A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2 RK2 is a bacterial plasmid of incompatibility group P-1 that is very similar, if not identical, to plasmids having the designation RP1, RP4, and R68 (1). It confers resistance to the antibiotics ampicillin, tetracycline, and kanamycin and exists at approximately five to eight copies per chromosomal equivalent in Escherichia coli (2). A general feature of P-I plasmids is their extensive host range. Such plasmids are capable of conjugal self-transfer to a wide variety of Gram-negative bacteria (3,4). This unique property has been used as the basis for development of a plasmid cloning system in E. coli with widespread applicability. Although native RK2 DNA can be used directly as a recombinant DNA cloning vector, its large size [56 kilobase pairs (kb)] is a serious drawback to routine use. In order to reduce the size and still retain overall broad host range transfer capability, a cloning system has been devised that separates RK2 transfer and replication functions onto separate plasmids. The tetracycline-resistant plasmid component of this system, pRK290, contains a functional RK2 replicon and can be mobilized at high frequency by using a helper plasmid, but is non-self-transmissible. pRK290 contains single EcoRI and Bgl II sites where DNA can be inserted without loss of essential functions. The kanamycin-resistant helper plasmid, pRK2013, consists of the RK2 transfer genes cloned onto a ColEl replicon (5). Its sole function in this system is to trans-complement the vector for mobilization. This paper describes the construction of pRK290, its properties as a cloning vector, and its use in constructing a gene bank of the agriculturally important bacterium Rhizobium meliloti. As an initial test of the gene bank, DNA containing the nitrogenase structural gene region of Klebsiella pneumoniae (6, 7) was used as a hybridization probe to identify clones carrying the nitrogenase region of R. meliloti. One of the members of the bank was found to contain a 26-kb insert with homology to this K. pneumoniae probe. Enzymes. Restriction endonuclease EcoRI was purified in our laboratory; Bgl II was a gift from C. Yanofsky; all other restriction enzymes were obtained from New England BioLabs. T4 DNA ligase was obtained from Bethesda Research Laboratories (Rockville, MD), and was used at a concentration of 1 unit/ml for ligations. Bacterial alkaline phosphatase was obtained from Miles and was dialyzed into 10 mM glycine, pH 9.5/0.1 mM ZnCl2 for storage. DNA was treated with this enzyme at 65°C for 90 min in 10 mM Tris-HCl (pH 9.5). The reaction was terminated by phenol extraction.Bacterial Matings. Matings were performed by mixing 109 cells each of the donor and recipient and filtering the suspension onto 0.45-,um Millipore filters. The filters were incubated at 30°C on nonselective agar plates for 3-6 hr before the cells were resuspended a...

SUMMARYPlant axillary meristems are composed of highly organized, self-renewing stem cells that produce indeterminate branches or terminate in differentiated structures, such as the flowers. These opposite fates, dictated by both genetic and environmental factors, determine interspecific differences in the architecture of plants. The Cys 2 -His 2 zinc-finger transcription factor RAMOSA1 (RA1) regulates the fate of most axillary meristems during the early development of maize inflorescences, the tassel and the ear, and has been implicated in the evolution of grass architecture. Mutations in RA1 or any other known members of the ramosa pathway, RAMOSA2 and RAMOSA3, generate highly branched inflorescences. Here, we report a genetic screen for the enhancement of maize inflorescence branching and the discovery of a new regulator of meristem fate: the RAMOSA1 ENHANCER LOCUS2 (REL2) gene. rel2 mutants dramatically increase the formation of long branches in ears of both ra1 and ra2 mutants. REL2 encodes a transcriptional co-repressor similar to the TOPLESS protein of Arabidopsis, which is known to maintain apical-basal polarity during embryogenesis. REL2 is capable of rescuing the embryonic defects of the Arabidopsis topless-1 mutant, suggesting that REL2 also functions as a transcriptional co-repressor throughout development. We show by genetic and molecular analyses that REL2 physically interacts with RA1, indicating that the REL2/RA1 transcriptional repressor complex antagonizes the formation of indeterminate branches during maize inflorescence development. Our results reveal a novel mechanism for the control of meristem fate and the architecture of plants.

Symbiotically essential genes have been identified in Rhizobium melilodi that are structurally and functionally related to chromosomal virulence (chv) genes of Agrobacterium tumefaciens. Homologous sequences also exist in the genomes of other fast-growing rhizobia including Rhizobium trifolii, Rhizobium leguminosarum, and Rhizobium phaseoli. In Agrobacterium, the chvA and chvB loci are known to be essential for oncogenic transformation of The family Rhizobiaceae has classically been considered to contain only two genera, Agrobacterium and Rhizobium. Agrobacterium species are plant pathogens that induce tumorous growths on a wide variety of dicotyledonous plants, while Rhizobium species are agriculturally beneficial plant symbionts that induce nitrogen-fixing nodules on the roots of legumes. We report here what is to our knowledge the first instance in which a group of symbiotically required Rhizobium genes has been shown to be structurally and functionally related to the Agrobacterium genes that are required for pathogenesis.Most of the genes necessary for virulence (vir) of Agrobacterium tumefaciens have been localized to a unique endogenous plasmid called the Ti plasmid (1). Mutations in these vir genes prevent oncogenic transformation, presumably by interfering with the successful transfer to the plant of another region on the Ti plasmid called T-DNA, that encodes enzymes involved in phytohormone production (2)(3)(4). In addition to plasmid-encoded vir genes, two closely linked virulence loci have been found in the chromosome of Agrobacterium (5). These loci, designated chvA and chvB, have the interesting feature that mutations at either locus interfere with the ability of Agrobacterium to bind to plant cells. Little is known about how the chv gene products function, but chv mutants show pleiotropic effects likely to be related to cell envelope changes (6, 7).The data presented here show that chvA and chvB are homologous to DNA sequences in the genomes of four different fast-growing Rhizobium species and that in the case ofRhizobium meliloti, the corresponding genes can functionally complement Agrobacterium chv mutants. R. meliloti mutants in the chv-equivalent loci are still able to induce nodule-like structures on alfalfa, but such nodules do not show normal bacterial invasion and differentiation. MATERIALS AND METHODSStrains and Plasmids. The following Rhizobium strains were used in this study: R. meliloti 102F34 (8), 1021 (9) and 41 (10); Rhizobium phaseoli 8002 (11) and its sym plasmidcured derivative 8400 (11); Rhizobium leguminosarum 128C53 (12) and its sym plasmid-cured derivative B151 (12); Rhizobium trifolii 162X68, from Nitragin (Milwaukee, WI), and RS 800 (13); Rhizobium japonicum USDA 110 (14). Agrobacterium strains have previously been described: A348 is A. tumefaciens C58 chromosome carrying pTiA6NC (15); Tn5 and Tn3HoHol insertion mutants were used for complementation studies (5). Escherichia coli strains were HB101 (pro, leu, thi, lacY, endoI, recA, hsdR, hsdM, str?) and HB1O1::Tn5....

The ndvA locus of Rhizobium meliloti is homologous to and can substitute for the chvA locus ofAgrobacterium tumefaciens. We have previously shown that an ndvA mutant exhibited reduced motility and formed small, white, empty nodules on alfalfa roots. Here we show that this ndvA mutant is defective in the production of the cyclic extracellular polysaccharide 0-(1---*2)glucan, even (1--2)glucan, we propose that NdvA is involved in export of P-(1-*2)glucan from the cell and that this process is fundamentally important for normal alfalfa nodule development.Rhizobium and Agrobacterium are closely related bacterial genera that are well known for their ability to interact with higher plants. Rhizobium species are symbionts that induce nitrogen-fixing nodules on the roots of legumes, while Agrobacterium species are pathogens that induce tumors on dicotyledonous plants. The production of nitrogen-fixing nodules by Rhizobium meliloti and the induction of tumors by Agrobacterium tumefaciens require a related set of closely linked chromosomally located genes known as ndvA and ndvB in R. meliloti (11) and chvA and chvB in A. tumefaciens (10). These two sets of genes are functionally equivalent; the ndvA and B loci of R. meliloti are able to complement chvA and chvB mutants, respectively, of A. tumefaciens (11).Mutations in either ndvA or ndvB of R. meliloti result in the delayed formation of numerous small white nodules that are distributed throughout the root system. These nodules are not invaded by the mutant bacteria and consequently do not fix nitrogen. Defects in either the chvA or chvB locus result in loss of the ability to induce plant tumors. How defects in ndv and chv genes interfere with nodulation or tumor formation is unknown, but mutations in these loci are pleiotropic, causing reductions in attachment and motility (2, 10; T. Dylan and S. Stanfield, unpublished data). While chvB mutants were also reported to lack the ability to produce the unique extracellular polysaccharide ,-(1-+2)glucan (22), it was questionable whether this deficiency was directly involved in the loss of virulence, since chvA mutants were

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