Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Identification and Characterization of Menadione and Benzethonium Chloride as Potential Treatments of Pierce's Disease of Grapevines.

Xylella fastidiosa infects a wide range of plant hosts and causes Pierce's disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naphthoquinone, vitamin K) from the phytochemical library and benzethonium chloride (a topical disinfectant) from the chemical library both showed significant bactericidal activity against X. fastidiosa. Both menadione and benzethonium chloride foliar spray (15 and 5 mM, respectively) and soil drench (5 and 25 mM, respectively) treatments were equally effective in reducing PD symptoms by 54 to 59% and revealed that the effects of both chemical treatments became systemic. However, menadione was phytotoxic when applied as a foliar spray at effective concentrations, causing significant loss of photosynthetic capacity.

Bacterial Brown Leaf Spot of Citrus, a New Disease Caused by Burkholderia andropogonis.

A new bacterial disease of citrus was recently identified in Florida and is here named bacterial brown leaf spot (BBLS) of citrus. BBLS-infected citrus leaves from the field displayed circular, brownish, flat lesions with slightly raised and water-soaked margins surrounded by a chlorotic halo. Based on Biolog carbon source metabolic "fingerprinting", fatty acid analysis, and sequence analysis of partial 16S rDNA, gyrB, and rpoD genes, the causal agent of the disease was identified as Burkholderia andropogonis. Pathogenicity of these B. andropogonis isolates taken from multiple citrus leaves with BBLS was tested by various inoculation methods on three species of citrus as well as on carnation, corn, and sorghum. All isolates infected carnation, corn, and sorghum with varying degrees of pathogenicity. Variation among citrus isolates in pathogenicity was also observed in high titer (108 CFU/ml) inoculations of citrus leaves, ranging from a hypersensitive-like response to canker-like lesions. When the inoculum concentration was low (106 CFU/ml), only necrotic spots or small lesions slowly developed with all strains. Growth of B. andropogonis in citrus was relatively slow, tissue wounding appeared necessary for symptom appearance with many isolates, and field samples were recovered only after severe storms, indicating that this wide-host-range bacterium is a weak, opportunistic pathogen of citrus.

First Report of Dodder Transmission of 'Candidatus Liberibacter asiaticus' to Tomato (Lycopersicon esculentum).

Citrus huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The disease is associated with three different species of fastidious α-proteobacteria, namely 'Candidatus Liberibacter asiaticus', Ca. L. americanus, and Ca. L. africanus (1). 'Ca. L. asiaticus' was first detected in South Florida in 2005 and has spread throughout the citrus-growing areas of Florida. 'Ca. L. asiaticus' is transmitted naturally by the Asian citrus psyllid, Diaphorina citri, and can also be transmitted by graft propagation and via various species of dodder (Cuscuta). HLB affects most if not all citrus and citrus relatives within the family Rutaceae (2), including the ornamental shrub Murraya paniculata (4). In addition, 'Ca. L. asiaticus' and 'Ca. L. americanus' can infect tobacco (Nicotiana xanthi) and periwinkle (Catharanthus roseus) (1,4). Here we report that 'Ca. L. asiaticus' can infect tomato (Lycopersicon esculentum) cvs. Manapal and FL47. Manapal and FL 47 plants grown from seed were placed adjacent to 'Ca. L. asiaticus'-infected sweet orange (Citrus sinensis) plants with dodder (Cuscuta pentagona) already well established on them. Young dodder shoots still connected to the citrus were draped over the tomato plants and subsequently also became attached to the tomato stems. After 1 month, the tomato plants were detached from the citrus and most of the dodder removed. One month later, these tomato plants started to show vein clearing and subsequently the mature leaves became thicker and leathery. Some leaves showed blotchy mottle symptoms and some fruits became lopsided in a manner similar to HLB symptom expression on citrus. PCR amplification of the 'Ca. L. asiaticus' 16S rDNA with primers OI1/OI2c and the ß-operon with primers A2/J5 (1) revealed the presence of 'Ca. L. asiaticus' DNA. Sequence analysis confirmed that the sequences of the cloned amplicons were identical to those from the HLB-infected citrus source plant. Both conventional and quantitative real-time PCR (3) revealed a much lower abundance of 'Ca. L. asiaticus' DNA in tomato as compared with 'Ca. L. asiaticus'-infected citrus or periwinkle, indicating that 'Ca. L. asiaticus' bacteria multiplied at a lower titer in these tomato cultivars. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) S. E. Halbert et al. Fla. Entomol. 87:330, 2004. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) L. Z. Zhou et al. Plant Dis. 91:227, 2007.

In planta horizontal transfer of a major pathogenicity effector gene.

Xanthomonas citri pv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus, X. citri pv. aurantifolii, arose more recently in South America. Genomes of X. citri pv. aurantifolii strains carry two DNA fragments that hybridize to pthA, an X. citri pv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed that X. citri pv. aurantifolii strain B69 carried one functional pthA homolog, designated pthB, that was required to cause cankers on citrus. Gene pthB was found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenic X. citri pv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating that pthB conferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.

First Report of Dodder Transmission of Huanglongbing from Naturally Infected Murraya paniculata to Citrus.

Huanglongbing (HLB) or "greening" disease of citrus is caused by phloem-limited, uncultured bacteria in the genus "Candidatus Liberibacter". HLB is one of the most destructive diseases of citrus worldwide and is considered so dangerous to a U.S. citrus production that the USDA has listed "Ca. Liberibacter species" as a Select Agent. HLB is spread by the Asian citrus psyllid, Diaphorina citri, which was intercepted 40 times by APHIS/PPQ at U.S. ports between 1985 and 1998, became established in Florida by 1998, and more recently in Texas (1). HLB was first detected in the United States near Miami, FL during August 2005, and to date has been confirmed to have spread to 12 Florida counties. In addition to citrus, Murraya paniculata (orange jasmine) is a preferred host of D. citri, and retail trade in this ornamental shrub is strongly implicated in the distribution of D. citri (1). M. paniculata is reported to be a cryptic or largely asymptomatic host of "Ca. Liberibacter" (4), but another report concludes that the bacteria cannot replicate in M. paniculata (2). The epidemiological significance of murraya as a host for the HLB pathogen is therefore unclear. We report here the transmission of "Ca. Liberibacter asiaticus" from M. paniculata to citrus. Two M. paniculata plants, suspected of harboring "Ca. Liberibacter" because of their proximity to HLB-infected citrus and infested with D. citri, were removed from the field, treated with insecticide, and transferred to a quarantine facility. Both plants tested positive for "Ca. Liberibacter" by nested PCR using primers OI1 and OI2 (3) as the first set and primers CGO3F (RGG GAA AGA TTT TAT TGG AG) and CGO5R (GAA AAT AYC ATC TCT GAT ATC GT) as the second set. Two, young, sweet orange plants (Citrus sinensis) grown and maintained in psyllid-free greenhouses in Gainesville, FL were infected by dodder (Cuscuta pentagona) grown from seed. After the dodder had become well established on the orange plants, the orange plants were moved adjacent to the two murraya plants and the dodder from the citrus was draped over the murraya. Coinfection of murraya by dodder occurred within a few days. Sixty days later, both murraya plants, both sweet orange plants, and the connecting dodder all repeatedly tested positive for "Ca. Liberibacter" by nested PCR. Beginning 2 weeks later, the orange plants tested positive by standard PCR using primer set OI1 and OI2 or CGO3F and CGO5R, but remained without typical greening symptoms. Sequencing of the PCR products confirmed amplification of "Ca. L. asiaticus" DNA. We conclude that M. paniculata can serve as an infection source of a Select Agent since it can host the HLB pathogen for at least 2 months and the HLB pathogen can be transmitted to sweet orange during this time. References: (1) S. E. Halbert and K. L. Manjunath. Florida Entomol. 87:330, 2004. (2) T. H. Hung et al. J. Phytopathol. 148:321, 2000. (3) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996. (4) T. Li and C. Ke. Acta Phytophylacica Sin. 29:31, 2002.

The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison.

The gene (denoted aroQp.pheA) encoding the bifunctional P-protein (chorismate mutase-P/prephenate dehydratase) from Xanthomonas campestris was cloned. aroQp.pheA is essential for L-phenylalanine biosynthesis. DNA sequencing of the smallest subclone capable of functional complementation of an Escherichia coli phenylalanine auxotroph revealed a putative open reading frame (ORF) of 1200 bp that would encode a 43,438-Da protein. AroQp.PheA exhibited 51% amino acid identity with a Pseudomonas stutzeri homologoue and greater than 30% identities with AroQp.PheA proteins from Haemophilus influenzae, Neisseria gonorrhoeae, and a number of enteric bacteria. AroQp.PheA from X. campestris, when expressed in E. coli, possesses a 40-residue amino-terminal extension that is lysine-rich and that is absent in all of the AroQp.PheA homologues known at present. About 95% of AroQp.PheA was particulate and readily sedimented by low-speed centrifugation. Soluble preparations of cloned AroQp.PheA exhibited a native molecular mass of 81,000 Da, indicating that the active enzyme species is a homodimer. These preparations were unstable after purification of about 40-fold, even in the presence of glycerol, which was an effective protectant before fractionation. When AroQp.PheA was overproduced by a T7 translation vector, unusual inclusion bodies having a macromolecular structure consisting of protein fibrils were observed by electron microscopy. Insoluble protein collected at low-speed centrifugation possessed high catalytic activity. The single band obtained via SDS-PAGE was used to confirm the translational start via N-terminal amino acid sequencing. A perspective on the evolutionary relationships of monofunctional AroQ and PheA proteins and the AroQp.PheA family of proteins is presented. A serC gene located immediately upstream of X. campestris aroQp.pheA appears to reflect a conserved gene organization, and both may belong to a single transcriptional unit.

Cloning and Characterization of a Member of the Xanthomonas avr/pth Gene Family That Evades All Commercially Utilized Cotton R Genes in the United States.

ABSTRACT The highly virulent African strains of Xanthomonas campestris pv. malvacearum are quarantined pathogens in the United States and can evade or overcome all commercially utilized resistance (R) genes in cotton grown in the United States including the entire set of host differential lines used to distinguish 19 races of the pathogen. Nevertheless, the African strains carry multiple DNA fragments that strongly hybridize with members of the Xanthomonas avirulence (avr)/pathogenicity (pth) gene family. Since all previously tested members of the gene family confer avirulence against one or more R genes in cotton, strains carrying multiple members might not be expected to evade so many different R genes. The hybridizing DNA fragments were cloned from African strain XcmN and found to confer water-soaking ability to a nearly asymptomatic mutant strain of the pathogen. Restriction mapping, Southern hybridization, and DNA sequencing of the cloned fragments from XcmN were used to identify two water-soaking genes, pthN and pthN2, as new members of the Xanthomonas avr/pth gene family. The complete DNA sequence of pthN was obtained, and it is >94% identical with all other sequenced members of the gene family. Gene fusions of pthN with avrb6 (another family member) and other experiments revealed that the ability of African strain XcmN to water-soak cotton and avoid recognition by commercially used cotton R genes is determined by the specific repeats of multiple functional members of the Xanthomonas avr/pth gene family.

At least two separate gene clusters are involved in albicidin production by Xanthomonas albilineans.

Transposon mutagenesis was used to obtain mutations affecting production of the toxin albicidin in Xanthomonas albilineans, which causes leaf scald disease of sugarcane and is also pathogenic to corn. Transposon Tn5-gusA inserted randomly into genomic DNA of X. albilineans Xa23R1 at a frequency of 10(-4) to 10(-5) per recipient after conjugal transfer from Escherichia coli. Fifty prototrophic mutants defective in albicidin production were isolated from 7,100 Tn5-gusA insertional derivatives tested for toxin production by an antibiosis bioassay. EcoRI fragments containing Tn5 flanking sequences from two mutants (AM15 and AM40) were cloned and used to probe a wild-type Xa23R1 DNA library by colony hybridization. Nine cosmids showed homology to the AM15 probe, and six showed homology to the AM40 probe. Four cosmid clones hybridized to both probes. Forty-five of the 50 defective mutants were restored to albicidin production with two overlapping cosmid clones. Restriction mapping showed that these mutants span a genomic region of about 48 kb. At least one other gene cluster is also involved in albicidin production in Xa23R1. DNA fragments from the 48-kb cluster proved to be very specific to X. albilineans. Some mutants affected in albicidin production retain their ability to colonize sugarcane cultivated in vitro.

Intragenic recombination of a single plant pathogen gene provides a mechanism for the evolution of new host specificities.

Gene pthA is required for virulence of Xanthomonas citri on citrus plants and has pleiotropic pathogenicity and avirulence functions when transferred to many different xanthomonads. DNA sequencing revealed that pthA belongs to a family of Xanthomonas avirulence/pathogenicity genes characterized by nearly identical 102-bp tandem repeats in the central region. By inserting an nptI-sac cartridge into the tandemly repeated region of pthA as a selective marker, intragenic recombination among homologous repeats was observed in both Xanthomonas spp. and Escherichia coli. Intragenic recombination within pthA created new genes with novel host specificities and altered pathogenicity and/or avirulence phenotypes. Many pthA recombinants gained or lost avirulence function in pathogenicity assays on bean, citrus, and cotton cultivars. Although the ability to induce cell division (hyperplastic cankers) on citrus could be lost, this ability was not acquired on cotton or bean plants. Intragenic recombination therefore provides a genetic mechanism for the generation of multiple, different, and gratuitous avirulence genes from a single, required, host-specific pathogenicity gene.

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The opsX locus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide.

Xanthomonas campestris pv. citrumelo strain 3048 is the causal agent of citrus bacterial leaf spot disease and has a wide host range that includes rutaceous and leguminous plants. A spontaneous prototrophic mutant of strain 3048 (strain M28) that had lost virulence on citrus but retained virulence on bean plants was recovered. Growth studies in planta showed that M28 cells died rapidly in citrus leaves but grew normally in bean leaves. In addition to the loss of citrus-specific virulence, M28 displayed the following mutant phenotypes in culture: decreased growth rate, reduction of the amount of exopolysaccharide (to ca. 25% of the amount in 3048), loss of capsules, and significant alterations of the two 3048 lipopolysaccharide (LPS) bands visualized by silver stain on polyacrylamide gels, consistent with a defect(s) in LPS assembly. A 38-kb DNA fragment from a 3048 total DNA library that complemented the mutant phenotypes of M28 was identified. The 38-kb fragment did not hybridize to two similarly sized fragments carrying different hrp (hypersensitive response and pathogenicity) genes cloned from 3048. Subcloning, DNA sequence analyses, and gene disruption experiments were used to identify a single gene, opsX (for outer-membrane polysaccharide), responsible for the mutant phenotypes of M28. At least one other gene downstream from opsX also affected the same phenotypes and may be part of a gene cluster. We report here the DNA sequence and transcriptional start site of opsX. A search of protein sequence data bases with the predicted 31.3-kDa OpsX sequence found strong similarity to Lsi-1 of Neisseria gonorrhoeae and RfaQ of Escherichia coli (both are involved in LPS core assembly). The host-specific virulence function of opsX appears to involve biosynthesis of the extracellular polysaccharide and a complete LPS. Both may be needed in normal amounts for protection from citrus, but not bean, defense compounds.

Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes.

Six plasmid-borne avirulence (avr) genes were previously cloned from strain XcmH of the cotton pathogen, Xanthomonas campestris pv. malvacearum. We have now localized all six avr genes on the cloned fragments by subcloning and Tn5-gusA insertional mutagenesis. None of these avr genes appeared to exhibit exclusively gene-for-gene patterns of interactions with cotton R genes, and avrB4 was demonstrated to confer avr gene-for-R genes (plural) avirulence to X. c. pv. malvacearum on congenic cotton lines carrying either of two different resistance loci, B1 or B4. Furthermore, the B1 locus appeared to confer R gene-for-avr genes resistance to cotton against isogenic X. c. pv. malvacearum strains carrying any one of three avr genes: avrB4, avrb6, or avrB102. Restriction enzyme, Southern blot hybridization, and DNA sequence analyses showed that the XcmH avr genes are all highly similar to each other, to avrBs3 and avrBsP from the pepper pathogen X. c. pv. vesicatoria, and to the host-specific virulence gene pthA from the citrus pathogen X. citri. The XcmH avr genes differed primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central portions of the genes, repeated from 14 to 23 times in members of this gene family. The complete nucleotide sequence of avrb6 revealed that it is 97% identical in DNA sequence to avrB4, avrBs3, avrBsP, and pthA and that 62-bp inverted terminal repeats mark the boundaries of homology between avrb6 and all members of this Xanthomonas virulence/avirulence gene family sequenced to date. The terminal 38 bp of both inverted repeats are highly similar to the 38-bp consensus terminal sequence of the Tn3 family of transposons. Up to 11 members of the avr gene family appear to be present in North American strains of X. c. pv. malvacearum, including XcmH. The high level of homology observed among these avr genes and their presence in multiple copies may explain the gene-for-genes interactions and also the observed high frequencies (10(-3) to 10(-4) per locus) of X. c. pv. malvacearum race change mutations. Five spontaneous race change mutants of XcmH suffered avr locus deletions, strongly indicating intergenic recombination as the primary mechanism for generating new races in X. c. pv. malvacearum.

An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts.

The pathogenicity gene, pthA, of Xanthomonas citri is required to elicit symptoms of Asiatic citrus canker disease; introduction of pthA into Xanthomonas strains that are mildly pathogenic or opportunistic on citrus confers the ability to induce cankers on citrus (S. Swarup, R. De Feyter, R. H. Brlansky, and D. W. Gabriel, Phytopathology 81:802-809, 1991). The structure and the function of pthA in other xanthomonads and in X. citri were further investigated. When pthA was introduced into strains of X. phaseoli and X. campestris pv. malvacearum (neither pathogenic to citrus), the transconjugants remained nonpathogenic to citrus and elicited a hypersensitive response (HR) on their respective hosts, bean and cotton. In X. c. pv. malvacearum, pthA conferred cultivar-specific avirulence. Structurally, pthA is highly similar to avrBs3 and avrBsP from X. c. pv. vesicatoria and to avrB4, avrb6, avrb7, avrBIn, avrB101, and avrB102 from X. c. pv. malvacearum. Surprisingly, marker-exchanged pthA::Tn5-gusA mutant B21.2 of X. citri specifically lost the ability to induce the nonhost HR on bean, but retained the ability to induce the nonhost HR on cotton. The loss of the ability of B21.2 to elicit an HR on bean was restored by introduction of cloned pthA, indicating that the genetics of the nonhost HR may be the same as that found in homologous interactions involving specific avr genes. In contrast with expectations of homologous HR reactions, however, elimination of pthA function (resulting in loss of HR) did not result in water-soaking or even moderate levels of growth in planta of X. citri on bean; the nonhost HR, therefore, may not be responsible for the "resistance" of bean to X. citri and may not limit the host range of X. citri on bean. The pleiotropic avirulence function of pthA and the heterologous HR of bean to X. citri are both evidently gratuitous.

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.

Small, stable shuttle vectors for use in Xanthomonas.

Plasmids from three broad-host-range (bhr) incompatibility groups (Inc) were evaluated for use as cloning vectors in Xanthomonas campestris pv. malvacearum (Xcm), the causal agent of bacterial blight of cotton. The IncP vectors pLAFR3 and pVK102 could not be introduced into Xcm at a significant frequency (less than 1 x 10(-10] and IncQ vectors such as pKT210 were unstable in their maintenance and tended to delete cloned inserts. IncW vectors such as pSa747 also were lost readily from Xcm in the absence of selection pressure. We constructed two plasmids, pUFR027 and a cosmid derivative, pUFR034, which have proven useful as cloning vectors in Xcm and other xanthomonads. They contain the pSa origin of DNA replication, the partition locus parA from the Agrobacterium plasmid pTAR, a neomycin-resistance selection marker, and alacZ alpha cassette with cloning sites. pUFR027 is 9.3 kb, and pUFR034 is 8.7 kb in size. They can be mobilized by conjugation into Xcm at a frequency of approx. 1 x 10(-6) per recipient and are maintained stably (greater than 95% retention over 36 generations without selection pressure) in both broth culture and in planta. The plasmids were introduced and maintained stably in X. citri, and in X. campestris pathovars campestris, citrumelo, vesicatoria and translucens, and were moderately stable in X. phaseoli. No effects of the plasmids on pathogenicity have been observed.

Gene-for-gene interactions of five cloned avirulence genes from Xanthomonas campestris pv. malvacearum with specific resistance genes in cotton.

A total DNA clone bank of a strain of Xanthomonas campestris pv. malvacearum (Xcm) was constructed in the cosmid vector pSa747 and transfected into Escherichia coli. The Xcm strain carries at least nine identifiable avirulence (A) genes. Clones in E. coli were mated individually into a recombination-proficient Xcm isolate carrying no known A genes. Screening was for incompatibility on congenic cotton host lines that differ by single specific resistance (R) genes. Ten different cosmid clones conferring race-specific avirulence were recovered. In most cases, the same A gene clone was recovered independently several times. Using the congenic host lines and the merodiploid transconjugant pathogen strains, five of the A genes were shown to specifically interact, gene-for-gene, with individual R genes in the congenic cotton lines. Some A/R gene interactions appeared qualitatively different from others, suggesting that the physiological mechanism(s) of gene-for-gene specified incompatibility may be unique to the interactive gene pair. All A genes appeared to be chromosomally determined, three were found linked on a single 32-kilobase clone, and the rest were spaced more than 31 kilobases apart. Colinearity of the cosmid inserts with the Xcm recipient (carrying no known A genes) chromosome was demonstrated in two of the three tested. This and other evidence suggests that at least some A genes in bacteria may have the equivalent of virulence (a) alleles. The genetics of race specificity in this phytopathogenic bacterium appeared in all respects to be identical to that found in phytopathogenic fungi.