Characterization of a gene from a tomato pathogen determining hypersensitive resistance in non-host species and genetic analysis of this resistance in bean

Xanthomonas campestris pv. vesicatoria is the causal agent of leaf spot disease on pepper and tomato. On non-host plants, such as bean, soybean, cowpea, alfalfa, and cotton, X. campestris pv. vesicatoria is unable to cause disease, inducing instead a hypersensitive resistance response (HR). Since avirulence genes from X. campestris pv. vesicatoria specifically induce HR in several pepper cultivars, we investigated whether there were avirulence genes governing induction of resistance in non-host species. We report on the molecular cloning and characterization of a non-host avirulence gene from X. campestris pv. vesicatoria. A cosmid clone isolated from a library of DNA from X. campestris pv. vesicatoria tomato race 1 converted X. campestris pv. phaseoli to avirulence by inducing HR on the bean cultivar Sprite, but not on Bush Blue Lake. The HR-inducing activity was localized to a 2.1-kilobase Pst I fragment of DNA, designated avrRxv. In addition, we demonstrate that avrRxv inhibited disease production by several X. campestris pathovars on their normally susceptible hosts: glycines on soybean, vignicola on cowpea, alfalfae on alfalfa, holcicola on corn, and malvacearum on cotton. The HR resistance in bean induced by avrRxv segregated as a single incompletely dominant gene, designated Rxv. These results indicate that the avirulence gene avrRxv and the resistance gene Rxv partially control the outcome of the interaction between X. campestris pv. vesicatoria and the non-host bean.     

Cloned avirulence genes from the tomato pathogen Pseudomonas syringae pv. tomato confer cultivar specificity on soybean

Three different cosmid clones were isolated from a genomic library of the tomato pathogen Pseudomonas syringae pv. tomato, which, when introduced into the soybean pathogen P. syringae pv. glycinea, caused a defensive hypersensitive response (HR) in certain soybean cultivars. Each clone was distinguished by the specific cultivars that reacted hypersensitively and by the intensity of the HR elicited. Unlike wild-type P. syringae pv. tomato isolates, which elicit the HR on all soybean cultivars, all three clones exhibited cultivar specificities analogous to avirulence genes previously cloned from P. syringae pv. glycinea. However, the collective phenotypes of the three clones accounted for HRs on all tested soybean cultivars. One of the three P. syringae pv. tomato clones contained an avirulence gene homologous to avrA, which was previously cloned from P. syringae pv. glycinea race 6. The other two P. syringae pv. tomato clones expressed unique HR patterns on various soybean cultivars, which were unlike those caused by any known P. syringae pv. glycinea race or previously cloned P. syringae pv. glycinea avr gene. Further characterization of the second P. syringae pv. tomato clone indicated that the avirulence phenotype resided on a 5.6-kilobase HindIII fragment that, in Southern blot analyses, hybridized to an identical-size fragment in various P. syringae pathovars, including all tested glycinea races. These results demonstrate that avirulence genes may be distributed among several P. syringae pathovars but may be modified so that the HR is not elicited in a particular host plant. Furthermore, the data raise the possibility that avirulence genes may function in host-range determination at levels above race—cultivar specificity.     

Cloned avirulence gene of Pseudomonas syringae pv. glycinea determines race-specific incompatibility on Glycine max (L.) Merr

A genomic library of Pseudomonas syringae pv. glycinea race 6 DNA was constructed in the mobilizable cosmid vector pLAFR1 and maintained in Escherichia coli HB101. Completeness of the library was estimated by assaying clones for the expression of ice-nucleating activity in E. coli. Ice-nucleation activity was represented approximately once in every 600 clones. Six hundred eighty random race 6 cosmid clones were mobilized from E. coli by plasmid pRK2013 in individual conjugations to a race 5 strain of P. s. glycinea. A single clone (pPg6L3) was detected that changed the race specificity of race 5 from virulent (compatible) to avirulent (incompatible) on the appropriate soybean cultivars. The clone was also mobilized from E. coli into race 1 and race 4 strains of P. s. glycinea, and it conferred on these transconjugants the same host range incompatibility as the wild-type race 6 strain. The cosmid clone was mapped by restriction endonucleases, and two adjacent EcoRI fragments were identified by transposon Tn5 mutagenesis to be important in determining race specificity. Southern blot analysis showed that the two EcoRI fragments are unique to race 6 and are not present in the other races tested. The cosmid clone pPg6L3 was also mobilized to Pseudomonas fluorescens and Rhizobium japonicum. However, neither these isolates nor E. coli harboring pPg6L3 elicited a hypersensitive reaction in soybean leaves.     

Isolation of genes involved in nodulation competitiveness from Rhizobium leguminosarum bv. trifolii T24

Rhizobium leguminosarum bv. trifolii T24 produces a potent anti-rhizobial compound, trifolitoxin, and exclusively nodulates clover roots when in mixed inoculum with trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii [Schwinghamer, E. A. & Belkengren R. P. (1968) Arch. Mikrobiol. 64, 130-145]. In the present study, the isolation of trifolitoxin production and resistance genes is described. A cosmid genomic library of T24 was prepared in pLAFR3. No trifolitoxin expression was observed in the resulting Escherichia coli cosmid clones. One cosmid clone was identified that restored trifolitoxin production and nodulation competitiveness in three nonproducing mutants of T24. The recombinant plasmid from this cosmid clone, pTFX1, also conferred trifolitoxin production and resistance when transferred to symbiotically effective strains of R. leguminosarum bvs. trifolii, phaseoli, and viceae. Cosmid pTFX1 also conferred expression of trifolitoxin production when present in strains of Rhizobium meliloti and Agrobacterium tumefaciens. No trifolitoxin expression was observed in strains of Bradyrhizobium japonicum or Rhizobium sp. (cowpea) with pTFX1. Southern blot analysis with a biotinylated pTFX1 probe did not suggest that these genes were plasmid-borne. Transfer of pTFX1 to T24 or its derivatives resulted in 6- to 10-fold higher level of trifolitoxin production than wild-type T24.     

From Player to Pawn: Viral Avirulence Factors Involved in Plant Immunity

In the plant immune system, according to the ‘gene-for-gene’ model, a resistance (R) gene product in the plant specifically surveils a corresponding effector protein functioning as an avirulence (Avr) gene product. This system differs from other plant–pathogen interaction systems, in which plant R genes recognize a single type of gene or gene family because almost all virus genes with distinct structures and functions can also interact with R genes as Avr determinants. Thus, research conducted on viral Avr-R systems can provide a novel understanding of Avr and R gene product interactions and identify mechanisms that enable rapid co-evolution of plants and phytopathogens. In this review, we intend to provide a brief overview of virus-encoded proteins and their roles in triggering plant resistance, and we also summarize current progress in understanding plant resistance against virus Avr genes. Moreover, we present applications of Avr gene-mediated phenotyping in R gene identification and screening of segregating populations during breeding processes.     

Inheritance and expression of foreign genes in transgenic soybean plants

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R₁) and second (R₂) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for β-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for β-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R₁ generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R₂ generation. R₁ plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R₂ generation, demonstrating recovery of both homozygous and heterozygous R₁ plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.     

Characterisation of Campylobacter jejuni genes potentially involved in phosphonate degradation

Potential biological roles of the Campylobacter jejuni genes cj0641, cj0774c and cj1663 were investigated. The proteins encoded by these genes showed sequence similarities to the phosphonate utilisation PhnH, K and L gene products of Escherichia coli. The genes cj0641, cj0774c and cj1663 were amplified from the pathogenic C. jejuni strain 81116, sequenced, and cloned into pGEM-T Easy vectors. Recombinant plasmids were used to disrupt each one of the genes by inserting a kanamycin resistance (Km ^R) cassette employing site-directed mutagenesis or inverse PCR. Campylobacter jejuni 81116 isogenic mutants were generated by integration of the mutated genes into the genome of the wild-type strain. The C. jejuni mutants grew on primary isolation plates, but they could not be purified by subsequent passages owing to cell death. The mutant C. jejuni strains survived and proliferated in co-cultures with wild-type bacteria or in media in which wild-type C. jejuni had been previously grown. PCR analyses of mixed wild-type/mutant cultures served to verify the presence of the mutated gene in the genome of a fraction of the total bacterial population. The data suggested that each mutation inactivated a gene essential for survival. Rates of phosphonate catabolism in lysates of E. coli strain DH5α were determined using proton nuclear magnetic resonance spectroscopy. Whole-cell lysates of the wild-type degraded phosphonoacetate, phenylphosphonate and aminomethylphosphonate. Significant differences in the rates of phosphonate degradation were observed between lysates of wild-type E. coli, and of bacteria transformed with each one of the vectors carrying one of the C. jejuni genes, suggesting that these genes were involved in phosphonate catabolism.     

Efficient cloning of genes of Neurospora crassa

We have constructed a genomic library of Neurospora crassa DNA in a cosmid vector that contains the dominant selectable marker for benomyl resistance. The library is arranged to permit the rapid cloning of Neurospora genes by either sib-selection or colony-hybridization protocols. Detailed procedures for the uses of the library are described. By use of these procedures, a modest number of unrelated genes have been isolated. The cloning of trp-3, the structural gene for the multifunctional enzyme tryptophan synthetase (tryptophan synthase, EC 4.2.1.20), is reported in detail; its identity was verified by restriction fragment length polymorphism mapping. The strategies described in this paper should be of use in the cloning of any gene of Neurospora, as well as genes of other lower eukaryotes.     

A cosmid for selecting genes by complementation in Aspergillus nidulans: Selection of the developmentally regulated yA locus

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage λ cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC⁺ gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, BglII, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA^-) pabaA^-trpC^-Aspergillus strain (FGSC237) to trpC⁺ at frequencies of approximately 10 transformants per μg of DNA. Three of approximately 1000 trpC⁺pabaA^- colonies obtained were putative yA⁺ transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a λ packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded trpC⁺ transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.     

Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant

The cell death response known as the hypersensitive response (HR) is a central feature of gene-for-gene plant disease resistance. A mutant line of Arabidopsis thaliana was identified in which effective gene-for-gene resistance occurs despite the virtual absence of HR cell death. Plants mutated at the DND1 locus are defective in HR cell death but retain characteristic responses to avirulent Pseudomonas syringae such as induction of pathogenesis-related gene expression and strong restriction of pathogen growth. Mutant dnd1 plants also exhibit enhanced resistance against a broad spectrum of virulent fungal, bacterial, and viral pathogens. The resistance against virulent pathogens in dnd1 plants is quantitatively less strong and is differentiable from the gene-for-gene resistance mediated by resistance genes RPS2 and RPM1. Levels of salicylic acid compounds and mRNAs for pathogenesis-related genes are elevated constitutively in dnd1 plants. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. Although cell death may contribute to defense signal transduction in wild-type plants, the dnd1 mutant demonstrates that strong restriction of pathogen growth can occur in the absence of extensive HR cell death in the gene-for-gene resistance response of Arabidopsis against P. syringae.     

Rapidly evolving R genes in diverse grass species confer resistance to rice blast disease

We show that the genomes of maize, sorghum, and brachypodium contain genes that, when transformed into rice, confer resistance to rice blast disease. The genes are resistance genes (R genes) that encode proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains (NBS–LRR proteins). By using criteria associated with rapid molecular evolution, we identified three rapidly evolving R-gene families in these species as well as in rice, and transformed a randomly chosen subset of these genes into rice strains known to be sensitive to rice blast disease caused by the fungus Magnaporthe oryzae. The transformed strains were then tested for sensitivity or resistance to 12 diverse strains of M. oryzae. A total of 15 functional blast R genes were identified among 60 NBS–LRR genes cloned from maize, sorghum, and brachypodium; and 13 blast R genes were obtained from 20 NBS–LRR paralogs in rice. These results show that abundant blast R genes occur not only within species but also among species, and that the R genes in the same rapidly evolving gene family can exhibit an effector response that confers resistance to rapidly evolving fungal pathogens. Neither conventional evolutionary conservation nor conventional evolutionary convergence supplies a satisfactory explanation of our findings. We suggest a unique mechanism termed “constrained divergence,” in which R genes and pathogen effectors can follow only limited evolutionary pathways to increase fitness. Our results open avenues for R-gene identification that will help to elucidate R-gene vs. effector mechanisms and may yield new sources of durable pathogen resistance.Ascomycete fungi of the Magnaporthe oryzae species complex are ancient pathogens of grasses first described more than a century ago (1). The agent of rice blast disease, M. oryzae, is the most devastating pathogen of rice, and it can also infect other important crops, including wheat and barley. The pathogen evolves rapidly and exhibits a multitude of species-specific and cultivar-specific races (2–5). Species in the M. oryzae complex have therefore become a leading model for the study of interactions between plants and their pathogens (3).Plants have several layers of defense against pathogens. The first consists of passive structural barriers including cell walls and a waxy cuticle. The second line of defense is an active response triggered by transmembrane pathogen-associated molecular pattern recognition receptors, which act against slowly evolving protein motifs that may be shared among multiple different types of pathogens (6, 7). The third level of response defends against specific pathogen races. In this process, pathogen effectors activate host resistance genes (R genes). The main class of R genes encodes proteins containing a nucleotide-binding site (NBS) along with leucine-rich repeats (LRRs). The NBS domains contain conserved motifs that have been demonstrated to bind and hydrolyze ATP and GTP, and the LRR motif is typically involved in protein–protein interactions. Such NBS–LRR proteins play an important role in the recognition and resistance to diverse pathogens ranging from viruses, bacteria, and fungi to insects and nematodes (8).NBS–LRR proteins can act through variety of mechanisms. One mechanism acts via direct interaction with pathogen effectors to render them ineffective (the gene-for-gene hypothesis) (9). Another mechanism features indirect protection of host plant molecules targeted by the pathogen effectors, wherein effector modification of the protected protein activates the R-gene response. Indirect protection may secure host plant molecules that are essential (the guard hypothesis) (8) or host plant molecules that mimic those that are essential (the decoy hypothesis) (10).R genes encoding NBS–LRR proteins are among the most highly amplified gene families in plants. Arabidopsis thaliana has approximately 150 NBS–LRR genes, and Oryza sativa has more than 400 (8). Amplification of R genes often involves tandem or segmental duplication, and hence the genes tend to be clustered. The rate of evolution of R genes varies according to subfamily (6, 8). Some evolve relatively slowly, whereas others exhibit typical features of rapid evolution, including multiple and variable copy number, short branches in the phylogenetic gene tree, notably low divergence between paralogs within a genome, a high ratio of nonsynonymous to synonymous substitutions (K_a/K_s ratio), and high levels of within-species polymorphism (11, 12).It has been suggested that the allele frequencies of rapidly evolving, highly polymorphic pathogen effectors and those of their corresponding R genes might undergo cyclic, out-of-phase changes (6). A caricature of this process is as follows: When the effector(+) allele is at high frequency, the corresponding R(+) allele is favored and increases in frequency, whereupon the effector(−) allele becomes favored and increases in frequency at the expense of effector(+); this favors an intrinsically more fit R(−) allele at the expense of R(+), and around and around they go (6).We reasoned that, if this cyclical model were correct, the cycles may persist for many generations, and perhaps even through speciation of the host plant and the pathogen (barring severe population bottlenecks). Were this the case, one might discover that rapidly evolving R-gene families in any plant species might include alleles that confer resistance to an effector present in some races of a pathogen that affects a different but related species.To test this prediction, we characterized R-gene families encoding NBS–LRR proteins in the genomes of maize, sorghum, and brachypodium, and identified those subfamilies undergoing rapid evolution. We cloned a subset of genes from these subfamilies and transferred them into rice lines susceptible to rice blast disease. We found that 25% of the cloned genes conferred resistance to at least one of 12 independent isolates of M. oryzae. These results demonstrate that a diverse repertoire of R genes for rapidly evolving pathogens occurs not only within species but also among species and that there is functional similarity among NBS–LRR proteins undergoing rapid evolution in different species. Exploiting this diversity could be an important source of R genes for rice blast disease and perhaps other plant pathogens.     

Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic

Andrimid is a hybrid nonribosomal peptide-polyketide antibiotic that blocks the carboxyl-transfer reaction of bacterial acetyl-CoA carboxylase (ACC) and thereby inhibits fatty acid biosynthesis with submicromolar potency. The andrimid biosynthetic gene cluster from Pantoea agglomerans encodes an admT gene with homology to the acetyl-CoA carboxyltransferase (CT) β-subunit gene accD. Escherichia coli cells overexpressing admT showed resistance to andrimid. Co-overproduction of AdmT with E. coli CT α-subunit AccA allowed for the in vitro reconstitution of an active heterologous tetrameric CT A₂T₂ complex. A subsequent andrimid-inhibition assay revealed an IC₅₀ of 500 nM for this hybrid A₂T₂ in contrast to that of 12 nM for E. coli CT A₂D₂. These results validated that AdmT is an AccD homolog that confers resistance in the andrimid producer. Mutagenesis studies guided by the x-ray crystal structure of the E. coli A₂D₂ complex disclosed a single amino acid mutation of AdmT (L203M) responsible for 5-fold andrimid sensitivity (IC₅₀ = 100 nM). Complementarily, the E. coli AccD mutant M203L became 5-fold more resistant in the CT assays. This observation allowed for bioinformatic identification of several Vibrio cholerae strains in which accD genes encode the Met↔Leu switches, and their occurrences correlate predictively with sensitivities to andrimid in vivo.     

Transgenic rats reveal functional conservation of regulatory controls between the Fugu isotocin and rat oxytocin genes

We have asked whether comparative genome analysis and rat transgenesis can be used to identify functional regulatory domains in the gene locus encoding the hypothalamic neuropeptides oxytocin (OT) and vasopressin. Isotocin (IT) and vasotocin (VT) are the teleost homologues of these genes. A contiguous stretch of 46 kb spanning the Fugu IT-VT locus has been sequenced, and nine putative genes were found. Unlike the OT and vasopressin genes, which are closely linked in the mammalian genome in a tail-to-tail orientation, Fugu IT and VT genes are linked head to tail and are separated by five genes. When a cosmid containing the Fugu IT-VT locus was introduced into the rat genome, we found that the Fugu IT gene was specifically expressed in rat hypothalamic oxytocinergic neurons and mimicked the response of the endogenous OT gene to an osmotic stimulus. These data show that cis-acting elements and trans-acting factors mediating the cell-specific and physiological regulation of the OT and IT genes are conserved between mammals and fish. The combination of Fugu genome analysis and transgenesis in a mammal is a powerful tool for identifying and analyzing conserved vertebrate regulatory elements.     

Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles

We have employed microprojectiles to deliver genes involved in anthocyanin biosynthesis to cells within intact aleurone and embryo tissues of maize. Clones of the A1 or Bz1 genes were introduced into aleurone tissue that lacked anthocyanins due to mutations of the endogenous A1 or Bz1 gene. Following bombardment, cells within the aleurone developed purple pigmentation, indicating that the mutation in the a1 or bz1 genotypes was corrected by the introduced gene. To analyze the expression of these genes in different genetic backgrounds, chimeric genes containing the 5′ and 3′ regions of the A1 or Bz1 genes fused to a luciferase coding region were constructed. These constructs were introduced into aleurones of genotypes carrying either dominant or recessive alleles of the C1 and R genes, which are known to regulate anthocyanin production. Levels of luciferase activity in permissive backgrounds (C1, R) were 30- to 200-fold greater than those detected in tissue carrying one or both of the recessive alleles (c1, r) of these genes. These results show that genes delivered to intact tissues by microprojectiles are regulated in a manner similar to the endogenous genes. The transfer of genes directly to intact tissues provides a rapid means for analyzing the genetic and tissue-specific regulation of gene expression.     

Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: A new family of genes responsible for autoinducer production

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor–autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5α does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxS_V.h., luxS_S.t., and luxS_E.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5α can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5α luxS_E.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxS_E.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes.     

Efficient cloning of single-copy genes using specialized cosmid vectors: isolation of mutant dihydrofolate reductase genes.

A method for the efficient cloning of single-copy genes from restriction digests of mammalian DNA is described. The method is illustrated by the cloning of several mutant genes as well as the wild-type gene for Chinese hamster dihydrofolate reductase (DHFR; 7,8-dihydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). This gene is isolated within a 41-kilobase Bgl I fragment by using cosmid (plasmids containing a cohesive-end site) vectors that have been constructed especially for this purpose. Two cosmids are used: one contains a short region from the 5' flanking region of the dhfr gene, and the other contains a short region from the 3' flanking region. These two regions contain the Bgl I sites that bound the dhfr gene. Bgl I leaves staggered ends that are different depending on the DNA sequence within the enzyme binding site. When these cosmids are cut with Bgl I and hybridized with total Bgl I-cut genomic DNA, they preferentially associate with the fragment bearing the dhfr gene, since it has the same Bgl I ends. An approximately 500-fold enrichment for the dhfr gene in cosmid libraries from Chinese hamster ovary cells was achieved by using this method coupled with a single-step size fractionation. As a result, only several hundred cosmid colonies need to be screened in order to clone a dhfr gene from a particular mutant Chinese hamster ovary cell. This method should facilitate the repetitive cloning of any gene or gene fragment.     

Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a Rhizobium species. The bacterial strain RP501 is a slow-growing strain of Rhizobium isolated from Parasponia nodules. Strain RP501 also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb) EcoRI fragment that complemented insertion and point mutations in three contiguous nodulation genes (nodABC) of Rhizobium meliloti, the endosymbiont of alfalfa (Medicago sativa). The complemented R. meliloti nod mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or Parasponia. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the R. meliloti nodABC genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb EcoRI fragment prevented complementation of R. meliloti nodABC mutations.