Cloning Of Resistance Genes Research Articles

Intergeneric transfer and functional expression of the tomato disease resistance gene Pto.

Plant disease resistance loci have been used successfully in breeding programs to transfer traits from resistant germplasm to susceptible plant cultivars. The molecular cloning of plant disease resistance genes now permits the transfer of such traits across species boundaries by genetic transformation of recipient hosts. The tomato disease resistance gene Pto confers resistance to strains of the bacterial pathogen Pseudomonas syringae pv tomato expressing the avirulence gene avrPto. Transformation of Nicotiana benthamiana with Pto results in specific resistance to P. s. pv tabaci strains carrying avrPto. The resistant phenotype is manifested by a strong inhibition of bacterial growth and the ability to exhibit a hypersensitive response. Resistance cosegregates with the Pto gene in transgene selfings and testcrosses. Our results demonstrate the conservation of disease resistance functions across genus boundaries and suggest that the utility of host-specific resistance genes can be extended by intergeneric transfer.

A gene from Alcaligenes denitrificans that confers albicidin resistance by reversible antibiotic binding.

Albicidin antibiotics specifically block prokaryote DNA replication. The albicidin resistance gene (albB) cloned from a soil isolate of Alcaligenes denitrificans encodes a 23 kDa protein capable of detoxifying albicidin by reversible binding. This mechanism operates intracellularly to protect DNA replication in albicidin-sensitive Escherichia coli expressing the cloned resistance gene, which can be induced fivefold in the presence of 1.5 micrograms albicidin ml-1 in the surrounding medium. The coding region of 621 bp has regions with partial DNA sequence homology to an albicidin resistance gene (albA) from Klebsiella oxytoca, but with rearrangements and frame-shifts resulting in loss of protein homology. There is a short region of N-terminal homology between the albicidin resistance (Albr) proteins from A. denitrificans and K. oxytoca, although the two genes use different codons for shared amino acids. The N-terminal homology suggested a common functional domain; this was confirmed by deletion analysis, translational fusions and albicidin binding by a synthetic oligopeptide. Antibiotic binding provides a high level of albicidin resistance in E. coli. The gene appears to be a useful candidate for transfer to plants to protect plastid DNA replication from inhibition by albicidin phytotoxins involved in sugarcane leaf scald disease.

Isolation of disease resistance genes from crop plants

The recent cloning of several resistance genes from diverse plant species, in combination with various technical advances, has provided new opportunities for accessing the great diversity of disease resistance genes in crop plants. Many resistance genes probably belong to clusters of large multigene families encoding receptor-like proteins that have evolved to have different specificities. The isolation of genes from crop species is being facilitated by continuing technical improvements to methods for the saturation of markers within genomic regions containing resistance genes, for the cloning and characterization of large genomic fragments, and for efficient complementation. The primary limitation to cloning resistance genes with known specificities will be the genetic definition of the targeted gene.

Perception and response in plant disease resistance

Plants express sophisticated mechanisms for recognizing pathogens. The functionally defined repertoire of non-self perception is large; the number and nature of subsequent molecular events required for resistance is unknown. Recent cloning of disease resistance genes, and genetic identification of loci required for their function, allows dissection of the structure, evolution, and deployment within populations of pathogen-perception mechanisms. Role for reactive oxygen species and programmed cell death in resistance have also been suggested recently. New results document a role for salicylic acid as a lynchpin in the establishment and maintenance of the ‘effector functions’ of disease resistance, and strategies for engineered plant protection are moving closer to reality.

A series of integrative plasmids for Bacillus subtilis containing unique cloning sites in all three open reading frames for translational lacZ fusions.

Two sets of three plasmids each were constructed based on integrative plasmids for Bacillus subtilis. Each set encodes resistance to either chloramphenicol or kanamycin. The plasmids contain a cassette consisting of the resistance gene, BamHI and SmaI cloning sites, and the promoterless lacZ gene. The cassette is flanked by the 3' and 5' ends of the amyE gene (encoding amylase) allowing integration of the cassette into that locus in the B. subtilis chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of replication and the beta-lactamase-encoding gene. Within a set, each of the three plasmids carries the unique SmaI and BamHI restriction sites in a different reading frame relative to the 'lacZ gene. These sets of plasmids allow the easy construction of translational fusions with lacZ and single-copy integration at the amyE locus of B. subtilis.

Cadmium resistance acquirement by IS1 transposition into Escherichia coli C600

Abstract A 5.1 kb plasmid, pCDR1, which conferred cadmium resistance on Escherichia coli C600, was obtained during the cloning of the cadmium resistance gene from pGU100 harbored by cadmium-resistant Pseudomonas putida GAM-1, pCDR1 was constructed with pBR322 as vector and an about 800 bp inserted fragment. The nucleotide sequence revealed that the inserted DNA fragment was IS1. The cadmium resistance of this transformant was maintained even though the plasmid was cured. Southern analysis showed that the mobility of one band was changed, indicating that a chromosomal rearrangement occurred in the cadmium-resistant strain.

The enigmatic avirulence genes of phytopathogenic bacteria.

The previous chapters have discussed how phytopathogenic bacteria can sense and respond to conditions present in a variety of microenvironments: soil, water, plant cell surfaces, and intracellular spaces. The switch from epiphyte to pathogen is apparently accompanied by fundamental reprogramming of gene activity and attendant function, as evidenced by induction of hrp genes and subsequent production of various virulence and pathogenicity factors, some of which are hostspecific, some not. This reprogramming switch between epiphytic and pathogenic growth strategies, “the pathogenic cusp” (DANGL 1994), is the point at which not only the potential pathogen but also the host first sense and respond to each other. A successful plant defense response should be based on surveillance and interdiction before the pathogen has a chance to establish production of the armory of factors which determine successful colonization of that host. It is incumbent on each potential plant host, then, to evolve mechanisms to recognize some factor, preferably one produced at this pathogenic cusp, and to base resistance strategies on early recognition. Thus, an evolutionary tug-of-war is begun: plants evolve to “recognize” a particular isolate of a particular pathogen; variants arise in the pathogen population which are no longer recognized; variants arise in the host population which recognize the new pathogen variant, and the familiar game is afoot. This scenario has apparently given rise to systems of plantpathogen interactions explained genetically by the now well established “gene-for-gene” hypothesis (reviewed in CRUTE 1985; DANGL 1992; ELLINGBOE 1981; FLOR 1971; GABRIEL and ROLFE 1990; KEEN 1990; KEEN and STASKAWICZ 1988) . During plantmicrobe interactions of this sort, the products, either direct or indirect, of pathogen avirulence (avr) genes trigger a successful host resistance reaction through the action of the product of a particular host resistance (R) gene. These interactions are thus allele-specific: each partner is defined only by the simultaneous presence of the other. The complexity of resistance loci defined genetically is astounding and the paucity of knowledge regarding their structure and function is beginning to be resolved, as evidenced by the recent cloning of the first plant resistance gene of this class (MARTIN et al. 1993) . In contrast, a plethora of avirulence genes from pseudomonads and xanthomonads have been cloned, as discussed in the remainder of this review.

Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.

Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti: Conservation of the gene and resistance associated mutation with Drosophila

In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila. In D. melanogaster, resistance to cyclodienes and picrotoxinin is caused by a single amino acid substitution (alanine to serine) in the putative channel lining of a γ-aminobutyic acid gated chloride ion channel. We report that the mosquito gene not only shows high homology to that of Drosophila but also that resistant strains display substitution of the same amino acid. The significance of this result in relation to the evolution of pesticide resistance, the use of Drosophila as a model insect for resistance studies and the potential use of this gene as a selectable marker in the genetic transformation of non-Drosophilids is discussed.

Transposable Elements as Initiators of Insecticide Resistance

Genetic change that leads to insecticide resistance is poorly understood for most resistance genes. Transposable elements have been shown to be powerful mutagenic agents in Drosophila melanogaster (Meigen), and they potentially can create genetic change leading to resistance in pest insect populations. Here the characteristic of transposable elements and the consequences of their movement in D. melanogaster and other insects are discussed, and reports of their involvement in insecticide resistance are examined. I conclude that transposable elements must be considered as mutagenic agents that can lead to resistance. Their involvement needs to be directly addressed in studies of resistant insects, particularly by investigators involved in cloning resistance genes.

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans.

A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg-) mutant of Streptomyces lividans 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cms Arg- mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.

Emerging strategies for enhancing crop resistance to microbial pathogens.

There are marked differences in the pattern of host gene expression in incompatible plant:microbial pathogen interactions compared with compatible interactions, associated with the elaboration of inducible defenses. Constitutive expression of genes encoding a chitinase or a ribosome-inactivating protein in transgenic plants confers partial protection against fungal attack, and a large repertoire of such antimicrobial genes has been identified for further manipulation. In addition, strategies are emerging for the manipulation of multigenic defenses such as lignin deposition and synthesis of phytoalexin antibiotics by overexpression of genes encoding rate determining steps, modification of transcription factors or other regulatory genes, and engineering production of novel phytoalexins by interspecies transfer of biosynthetic genes. The imminent cloning of disease resistance genes, further molecular dissection of stress signal perception and transduction mechanisms, and identification of genes that affect symptom development will provide attractive new opportunities for enhancing crop protection. Combinatorial integration of these novel strategies into ongoing breeding programs should make an important contribution to effective, durable field resistance.

Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin

The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5′ to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.

Cloning of genes for spermine resistance in Saccharomyces cerevisiae and their effects on [formula omitted] accumulation

Among the polyamines tested, spermine strongly inhibited the growth of Saccharomyces cerevisiae DKD-5D-H. Two kinds of DNA fragments that confer strong and weak resistances to spermine were cloned onto a vector plasmid, YEp13. The restriction map of the DNA fragment conferring strong resistance was the same as that of a DNA fragment responsible for ethionine resistance in the same yeast cells. (Shiomi et al., Appl. Microbiol. Biotechnol., 29, 302–304, 1988) The yeast cells with the DNA fragment conferring strong resistance to spermine were resistant to ethionine and accumulated S- adenosyl- l-methionine intracellularly.

The cloning of MLS antibiotics inducible resistance gene

Four bacterial strains having inducible resistance to erythromycin were isolated from soil samples in Korea and characterized. MLS inducibility was checked in each strain. Cloning of inducible resistance gene(s) has been tried. Four isolates were identified asB. anthracis, Micrococcus luteus, Streptococcus faecium andB. licheniformis, in which erythromycin, oleandomycin, cirramycin and carbomycin acted as resistance inducers respectively. The resistance gene cloned fromB. licheniformis 597 strain using pBS 42 vector was found to have a 3.2 kb insert.

Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803

The facultative heterotrophic cyanobacterium Synechocystis sp. strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome. A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments. Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants. Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites. In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium. Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis. Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis.

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus.

The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic production in strains otherwise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.

Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca.

A DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated from Klebsiella oxytoca and shown to be expressed in Escherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin. In vivo translation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25kD protein. The DNA sequence was determined for a 654-base-pair open reading frame contained within a 1.2kb subcloned DNA fragment encoding albicidin resistance. The predicted molecular weight of the polypeptide translated from the open reading frame was 25.8kD. A putative Shine-Dalgarno sequence precedes the open reading frame but a potential promoter sequence was not detected. A possible rho-independent transcription termination signal was found directly following the stop codon. The functional protein for albicidin resistance was isolated and purified. Both the molecular weight and NH2-terminal amino acid sequence of this protein correspond with that predicted from the DNA sequence of the open reading frame. The cloned albicidin resistance gene had no effect on the tsx (nupA) nucleoside uptake gene associated with spontaneous albicidin resistance in E. coli; also, it did not complement any of a range of E. coli DNAts mutants at restrictive temperatures. The cloned resistance gene product remained intracellular in exponential cultures of K. oxytoca and E. coli. Cell-free extracts from E. coli containing the resistance gene protected a sensitive strain of E. coli from inhibition by albicidin, as did the purified albicidin resistance protein. The mechanism of this albicidin resistance protein involved binding to albicidin to form a complex without antibiotic activity, but without catalysing further chemical modification of the antibiotic.

Analysis of the nourseothricin-resistance gene ( nat) of Streptomyces noursei

A gene ( nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNATl. The nat gene was localized on a 1-kb SalI- MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of Nc R in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.

A Genetic Map of Lettuce (Lactuca sativa L.) With Restriction Fragment Length Polymorphism, Isozyme, Disease Resistance and Morphological Markers

A detailed linkage map of lettuce was constructed using 53 genetic markers including 41 restriction fragment length polymorphism (RFLP) loci, five downy mildew resistance genes, four isozyme loci and three morphological markers. The genetic markers were distributed into nine linkage groups and cover 404 cM which may be 25-30% of the lettuce genome. The majority (31 of 34) of the RFLP probes detected single segregating loci, although seven of these may have been homologous to further monomorphic loci. When several loci were detected by a single probe, the loci were generally linked, suggesting tandem duplications. One probe, however, detected loci in three linkage groups suggesting translocations. The five downy mildew resistance genes (Dm1, Dm3, Dm4, Dm5/8 and Dm13), segregating in the Calmar x Kordaat cross, represented each of the four resistance gene linkage groups. Dm5/8 is flanked by two cDNA loci, each located 10 cM away. These flanking markers will be used to study the source of variation in downy mildew genes and are also part our strategy to clone resistance genes.