Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Summary The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.     

Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

Electrotransformation of three pathovars of Xanthomonas campestris

Procedures for electrotransformation have been adapted for three pathovars of Xanthomonas campestris: campestris, vesicatoria and manihotis. Three differently sized plasmids (51, 9.3 and 3.3 kb) at different concentrations (10, 30 and 50 ng/sample) and different field strengths (10, 12, 14 and 18 kV/cm) were used. The efficiency of transformation was dependent on the recipient strain and the plasmid introduced. In general, a field strength of 14 kV/cm as well as a concentration of 30 ng plasmid DNA/sample seemed to be adequate for most conditions. Only X. campestris pv. vesicatoria strain 479 required a higher field strength for better efficiency. The plasmid size was inversely related to the efficiency of transformation; up to 1.6 × 10¹⁰, 5.3 × 10⁷ and 3.7 × 10⁵ transformants/g DNA were obtained using the pXG31 (3.3 kb), pUFR027 (9.3 kb) and RP4 (51 kb) plasmids, respectively.     

Inhibition of Seed Germination and Root Development Caused by Xanthomonas campestris pv. vesicatoria in Pepper and Tomato

Inoculation of pepper seeds with the leaf pathogen Xanthomonas campestris pv. vesicatoria inhibited pepper germination. The inhibitory effect, which was stronger in non-sterilized light textured soils, decreased with time, and after 20, days or more, there was no difference between inoculated and non-inoculated seeds. Inhibitory substance(s) within the cytoplasmatic fraction of pathogen cells inhibited the germination of non-host tomato seeds. No relationship between pathogenicity to pepper leaves and inhibition of pepper seed germination was detected. The inhibitory substance(s) was found in two out of four X. campestris pv. vesicatoria strains. Heat-killed bacteria suppressed growth of pepper but not tomato seedlings. It is, therefore, suggested that the inhibition of seed germination and the decrease in root development are different modes of X. campestris pv. vesicatoria pathogenesis toward pepper plants.     

Xanthan gum production by altered pathogenicity variants of Xanthomonas campestris

Summary Several morphologically different isolates of Xanthomonas campestris pv. campestris were obtained by treatment with N-methyl-N-nitro-N-nitrosoguanidine. These variants were used to infect Brassica plants where several degrees of virulence were found. The strains were cultured in order to produce polysaccharide, which was recovered by precipitation and subjected to physical and chemical characterization. A relationship was noted between virulence and parameters such as the final viscosity of the culture, the viscosifying capacity of the polymer and the amount of acetyl substituents in the gum. Infrared spectral analysis revealed that intramolecular interaction of gum constituents could play a significant role in virulence. It is suggested that the degree of virulence could be used as a criterion for selecting and isolating producers of high quality xanthan gum.     

Transfer of resistance to Xanthomonas campestris pv campestris into Brassica oleracea L. by protoplast fusion

Black rot caused by the bacterium Xanthomonas campestris pv campestris is one of the most serious diseases of Brassica oleracea. Since sources of resistance to the disease within B. oleracea are insufficient and control means are limited, the development of resistant breeding lines is extremely desirable. Certain lines of B. napus contain very high resistance controlled by a dominant gene, but crossing the two species sexually is very difficult. Therefore, somatic hybrids were produced by protoplast fusion between rapid cycling B. oleracea and a B. napus line highly resistant to X. campestris pv campestris. Hybrid identity was confirmed by morphological studies, flow cytometric estimation of nuclear DNA content, and analysis of random amplified polymorphic DNA (RAPD). Inoculations with the pathogen identified four somatic hybrids with high resistance. The resistant hybrid plants were fertile and set seed when selfed or crossed reciprocally to the bridge line 15 (Quazi 1988). Direct crosses to B. oleracea were unsuccessful, but embryo rescue facilitated the production of a first-backcross generation. The BC₁ plants were resistant to the pathogen. Progeny from the crosses to line 15 were all susceptible. Embryo rescue techniques were not obligatory for the development of a second-backcross generation, and several resistant BC₂ plants were obtained.     

Rearrangement,amplification, and assortment of mitochondrial DNA molecules in cultured cells of Brassica campestris

Summary We compared Brassica campestris mitochondrial and chloroplast DNAs from whole plants and from a 2-year-old cell culture. No differences were observed in the chloroplast DNAs (cpDNAs), whereas the culture mitochondrial DNA (mtDNA) was extensively altered. Hybridization analysis revealed that the alterations are due entirely to rearrangement. At least two inversions and one large duplication are found in the culture mtDNA. The duplication element is shown to have the usual properties of a plant mtDNA high frequency recombination repeat. The culture mtDNA exists as a complex heterogeneous population of rearranged and unrearranged molecules. Some of the culture-associated rearranged molecules are present in low levels in native plant tissue and appear to have sorted out and amplified in the culture. Other mtDNA rearrangements may have occurred de novo. In addition to alterations of the main mitochondrial genome, an 11.3 kb linear mtDNA plasmid present in whole plants is absent from the culture. Contrary to findings in cultured cells of other plants, small circular mtDNA molecules were not detected in the B. campestris cell culture.     

Initiation of primexine in freeze-substituted microspores of Brassica campestris

The processes involved in initiating the primexine were investigated during development of tetrads of microspores in Brassica campestris anthers using rapid freeze-substitution technology. The first event is the appearance of the primexine matrix. The second event is convolution of the microspore plasma membrane, followed by insertion of an electron opaque spacer into the plasma membrane crypts. A convoluted microspore plasma membrane is only recorded in those species where the final exine pattern is reticulate, comprising foot layer, bacula, and tectum. Our hypothesis is that the spacers demarcate the future interbacular cavities of the exine, so that the membrane peaks are the sites for probacula formation.     

The pigment ofXanthomonas populi is a nonbrominated aryl-heptaene belonging to xanthomonadin pigment group 11

Based on mass spectrometry and electronic absorption spectroscopy, the main pigment fromXanthomonas populi (synonym:Aplanobacter populi) was identified as a nonbrominated aryl-heptaene. TheX. populi pigment was indistinguishable—on the basis of cochromatography and electronic absorption spectroscopy—from an authentic sample of a xanthomonadin belonging to Pigment Group 11, previously found as minor components in strains belonging to theXanthomonas campestris taxospecies (specifically in theXanthomonas pruni andXanthomonas vesicatoria nomenspecies). Possession of a xanthomonadin pigment confirms the placement ofX. populi in the genusXanthomonas and this particular pigment serves to distinguishX. populi from the five other taxospecies presently assigned to that genus. TheXanthomonas sp. isolated fromSalix, which purportedly shows affinities toX. populi, forms a monobrominated aryl-polyene pigment and —on that basis—is unlikeX. populi.     

Ultrastructure of light cells in the dolphin thyroid

Summary The ultrastructural appearance of the light cells is described in the thyroid gland of the common (Pacific) dolphinDelphinus bairdi. The appearances of active light cells are discussed in relation to the production of thyrocalcitonin and the special osteological characteristics of marine mammals. The presence of nerve processes possibly associated with light cells is discussed.Also known as parafollicular cells (Nonidez, 1932) and C cells (Pearse, 1966).     

Molecular evolution of virulence in natural field strains of Xanthomonas campestris pv. vesicatoria

The avrBs2 avirulence gene of the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria triggers disease resistance in pepper plants containing the Bs2 resistance gene and contributes to bacterial virulence on susceptible host plants. We studied the effects of the pepper Bs2 gene on the evolution of avrBs2 by characterizing the molecular basis for virulence of 20 X. campestris pv. vesicatoria field strains that were isolated from disease spots on previously resistant Bs2 pepper plants. All field strains tested were complemented by a wild-type copy of avrBs2 in their ability to trigger disease resistance on Bs2 plants. DNA sequencing revealed four mutant alleles of avrBs2, two of which consisted of insertions or deletions of 5 nucleotides in a repetitive region of avrBs2. The other two avrBs2 alleles were characterized by point mutations with resulting single amino acid changes (R403P or A410D). We generated isogenic X. campestris pv. vesicatoria strains by chromosomal avrBs2 gene exchange to study the effects of these mutations on the dual functions of avrBs2 in enhancing bacterial virulence and inducing plant resistance by in planta bacterial growth experiments. The deletion of 5 nucleotides led to loss of avrBs2-induced resistance on Bs2 pepper plants and abolition of avrBs2-mediated enhancement of fitness on susceptible plants. Significantly, the point mutations led to minimal reduction in virulence function of avrBs2 on susceptible pepper plants, with either minimal (R403P allele) or an intermediate level of (A410D allele) triggering of resistance on Bs2 plants. Consistent with the divergent selection pressures on avrBs2 exerted by the Bs2 resistance gene, our results show that avrBs2 is evolving to decrease detection by the Bs2 gene while at the same time maintaining its virulence function.     

ThegrpD55 locus ofEscherichia coli appears to be an allele ofdnaB

Attempts to characterize thegrpD55 mutation ofEscherichia coli have led us to conclude that the gene had been assigned an incorrect map position. The mutation was found to cotransduce withmalF3089:: Tn10 (at 91.5 min) and adnaB-expressing plasmid was able to complement fully thegrpD55 defect in replication. These studies strongly suggest thatgrpD55 is an allele ofdnaB and is localized near 92 min on theE. coli linkage map.     

Genetic mapping and functional analysis of the tomato Bs4 locus governing recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 protein

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.     

Amplified Fragment Length Polymorphism (AFLP) reveals no genetic divergence of the Eastern Alpine endemic Oxytropis campestris subsp. tiroliensis (Fabaceae) from widespread subsp. campestris

Applying Amplified Fragment Length Polymorphism, we explored genetic differences between widespread Oxytropis campestris subsp. campestris and O. campestris subsp. tiroliensis, a presumed glacial relict restricted to a small area along the main chain of the Eastern Alps. We could not find genetic differences between the two taxa. Neither do the morphological characters given in the literature discriminate between them. Therefore Oxytropis campestris subsp. tiroliensis is unlikely a glacial relict that survived Pleistocene glaciations on nunataks, but rather a genetically insignificantly differentiated phenotype that arose in the course of postglacial recolonisation. There is no phylogeographical structure in O. campestris s.l. in the Alps most probably due to the fact that the taxon did not survive the cold stages of the Pleistocene in the interior of the Alps but immigrated to that region at a later date.     

AvrAC(Xcc8004), a type III effector with a leucine-rich repeat domain from Xanthomonas campestris pathovar campestris confers avirulence in vascular tissues of Arabidopsis thaliana ecotype Col-0

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC_Xcc8004, functions as an avirulence gene whose product seems to be recognized in vascular tissues.     

The tryptic peptide composition of the beta chains of hemoglobins A and B of the domestic cat (Felis catus)

Summary The tryptic peptides from the ^A and ^B chains of cat hemoglobins A and B have been isolated and the amino acid compositions determined. Differences between the two chains were found in two peptides,T-1 (GlySer) andT-14 (AsnSer and LysArg). The GlySer and LysArg substitutions are placed at-1 and-144 respectively from earlier work, and the third substitution, AsnSer at-139 is suggested from this work. In addition, the presence of a blocked amino terminus in ^B has been confirmed. Tentative sequences constructed by homology with known-chain structures suggest the occurrence of substitutions at _{1 1} contacts in ^A and ^B that may be functionally significant. There are at least 18 differences in amino acid composition between cat ^A and dog-chains and 22 differences between cat ^A and normal adult human-chains.     

Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. III. Dose-response pattern of mutation induction in UV-irradiated rev2ts cells

Summary Recent studies regarding the influence of cycloheximide on the temperature-dependent increase in survival and mutation frequencies of a thermoconditional rev2 mutant lead to the suggestion that the REV2-coded mutagenic repair function is UV-inducible. In the present study we show that stationary-phase rev2 ^ts cells are characterized by a biphasic linear-quadratic dose-dependence of mutation induction (mutation kinetics) of ochre alleles at 23° C (permissive temperature) but linear kinetics at the restrictive temperature of 36° C. Mathematical analysis using a model based on Poisson statistics and a further mathematical procedure, the calculation of apparent survival, support the assumption that the quadratic component of the reverse mutation kinetics investigated can be attributed to a UV-inducible component of mutagenic DNA repair controlled by the REV2 gene.