Sharon R. Long

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.

Calcium Spiking in Plant Root Hairs Responding to Rhizobium Nodulation Signals

SUMMARY Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development. The basis for signal transduction in the plant is not known. We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes. Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application. Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking. A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.

Rhizobium-legume nodulation: Life together in the underground

How good and how pleasant it is for organisms to dwell together in unity! And, probably, how common it is in the biological world. This review concerns a dramatic association, one of the few that has been studied in detail: the nitrogen fixing symbiosis between certain plants and microbes Rhizobium bacteria stimulate leguminous plants to develop root nodules, which the bacteria infect and inhabit. Ultimately, the two organisms establish metabolic cooperation: the bacteria reduce (fix) molecular nitrogen into ammonia, which they export to the plant for assimilation; the plant reduces carbon dioxide into sugars during photosynthesis and translocates these to the root where the bacteria use them as fuel. The plant family Leguminosae (Fabaceae) is the third largest family in the Angiosperms, spreads from the tropics to arctic regions, and includes forms varying from annual herbs to large trees. It doubtlessly owes at least some of this diversity and success to its ability to grow independently of often scarce soil nitrogen. Only one non-legume plant, Parasponia, has been found to form symbiotic root nodules with Rhizobium. The question of what makes the legumes unique is an important and provoking one. There is also considerable specificity of individual strains or species of Rhizobium for particular groups of plants, as shown in Table 1. The ecological and economic importance of nitrogen fixation has justly earned research attention for the Rhizobium-legume symbiosis. The system has an additional, fundamental attraction. During a complex series of developmental steps, the bacteria and the plant each influence in the other such fundamental activities as ceil division, gene expression, metabolic function, and cell morphogenesis. Analysis of the bacterial influence on these processes may lead to identification of otherwise elusive components that are parts of the indigenous plant systems for signal transduction, gene regulation, cell division, and cell wall formation. The driving forces for recent study of Rhizobium-plant symbioses include bacterial genetics, plant molecular biology, and detailed microscopy of the bacteria-plant interaction. This review highlights several questions of recent interest, with the focus on genetics and molecular biology; the references are representative, not exhaustive. A more complete view of the field can be found in two recent symposium volumes (Bothe et al., 1988; Verma and Palacios, 1988).

Rhizobium symbiosis: nod factors in perspective.

Rhizobium and its allies (Azorhizobium, Bradyrhizobium, and Sinorhizobium) are Gram-negative bacteria that cause the development of root (and sometimes stem) nodules on plant hosts, which the bacteria inhabit as nitrogen-fixing endosymbionts. The early stages of this process, including gene expression in the bacterium and cell growth, division, and differentiation in the host, are mediated by signal exchange between the eukaryotic host and the prokaryotic symbiont (Figure l A , left). The plant produces a signal, usually aflavonoid, that induces gene expression in the bacterium; the bacterium subsequently synthesizes a signal that triggers early nodule development on the plant. The developmental time line for nodulation has been described in several reviews and essays (Sprent, 1989; Truchet et al., 1989; Brewin, 1991; Brewin et al., 1992; Hirsch, 1992; Kijne et al., 1992; Ridge, 1992; Vijn et al., 1993) and is only considered briefly here. Nodules can take on several patterns during development, the form of the nodule being determined by the plant, not the bacterium. One major form is the indeterminate (also called meristematic or cylindrical) type, which develops on alfalfa, clover, and pea roots. A second major type is the spherical or determinate nodule, which is formed by soybean, Phaseolus, and Lofus. A comparison of these symbiotic nodules with those of nonlegumes is presented elsewhere in this issue (see Pawlowski and Bisseling, 1996). The twin hallmarks of early nodulation are its developmental complexity and its specificity. The developmental process in the plant involves architectural changes at the cell and organ levels (for example, root hair morphogenesis, cortical cell enlargement, and vascular patterning) as well as interna1 cellular differentiation that includes cytoplasmic activation, cell division, and new gene expression. Structural and developmental studies of nodule formation remain an important part of the overall Rhizobium research picture. Indeed, new views of infection thread formation, cell wall modifications, and intracellular rearrangements in root hairs and elsewhere have recently appeared (Kijne et al., 1992; van Brussel et al., 1992; van Spronsen et al., 1994; DeBoer and Djordjevic, 1995; Ridge, 1995). The exciting tools of video microscopy and image analysis are making it possible to obtain dynamic views of early plant reactions to Rhizobium signals (Allen et al., 1994; Sanchez et al., 1996). Thus, in addition to its inherent interest as a model for understanding plant-microbe interactions, the specificity and timing of early nodulation events make the Rhizobium-plant symbiosis an attractive model system for general plant cell biology studies. The specificity of nodulation is likewise remarkable: with one known exception, the Rhizobium nodulation habit is restricted to a single plant taxon, the Fabaceae, or legume family. Within this family, individual species, strains, or biovars of bacteria nodulate a restricted set of host plants that are usually but not always related. The signal model (Figure 1) provides an explanation for the species-leve1 pattern of host specificity. But we cannot yet answer the larger mechanistic and evolutionary question: Why only legumes? Because a short review cannot catalog complete lists of referentes, even recent ones, the focus of this article is to put selected papers in context: Why is it important for plant biologists to be concerned with bacterial genes, their regulation, and activities? Where do the questions lie in the study of the plant response? What new genetic and cellular methods are needed for their resolution?

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

Medicago truncatula NIN Is Essential for Rhizobial-Independent Nodule Organogenesis Induced by Autoactive Calcium/Calmodulin-Dependent Protein Kinase

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11∷GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

An ERF Transcription Factor in Medicago truncatula That Is Essential for Nod Factor Signal Transduction

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor–induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Ethylene Inhibits the Nod Factor Signal Transduction Pathway of Medicago truncatula

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.

Morphogenetic Rescue of Rhizobium meliloti Nodulation Mutants by trans-Zeatin Secretion.

The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria. We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37. This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures. Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation. This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses. Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

SUMMARY The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis, with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.