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The quest for nitrogen fixation in riceProceedings of a workshop, 9-12 August 1999, Los Baños Laguna, Philippines Edited by J.K. Ladha and P.M. Reddy Diazotrophic enterobacteria: What is their role in the rhizosphere of rice? Wilfredo L. Barraquio, Edelwisa M. Segubre, Mary Ann S. Gonzalez, Subhash C. Verma, Euan K. James, Jagdish K. Ladha, and Anil K. Tripathi The search for more interesting and potentially beneficial diazotrophic enterobacterial endophytes continues to be carried out as part of the "New Frontier Project on Nitrogen Fixation in Rice." Diverse diazotrophic enterobacteria, such as Enterobacter cloacae, Erwinia herbicola/Enterobacter agglomerans (Pantoea agglomerans), Klebsiella planticola, K. oxytoca, and Serratia marcescens inhabit the rhizosphere of rice plants growing in both tropical and temperate climates. The ubiquity of enterobacteria can perhaps be attributed to their great metabolic diversity and fast growth rates. Indeed, the widespread occurrence in the rhizosphere of high populations of r-strategist bacteria, such as free-living Klebsiella and endophytic Enterobacter and Serratia species, indicates that there is sufficient availability of organic carbon in and around roots to meet their metabolic needs. The possession by enterobacters of diverse traits, such as diazotrophy in the presence and absence of O2 and tolerance for heavy metals, probably also contributes enormously to their ability to survive and grow in diverse habitats, including the rhizosphere. In addition, many enterobacters have the ability to produce gums, siderophores, indole acetic acid, and metabolites active against phytopathogens, and they may also have phosphate-solubilizing, chitinase, cellulase, and pectinase activities. The potential role of these diazotrophs in plant growth is now being increasingly understood. Retrospective on biological nitrogen fixation Robert H. Burris In 1838, Boussingault reported that leguminous plants fixed atmospheric nitrogen, but it was not until the convincing 1888 report of Hellriegel and Wilfarth that the validity of this concept was generally accepted. The biochemistry of the process was not investigated seriously until the 1930s when the influence of nitrogen and oxygen pressures and the inhibitory action of hydrogen were established. The application of 15N as a tracer in the 1940s indicated that ammonia is the key intermediate in fixation. Achievement of consistent cell-free nitrogen fixation in the 1960s substantiated the role of ammonia and showed that both an Fe protein and a MoFe protein are involved in the fixation process with energy derived from ATP. In 1992, the tertiary structure of the MoFe protein was established. Recent investigations have emphasized genetic aspects of the nitrogenase system. Xylem colonization of rice and Arabidopsis by Azorhizobium caulinodans ORS571 Edward C. Cocking Our discovery that the xylem of the roots of Sesbania rostrata is colonized when inoculated with Azorhizobium caulinodans led us to investigate whether the xylem of rice and other nonlegumes could also be colonized. If so, this might provide a nonnodular niche for endophytic symbiotic nitrogen fixation in rice and other nonlegume crops, somewhat comparable to the naturally occurring nonnodular endophytic nitrogen fixation by diazotrophs in xylem vessels and intercellular spaces that other researchers have detected in sugarcane. Our earlier studies indicated the colonization of the xylem of lateral roots of wheat inoculated repeatedly with Azorhizobium caulinodans. We also observed the crack entry of A. caulinodans and intercellular colonization of the root systems of rice, wheat, and the model nonlegume Arabidopsis thaliana, and stimulation by flavonoids such as naringenin. Recently, using the LacZ reporter gene, we have shown, for the first time, in rice inoculated with Azorhizobium caulinodans, that the xylem of roots can be colonized by azorhizobia. We have also shown, for the first time, that the xylem of the root system of the model nonlegume dicot Arabidopsis thaliana is extensively colonized, when inoculated with Azorhizobium caulinodans. Using this ability to induce xylem colonization by azorhizobia in rice and Arabidopsis, we are now investigating factors, including flavonoids, that might influence the extent of xylem colonization, and also the extent to which xylem colonization by azorhizobia will provide a niche for nonnodular symbiotic nitrogen fixation. The potential role of biological nitrogen fixation in meeting future demand for rice and fertilizer David Dawe Rice is the most important crop in Asia, accounting for nearly half of caloric intake in many countries. Future demand for rice will increase at approximately the rate of population growth in the next 20 years, which is projected to be slightly more than 1% per year in Asia. Nitrogen is a key input in rice farming, but the cost of mineral nitrogen fertilizer relative to the value of rice production is typically low in both irrigated and rainfed systems (just 4-7%). This low share is not due to a preponderance of subsidies, which are much less common today than in the past. More important factors are the sharp decline in inflation-adjusted world prices of urea over the past 40 years and the fact that labor is by far the most important cost in rice farming. The low share of N fertilizer in gross returns implies that farmers will not adopt rice varieties with enhanced nitrogen fixation capacity if there is even a small yield penalty associated with such capacity. It also implies that the environmental benefits from reducing nitrogen runoff are likely to be more important than the financial benefits for farmers due to reduced mineral N consumption. Because a large share of rice is grown under irrigated conditions, or in rainfed environments with continuous flooding, it will be important to develop biological nitrogen fixation that can operate under flooded conditions. F.B. Dazzo, Y.G. Yanni, R. Rizk, F.J. de Bruijn, J. Rademaker, A. Squartini, V. Corich, P. Mateos, E. Martínez-Molina, E. Velázquez, J.C. Biswas, R.J. Hernandez, J.K. Ladha, J. Hill, J. Weinman, B.G. Rolfe, M. Vega-Hernández, J.J. Bradford, R.I. Hollingsworth, P. Ostrom, E. Marshall, T. Jain, G. Orgambide, S. Philip-Hollingsworth, E. Triplett, K.A. Malik, J. Maya-Flores, A. Hartmann, M. Umali-Garcia, and M.L. Izaguirre-Mayoral This chapter summarizes our collaborative project to search for natural, intimate associations between rhizobia and rice (Oryza sativa), assess their impact on plant growth, and ultimately exploit those that can enhance grain yield with less dependence on nitrogen fertilizer inputs. Two cycles of field and laboratory studies have indicated that diverse indigenous populations of the clover root-nodule symbiont, Rhizobium leguminosarum bv. trifolii, intimately colonize rice roots in cultivated fields of the Egyptian Nile Delta, where rice has been rotated successfully with berseem clover (Trifolium alexandrinum) since antiquity. Certain strain/variety interactions significantly expand rice root architecture, enhance the uptake of several plant nutrients, and increase plant biomass under laboratory and greenhouse conditions. Preliminary results indicating statistically significant increases in grain yield and agronomic fertilizer N-use efficiency following inoculation have been obtained. We are now examining various basic and applied aspects of this beneficial Rhizobium-rice association, such as its ecology, physiology, biochemistry, and molecular biology and the identification of the underlying mechanisms of plant growth promotion operative in this beneficial association. We are also further assessing selected rhizobial strains to perform as beneficial biofertilizer inoculants for rice under field conditions. Certain strains of these rice-adapted rhizobia colonize the surface and, within limited regions of the interior of lateral roots of rice seedlings, secrete indoleacetic acid and gibberellin phytohormones in vitro, and extracellularly solubilize precipitated phosphates. Various acetylene reduction assays and 15N-based studies do not support a role of biological nitrogen fixation in the positive plant growth-promotion response of this Rhizobium-rice association. This natural, intimate Rhizobium-rice association represents a unique experimental system suitable for both basic and applied studies on beneficial rice-bacteria interactions. This association of dissimilar organisms living together may also turn out to offer potential benefits to enhance the sustainable agriculture of rice, the most important cereal crop of the developing world. Prospects for constructing nitrogen-fixing cereals Ray Dixon, Qi Cheng, and Anil Day The engineering of autonomous nitrogen-fixing plants is undoubtedly a long-term goal because it requires the assembly of a complex enzyme and provision of appropriate physiological conditions in the absence of the environment normally provided by the prokaryotic cell. Here we briefly review the genetic, biochemical, and physiological requirements for nitrogen fixation in the context of engineering nitrogen-fixing plants and report some of our recent findings on the expression of one of the nitrogenase structural subunits within plastids of the model photosynthetic eukaryote Chlamydomonas reinhardtii. Frontier Project on nitrogen fixation in rice: looking ahead Kenneth S. Fischer Rice is the world's most important food, particularly for the poor. The future need for rice (more than a 30% increase by 2020) to be grown with less land in the intensive rice areas, and with new technologies in the rainfed systems, demands that science explore new frontiers. IRRI has begun a set of Frontier Projects that explore the feasibility of applying new knowledge and methods to transfer novel traits from other crops to rice. One of these projects is "Nitrogen Fixation in Rice." In 1992, a think-tank workshop concluded that IRRI, working closely with advanced research laboratories, should explore the feasibility of N2 fixation in rice in a few well-defined approaches: nonnodular symbiosis, nodular symbiosis, transferring nif genes, and CO2 fixation and N-use efficiency. A BNF (biological nitrogen fixation) working group for rice was formed to monitor research progress and the free exchange of information and materials. Significant scientific progress has been made in providing new knowledge. Yet this alone is not enough to maintain the project. The Frontier Projects must be periodically assessed on how such new knowledge has furthered our probability of success in meeting the long-term goal. This chapter describes the rationale for IRRI's support of these projects with a long-term, ambitious, and problematic outcome. Exploring genetic programs for root endosymbioses Clare Gough and Jean Dénarié The study of the interaction of plants with diazotrophic bacteria has clearly shown that it is only in intercellular, endosymbiotic interactions that significant amounts of fixed nitrogen are provided to the host plant. While only a minority of plants can establish such root endosymbioses with diazotrophic bacteria, most plants are able to establish endosymbiotic associations with arbuscular endomycorrhizal fungi. Therefore, a large majority of plants, including rice, wheat, and maize, possess the genetic information required to pave the way for successful symbiotic root infection, involving the reactivation of cortical cells in the presence of a microsymbiont, the accepted penetration of the symbiont in host cells, and the synthesis of a membranous interface with the symbiotic partner to facilitate metabolic exchange. We summarize the genetic and genomic approaches used on the model legume Medicago truncatula to identify the legume genetic program controlling root endosymbioses, that is, the genes involved in nodulation, in endomycorrhizae formation, and in both types of symbioses. We propose to generate and study expressed sequence tag libraries of mycorrhized roots in rice to identify rice genes involved in endomycorrhizae formation, that is, the rice genetic program for endosymbiotic interactions. A comparison of the M. truncatula and rice programs for root endosymbioses should allow a critical assessment of the feasibility of exploiting and improving the existing symbiotic genetic program of rice to make endosymbiotic associations possible between nitrogen-fixing bacteria and this major crop. Novel nitrogen-fixing bacteria associated with the root interior of rice Thomas Hurek, Zhiyuan Tan, Natarajan Mathan, Tanja Egener, Michaela Engelhard, Prasad Gyaneshwar, Jagdish K. Ladha, and Barbara Reinhold-Hurek We have for a long time been working with Kallar grass (Leptochloa fusca (L.) Kunth), a pioneer grass grown on salt-affected, flooded low-fertility soils in the Punjab of Pakistan, which gives high yields of hay without application of N fertilizer, as a model system for interactions between diazotrophs and grasses. A new genus of proteobacteria of the beta subgroup, Azoarcus spp., occurs in high numbers in the root interior of this grass. Some members of this genus share with other diazotrophs such as Acetobacter diazotrophicus a rather novel type of interaction with the plant: as "endophytes," they proliferate within the tissue without causing symptoms of plant disease, but they do not form an endosymbiosis inside living plant cells. Azoarcus spp. show similar colonization patterns in kallar grass and-in laboratory cultures-in rice seedlings: they are capable of infecting roots, spreading inside systemically, and multiplying within the aerenchyma and the stele. Therefore, we extended our studies to rice. Our lab focused on several different aspects of interactions of diazotrophs with graminaceous plants such as rice. First, for taxonomy and diversity of rice endophytes, we screened for abundant diazotrophs in cultivated and wild rice species, and detection and identification of abundant noncultured diazotrophs in rice by DNA- and RNA-based methods. Second, we studied the regulation of nif-gene expression in Azoarcus sp.: under certain culture conditions, the bacteria are "hyperinduced," that is, they fix nitrogen more efficiently and actively. Therefore, we are now unraveling the regulatory cascade for activation of nif genes in Azoarcus sp. Third, we analyzed bacterial gene regulation in association with rice roots: a way to analyze functions of bacteria inside roots is to study which specific genes are induced in their microhabitat, the root interior. We demonstrated recently, by using a reporter gene approach, that the interior of rice roots may provide suitable conditions for the expression of nitrogenase genes by Azoarcus sp. (low concentrations of O2 and combined N), thus representing a putative niche for nitrogen fixation. Fourth, we identified new endophytes of rice: taxonomy of a novel Serratia group associated with rice, and taxonomy of novel seed-associated putative endophytes of rice. Endophytic diazotrophs associated with rice Euan K. James, Prasad Gyaneshwar, Wilfredo L. Barraquio, Natarajan Mathan, and Jagdish K. Ladha Wetland rice receives a significant proportion of its nitrogen requirements from biological nitrogen fixation (BNF). This is partly provided by free-living photosynthetic diazotrophs, especially cyanobacteria, that live in the soil and floodwater. Another significant source of fixed nitrogen are the abundant heterotrophic bacteria in the rhizosphere, particularly species of Azospirillum, Burkholderia, and Pseudomonas, and bacteria belonging to the Enterobacteriaceae. Some rice varieties can obtain more fixed nitrogen from heterotrophic BNF than others, suggesting that they have a more "intimate" association with diazotrophs. In support of this, a wide range of diazotrophs appear to live within the tissues of the plants and it has been suggested that these endophytic diazotrophs are actually responsible for much of the N2 fixation. Some of these bacteria, such as Azoarcus spp., Herbaspirillum seropedicae, Pseudomonas stutzeri A15 (formerly Alcaligenes faecalis A15), Rhizobium leguminosarum bv. trifolii, and Serratia marcescens, have been confirmed to be endophytes by using microscopy allied with marker genes and/or immunogold labeling. Endophytic diazotrophs usually live within the root apoplast, that is, the intercellular spaces and/or the xylem vessels, and may enter the plants via the root tips or epidermal cracks at lateral root junctions. Some also colonize the aerial parts of the plants, as well as the seeds. No obvious "symbiotic" organs are present, and the mechanism by which these relatively unstructured associations operate in the plant has yet to be established. The challenge that now faces research on heterotrophic BNF by rice is to find the optimum combination(s) of plant genotypes and endophytic diazotrophs, and to establish beyond a doubt that they can fix significant quantities of N2. Ivan R. Kennedy, Lily Pereg-Gerk, Rosalind Deaker, Craig Wood, Kate Gilchrist, David McFadden, and Nazrul Islam The key features of an experimental model for achieving significant biological N2 fixation by associations between Azospirillum and cereals are suggested to include adequate colonization, endophytic in nature to ensure both access to carbon substrates and suitable microaerobic oxygen conditions, and a means of ensuring adequate transfer of newly fixed nitrogen to the host plant. In our research program, we have exploited the property of synthetic plant hormones such as 2,4-dichlorophenoxyacetic acid to enhance access of azospirilla to protected niches such as the base of modified lateral roots (para-nodules) and channels between cortical cells. This approach has demonstrated the possibility of achieving such colonization. By using nifH-lacZ fusions of A. brasilense strains, we have been able to show a relationship between nifH expression, oxygen pressure, and the magnitude of acetylene reduction rates in such associations. In addition, the significance of the flcA (controlling flocculation) gene in effective colonization by regulating the expression of exopolysaccharides and the conversion of vegetative cells of azospirilla to cysts has been shown by the use of flcA- mutants. We have studied the membrane potential and permeability properties of A. brasilense Sp7-S with a view to establishing the mechanism of retaining ammonia in free-living cells and the possibility of excretion in association with plants. Using an ammonia-excreting mutant (HM53) with adequate carbon substrate supplied as malate, the transfer of substantial quantities of newly fixed 15N2 from the diazotroph to model wheat seedlings, adequate to support significant growth, has been shown. Furthermore, a Tn5-site-directed flcA- mutant of HM53 supported solely by photosynthate from wheat seedlings grown in sand culture in air was able to accumulate significantly more dry weight in the roots and shoots than the ammonia-excreting mutant alone. We suggest that this result vindicates continuing our stepwise approach to achieving a genotype × environment interaction that may eventually provide a symbiosis between Azospirillum and cereals such as wheat and rice. Hiroshi Kouchi, Ken-ichi Takane, Rolando B. So, Jagdish K. Ladha, and Pallavolu M. Reddy We have isolated the ENOD40 gene homologues ObENOD40 and OsENOD40 from the wild and cultivated rice genotypes Oryza brachyantha and O. sativa, respectively. Rice ENOD40s contain two nucleotide sequence domains, regions I and II, which are highly conserved in all legume ENOD40s. Region I of rice ENOD40s potentially encodes an oligopeptide that is similar to those found in legume ENOD40s. The expression of OsENOD40 was detected only in stem vascular bundles. Detailed in situ hybridization studies revealed that transcription of OsENOD40 is confined to parenchyma cells surrounding the protoxylem during the early stages of development of lateral vascular bundles of the stem. The expression pattern of OsENOD40 promoter-GUS fusion in nodules developed on transgenic hairy roots of soybean was also restricted to peripheral cells of nodule vascular bundles, thus presenting evidence that the regulation mechanism of ENOD40 gene expression is well conserved through legumes and the very distantly related monocot, rice. These results strongly suggest that OsENOD40 and legume ENOD40s share common, if not identical, roles in differentiation or function of vascular bundles or both. Steps toward nitrogen fixation in rice Jagdish K. Ladha and Pallavolu M. Reddy Nitrogen is the most important nutrient input required for rice production. A major goal of biological nitrogen fixation (BNF) research has been to extend the nitrogen-fixing capacity to cereal plants such as rice. If a BNF system could be assembled in the rice plant, it could increase the potential for nitrogen supply because fixed nitrogen would be available to the plant directly, with little or no loss. Such a system could also enhance resource conservation and environmental security, besides freeing farmers from the economic burden of purchasing fertilizer nitrogen for crop production. To achieve nitrogen fixation in rice, the International Rice Research Institute (IRRI) launched a global collaborative initiative, the Frontier Project on Nitrogen Fixation in Rice, about six years ago. An international working group, consisting of research scientists with diverse backgrounds and approaches and committed to reducing the dependency of rice on mineral nitrogen resources, was established to review, share research results and materials, and catalyze research in the frontier project. The strategies enabling rice to fix its own nitrogen are complex and long-term in nature. Nonetheless, in the past six years, worldwide collaborative efforts have led to remarkable progress in the areas of rice-endophytic diazotroph associations and determination of the genetic predisposition of rice for symbiosis with rhizobia. In addition, concerted efforts are also under way to elucidate the genetic, biochemical, and physiological requirements for the assembly and function of the nitrogenase enzyme complex in plant cells. In the next five to ten years, we envisage even greater progress in achieving nitrogen fixation in rice. Beneficial effects of inoculated nitrogen-fixing bacteria on rice M. Sajjad Mirza, Ghulam Rasul, Samina Mehnaz, Jagdish K. Ladha, Rolando B. So, Sikander Ali, and Kauser A. Malik Nitrogen fixation in two rice varieties-Super Basmati and Basmati 385-was studied by the acetylene reduction (AR) assay and the 15N dilution technique. In the roots and submerged shoots of field-grown plants of both varieties, a higher acetylene reduction activity and population of diazotrophs were detected at the grain-filling stage than at the panicle initiation stage. Nitrogen-fixing bacterial isolates from rice and other plants were differentiated by using ERIC- and BOX-PCR. A unique banding pattern of the PCR products was obtained from each isolate, confirming that distinct bacterial strains have been isolated. The beneficial effects of the bacterial inoculations were studied on rice plants grown in pots filled with nonsterile soil. Super Basmati showed a better response (plant biomass and grain yield) to bacterial inoculations than did Basmati 385. Maximum fixation (59%Ndfa) in plants of Super Basmati was obtained when Azospirillum lipoferum N-4 was used as inoculum, whereas Herbaspirillum RR8 showed maximum fixation (39%Ndfa) in Basmati 385. Donald A. Phillips, Esperanza Martínez-Romero, Guo-Ping Yang, and Cecillia M. Joseph A new method of screening for biological N2 fixation (BNF) was tested with bacterial isolates from alfalfa and rice. Because agronomically useful BNF requires the transfer of bacterial N to the plant, the technique assessed N released from bacteria in N-free liquid medium using thin-layer chromatography (TLC). Many natural products with high N content (22-50% N) are lipophilic, and 7% of 21 mg N L-1 released by the alfalfa endophyte Pantoea agglomerans growing in N-free liquid medium was removed from the aqueous phase by C18 resin. Riboflavin was a minor lipophilic component (< 0.1%), but it served as an easily detected indicator molecule because small amounts (10 pmol) fluoresced visibly on TLC plates. Tests with 66 putative endophytes from Chinese rice culms found that 38 formed pellicles in N-free medium, and 34 of those released riboflavin. Acetylene reduction was detected in 31 of the 34 isolates that released riboflavin. The C2H2-reducing activity and release of riboflavin were correlated significantly in the 38 pellicle-forming isolates (r2 = 0.72, P < 0.001). Detailed studies of eight isolates showed that all released riboflavin during logarithmic growth in N-free medium and also later after increases in viable cell number ceased. Several endophytic isolates that reinfected rice had little effect on plant growth in the absence of combined N, but others impaired rice growth. Thus, riboflavin is an easily detected indicator of BNF in bacterial cultures, but beneficial interactions with plants must be confirmed. Realizing the genetic predisposition of rice for symbiotic nitrogen fixation Pallavolu M. Reddy, Jagdish K. Ladha, Hiroshi Kouchi, Gary Stacey, Rowena J. Hernandez-Oane, Marilou C. Ramos, Rolando B. So, Olivyn R. Angeles, V.S. Sreevidya, R. Bradley Day, Jonathan Cohn, and Serry Koh A long-standing goal of biological nitrogen fixation research has been to extend the nitrogen-fixing rhizobial symbioses of legumes to nonnodulated cereal plants such as rice. To formulate strategies for developing rice-rhizobia symbioses, adopting a systematic approach, we initially studied the extent of predisposition of rice to form an intimate association with rhizobia. The induction of Rhizobium nod genes by plant-produced flavonoids is essential for the infection of legume roots. Our studies indicated that the roots of certain rice cultivars exude compounds that are able to induce, albeit to a low extent, transcription of the nod genes of Rhizobium sp. NGR234. Neither rhizobia nor purified Nod factors, however, could elicit root hair deformation or cortical cell divisions leading to true nodule development in rice. Rhizobia primarily invade rice roots through cracks in the epidermis and fissures created during the emergence of lateral roots. This infection process, unlike in legumes, is nod-gene independent, and does not involve the formation of infection threads. Moreover, rhizobial invasion provokes a mild defense response localized in the vicinity of the colonization site. During legume nodulation, specific plant genes, known as early nodulin (ENOD) genes, are induced and are required for normal development of the nodule. We found that several legume ENOD genes hybridized to DNA from a variety of rice genotypes. This work was subsequently confirmed by the isolation of rice cDNAs showing considerable sequence homology to legume ENOD93 and ENOD40. Legume nodulation also requires the ability to respond to the rhizobial Nod signal, and the consequent signal-induced expression of the ENOD genes. Studies revealed that rhizobial Nod factors can induce the expression of legume ENOD12 promoter in rice, thus demonstrating that rice has a mechanism to perceive Nod factors and possesses a signal transduction chain that links such recognition to ENOD gene transcription. In addition, similar to the situation in legumes, the expression of the legume ENOD40 promoter is induced only in the vascular tissues in rice. Likewise, the expression of rice ENOD40 in soybean nodules is also restricted to the vascular bundles. These findings clearly suggest that legume and rice ENOD40s share a similar regulatory mechanism(s). Research makes it evident that rice possesses some developmental subprograms in its genome, which are similar to those that lead to the development of symbioses in legumes. It is therefore essential that studies be extended at the cellular and molecular levels to identify why symbiotic responses do not occur fully in rice in order to contemplate genetically engineering this major cereal crop to form a more intimate endosymbiotic association with rhizobia. Rhizobium nodulation and interaction with legumes and nonlegumes Barry G. Rolfe, Ulrike Mathesius, Joko Prayitno, Francine Perrine, Jeremy J. Weinman, John Stefaniak, Michael Djordjevic, Nelson Guerreiro, and Frank B. Dazzo Our research program has been investigating the signal exchanges that occur during the early stages of Rhizobium infection of clovers and the nonlegumes Parasponia and rice. We propose that, early during clover infection, the Rhizobium lipochitin-oligosaccharides cause the induction of chalcone synthase, the first enzyme of the flavonoid pathway in the inner cortical cells of the root. This is then followed by an alteration of auxin flow. Subsequently, the inner cortical cells accumulate specific flavonoids, followed by auxin accumulation in those cells that then divide to form a nodule primordium. In Bradyrhizobium infection of Parasponia, fluorescent compounds, whose properties are consistent with those of flavonoids, accumulate in dividing cells during nodule formation. Thus, flavonoids may not be the unique feature of legumes that enables legumes to form symbioses with Rhizobium. The rice-Rhizobium leguminosarum bv. trifolii interaction was studied by several approaches: first, by analyzing rice plants and showing that they produce various chemical signals, including flavonoids, that might interact with rhizobia; second, by investigating the effects of bacterial inoculation on rice seedling growth and finding that some Rhizobium strains inhibit while others can stimulate rice growth; and, third, by using proteome analysis to investigate the molecular genetic basis of Rhizobium strain ANU843 inhibition of rice plants. The possibility of establishing a more effective Rhizobium-nonlegume interaction is potentially available in rice because some of the phenylpropanoid pathway compounds that could interact with Rhizobium are also present in nonlegume roots. Colonization of rice and other cereals by Acetobacter diazotrophicus, an endophyte of sugarcane Myrna Sevilla and Christina Kennedy Our recent work established that Acetobacter diazotrophicus can colonize, fix N2 within, and promote growth of its natural host plant, sugarcane, after inoculation of sterile plantlets. In the new work presented here, the potential of A. diazotrophicus to colonize and promote the growth of rice, maize, and wheat was investigated using Nif + and Nif - strains tagged with the uidA gene. Three wide host range plasmids each expressing a different marker gene from a constitutive promoter were constructed for this work. While the uidA gene product GUS was detectable in A. diazotrophicus, the fluorescent products of the other two, cobA and gfp, were not. A. diazotrophicus was able to colonize maize, rice, and wheat, but the colonization was apparently restricted to root tissues. Differences in the colonization patterns and persistence of A. diazotrophicus inside these plants were also observed. Inoculation with A. diazotrophicus did not enhance the growth of wheat seedlings. Rice seedlings inoculated with the wild-type strain grew to be significantly taller 30 days after inoculation than plants inoculated with the Nif - mutant or uninoculated plants under N-deficient conditions. When N was not limiting, there were no differences between inoculated and uninoculated plants, indicating that, unlike in sugarcane, A. diazotrophicus may not benefit plant growth in the presence of sufficient N. Chitin perception in legumes and rice: what distinguishes a nodulating plant? Gary Stacey, R. Bradley Day, Pallavolu M. Reddy, Jonathan Cohn, Serry Koh, Mitsuo Okada, Yuki Ito, Naoto Shibuya, and Jagdish K. Ladha We are interested in using Rhizobium-legume symbiosis as a model for developing a comparable nitrogen-fixing symbiosis in rice. Our method is to first identify recognition steps critical to the development of the symbiosis between rhizobia and legumes. Once these steps are identified, we can then ask whether rice possesses the same ability as the legume. If we can identify traits that are unique to legume nodulation, we can develop a rational approach for creating a rice plant with the ability to form a symbiosis with rhizobia. This comparative approach has the added benefit of identifying those unique features of legumes that permit a nitrogen-fixing symbiosis. Nod signals are substituted lipo-chitin molecules produced by rhizobial nod gene products and are essential for nodulation. Previous work suggested that Nod signal recognition involves at least two recognition events, perhaps mediated by two different receptors. Our work in soybean suggests that one receptor can recognize the Nod signal but also responds to simple chitin oligomers. The second receptor appears to require the specific chemical structure of the Nod signal. Binding studies with 125I-labeled chitin oligomers identified a chitin-binding site on the plasma membrane of soybean and rice that shows similar specificity and binding parameters. Binding to this "receptor" correlates with the activation of plant defense responses (e.g., oxidative burst) in both soybean and rice. We have also isolated cDNA clones from a variety of legumes and rice whose sequence is similar to an apyrase recently shown to be a Nod signal-binding protein in Dolichos biflorus. The presence of this protein in rice could explain the ability of rice to recognize Nod signals, as evidenced by the induction of ENOD12-GUS fusion. Jon R. Stoltzfus and Frans J. de Bruijn Information about the nitrogen-fixing potential, diversity, and sites of colonization of endophytic bacteria from rice is needed to expand our understanding of microbial ecology and plant-microbe interactions in general and more specifically to lay the foundations for future studies aimed at using biologically fixed nitrogen to replace nitrogen fertilizers. Therefore, a collection of 142 bacteria isolated from mechanically abraded, surface-sterilized rice roots was studied. Polymerase chain reaction (PCR)-mediated gene amplification using degenerate primers derived from highly conserved regions of the nitrogenase nifD gene revealed 20 isolates harboring nifD gene sequences. Southern hybridization analysis confirmed the presence of nif genes in 19 of these isolates. The diazotrophic nature of these 19 isolates was confirmed using acetylene reduction assays (ARA). Examination of genetic diversity using amplified ribosomal DNA restriction analysis (ARDRA) and rep-PCR genomic fingerprinting in combination with computer-assisted pattern analysis revealed 56 unique ARDRA and 71 unique rep-PCR genomic fingerprints. Clusters of similar combined fingerprints consisting of 37, 15, 12, and 9 nondiazotrophic bacteria, as well as two clusters each containing 4 diazotrophic bacteria, were found. Analysis of partial small subunit (SSU) ribosomal RNA (rRNA) gene sequences revealed the presence of isolates with similarity to strains from the alpha, beta, and gamma subdivisions of the Proteobacteria and to members of the Bacillaceae and Microbacteriaceae. Many of the ARDRA fingerprints and/or SSU rRNA gene sequences of these bacteria were highly similar to those of other bacteria previously isolated from the rhizosphere of rice. Two isolates from the collection and Sinorhizobium meliloti, a control, were tagged with the biomarker gus or gfp, and the recolonization of rice tissue was examined. Visualization in situ of colonization of 3-wk-old inoculated rice seedlings revealed no significant endophytic colonization of rice tissue by these bacteria. Clumps of bacteria, as well as individual cells, however, could be visualized on the surface of the roots. On very rare occasions, an isolated epidermal cell filled with bacteria was observed. |
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Diazotrophic enterobacteria: What is their role in the rhizosphere of rice? Retrospective on biological nitrogen fixation Xylem colonization of rice and Arabidopsis by Azorhizobium caulinodans ORS571 The potential role of biological nitrogen fixation in meeting future demand for rice and fertilizer Prospects for constructing nitrogen-fixing cereals Frontier Project on nitrogen fixation in rice: looking ahead Exploring genetic programs for root endosymbioses Novel nitrogen-fixing bacteria associated with the root interior of rice Endophytic diazotrophs associated with rice Steps toward nitrogen fixation in rice Beneficial effects of inoculated nitrogen-fixing bacteria on rice Realizing the genetic predisposition of rice for symbiotic nitrogen fixation Rhizobium nodulation and interaction with legumes and nonlegumes Colonization of rice and other cereals by Acetobacter diazotrophicus, an endophyte of sugarcane Chitin perception in legumes and rice: what distinguishes a nodulating plant? |
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