kDa globulin antiserum detected no proteins from other cereals. Electron microscopic observation of thin sections of rice endosperm tissue stained with uranyl acetate and lead citrate revealed a complex internal organization of the glutelin protein bodies. Rice endosperm contains two morphologically distinct, spherical and irregularly shaped protein bodies. Double-label immunogold electron-microscopic localization indicated that both the 25-kDa and 16-kDa rice globulins are localized in discrete zones within the irregularly shaped protein bodies.

Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp.

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod‐box promoter from NGR234 are sufficient to confer this extended host‐range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod‐box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5′‐end of nodS of USDA257. Consequently, the deduced NodS protein of USDA2S7 is shorter than its counterpart from NGR234, and the distance between the nod‐box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod‐box and extends into the upstream leader sequence. Transcriptional fusions with both nod‐boxes confirmed that the promoter from NGR234 is flavonoid‐inducible, and that the nod‐box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS‐containing Xhol fragment as hybridization probe. Flavonoid‐induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRl fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives ofBradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild‐type, leucaena‐nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.

Early Detection and Mitigation of Resistance to Bt Maize by Western Corn Rootworm (Coleoptera: Chrysomelidae)

Abstract Transgenic Bt maize that produces less than a high-dose has been widely adopted and presents considerable insect resistance management (IRM) challenges. Western corn rootworm, Diabrotica virgifera virgifera LeConte, has rapidly evolved resistance to Bt maize in the field, leading to local loss of efficacy for some corn rootworm Bt maize events. Documenting and responding to this resistance has been complicated by a lack of rapid diagnostic bioassays and by regulatory triggers that hinder timely and effective management responses. These failures are of great concern to the scientific and agricultural community. Specific challenges posed by western corn rootworm resistance to Bt maize, and more general concerns around Bt crops that produce less than a high-dose of Bt toxin, have caused uncertainty around current IRM protocols. More than 15 years of experience with IRM has shown that high-dose and refuge-based IRM is not applicable to Bt crops that produce less than a high-dose. Adaptive IRM approaches and pro-active, integrated IRM-pest management strategies are needed and should be in place before release of new technologies that produce less than a high-dose. We suggest changes in IRM strategies to preserve the utility of corn rootworm Bt maize by 1) targeting local resistance management earlier in the sequence of responses to resistance and 2) developing area-wide criteria to address widespread economic losses. We also favor consideration of policies and programs to counteract economic forces that are contributing to rapid resistance evolution.

Elaboration of flavonoid-induced proteins by the nitrogen-fixing soybean symbiont Rhizobium fredii is regulated by both nodD1 and nodD2, and is dependent on the cultivar-specificity locus, nolXWBTUV

Genistein and other flavonoids from host legumes are known to stimulate cells of the nitrogen-fixing soybean symbiont Rhizobium fredii to synthesize Nod factors, which function as signals during nodule initiation. Flavonoids also trigger R. fredii to secrete a set of signal-responsive (SR) proteins into the environment. By insertion mutagenesis, we showed that secretion of SR proteins by this organism has an absolute dependence on the regulatory gene nodD1. We isolated and sequenced nodD1 and nodD2 of R. fredii USDA257 and constructed strains containing additional, plasmid-borne copies of these genes. Extra copies of nodD1 had no effect on secretion of SR proteins, but extra copies of nodD2 rendered the process constitutive. Extracts from seeds of the soybean cultivars McCall and Peking can substitute for purified flavonoids as inducers of SR proteins. The nolXWBTUV locus is known to control cultivar-specific nodulation of McCall soybean in a negative, flavonoid-dependent manner. Inactivation of any of these genes prevented SR proteins from accumulating in culture fluids. Protein secretion in response to host signals was a characteristic of nine out of ten R. fredii strains tested. Immunological probes failed to detect SR3 or SR5 in mature soybean or cowpea nodules. Although the functions of these proteins remain unknown, their potential role in symbiosis is strengthened by the discovery that their accumulation depends on nodD1, nodD2 and nolXWBTUV.