Virulence genes of the phytopathogen Rhodococcus fascians show specific spatial and temporal expression patterns during plant infection

The phytopathogenic bacterium Rhodococcus fascians provokes shoot meristem formation and malformations on aerial plant parts, mainly at the axils. The interaction is accompanied by bacterial colonization of the plant surface and tissues. Upon infection, the two bacterial loci required for full virulence, fas and att, were expressed only at the sites of symptom development, although their expression profiles differed both spatially and temporally. The att locus was expressed principally in bacteria located on the plant surface at early stages of infection. Expression of the fas locus occurred throughout infection, mainly in bacteria that were penetrating, or had penetrated, the plant tissues and coincided with sites of meristem initiation and proliferation. The implications for the regulation of virulence genes of R. fascians during plant infection are discussed.     

The plasmid-encoded chloramphenicol-resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins

The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.     

Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides

Summary A gene encoding acetolactate synthase was cloned from a chlorsulfuron-resistant mutant of Arabidopsis. The DNA sequence of the mutant gene differed from that of the wild type by a single base pair substitution. When introduced into tobacco by Ti plasmid-mediated transformation the gene conferred a high level of herbicide resistance. These results suggest that the cloned gene may confer agronomically useful levels of herbicide resistnace in other crop species, and that it may be useful as a selectable marker for plant transformation experiments.     

Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria.     

Biosynthesis of auxin by the gram-positive phytopathogen Rhodococcus fascians is controlled by compounds specific to infected plant tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

Biosynthesis of Auxin by the Gram-Positive Phytopathogen Rhodococcus fascians Is Controlled by Compounds Specific to Infected Plant Tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

Conjugal Transfer of a Virulence Plasmid in the Opportunistic Intracellular Actinomycete Rhodococcus equi

Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ∼81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.     

Preinfection treatment of resistant mice with CpG oligodeoxynucleotides renders them susceptible to friend retrovirus-induced leukemia

We recently reported that immunostimulatory oligodeoxynucleotides (CpG oligodeoxynucleotides [CpG-ODN]) were effective in postexposure treatment of retrovirus-induced disease (A. R. M. Olbrich et al., J. Virol. 76:11397-11404, 2002). We now show that the timing of treatment is a critical factor in treatment efficacy. In stark contrast to the success of postexposure treatments, we found that CpG treatment of susceptible mice prior to Friend retrovirus infection accelerated the development of virus-induced erythroleukemia. Furthermore, 70.8% of mice that were resistant to Friend virus-induced leukemia developed disease after inoculation of CpG-ODN before infection. The CpG pretreatment of these mice enhanced viral loads in their spleens and blood compared to controls that received ODN without CpG motifs. The main target cells of Friend virus, erythroid precursor cells and B cells, proliferated after CpG-ODN inoculation and provided an enlarged target cell population for viral infection. Our present findings together with our previous report demonstrate that CpG-ODN treatment of viral infections may be a double-edged sword that can result in an effective therapy but also in an acceleration of disease progression depending on the time point of treatment.     

Pretreatment of rats with anticollagen IgG renders them resistant to active type II collagen arthritis

Intravenous administration of 24 mg of affinity-purified rat anticollagen IgG induced a polyarthritis in recipient rats within 48 hr. This polyarthritis was transient and hind paw diameters returned to normal values within 12 days. IgG and C3 could be detected on the articular cartilage by immunofluorescence up to 16 days after antibody administration. Administration of 24 mg of rat anticollagen IgG to these antibody-treated rats did not induce a second phase of polyarthritis. In addition, recovered rats that had been pretreated with antibody were resistant to arthritis when Type II collagen was administered intradermally. In these rats, serum anticollagen IgG levels were significantly lower than in control rats which were not treated with antibody. Pretreatment of rats with anticollagen IgG did not have an effect on the severity or the incidence of adjuvant-induced arthritis. In addition, pretreatment of rats with anticollagen IgG did not have an effect on the development of a humoral response to ovalbumin.     

Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline

The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria. It has been shown previously (Burdett, V. (1986) J. Bacteriol. 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay. Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter. The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis. Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G. Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.     

Stimulation of the midkine/ALK axis renders glioma cells resistant to cannabinoid antitumoral action

Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas.     

Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol in vitro

Experimental evidence supports a role for E-cadherin in suppressing invasion, metastasis, and proliferation. Germline mutations of the E-cadherin represent the genetic cause of hereditary diffuse gastric cancer (HDGC). In this type of tumor, isolated cancer cells permeate the basal membrane and paradoxically survive in the gastric wall in the absence of contact with neighbor epithelial cells or with the extracellular matrix. This suggests that upon E-cadherin deregulation, cells acquired resistance to apoptosis. To test this hypothesis, CHO cells stably expressing either wild-type E-cadherin or the HDGC-related germline mutations T340A and V832M were seeded either on a thin layer of collagen type I or on plastic and then subjected to the apoptotic agent taxol. We found that in vitro functional E-cadherin renders cells more sensitive to the effect of taxol. Our results also indicate that this effect is associated to decreased level of the anti-apoptotic bcl-2 protein.     

Insulin renders diabetic rats resistant to acute ischemic stroke by arresting nitric oxide reaction with superoxide to form peroxynitrite

Background

The functions of free radicals on the effects of insulin that result in protection against cerebral ischemic insult in diabetes remain undefined. This present study aims to explain the contradiction among nitric oxide (NO)/superoxide/peroxynitrite of insulin in amelioration of focal cerebral ischemia–reperfusion (FC I/R) injury in streptozotocin (STZ)-diabetic rats and to delineate the underlying mechanisms. Long-Evans male rats were divided into three groups (age-matched controls, diabetic, and diabetic treated with insulin) with or without being subjected to FC I/R injury.

Results

Hyperglycemia exacerbated microvascular functions, increased cerebral NO production, and aggravated FC I/R-induced cerebral infarction and neurological deficits. Parallel with hypoglycemic effects, insulin improved microvascular functions and attenuated FC I/R injury in STZ-diabetic rats. Diabetes decreased the efficacy of NO and superoxide production, but NO and superoxide easily formed peroxynitrite in diabetic rats after FC I/R injury. Insulin treatment significantly rescued the phenomenon.

Conclusions

These results suggest that insulin renders diabetic rats resistant to acute ischemic stroke by arresting NO reaction with superoxide to form peroxynitrite.     

Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus

A reverse-genetics approach was applied to identify genes involved in Tomato yellow leaf curl virus (TYLCV) resistance, taking advantage of two tomato inbred lines from the same breeding program—one susceptible (S), one resistant (R—that used Solanum habrochaites as the source of resistance. cDNA libraries from inoculated and non-inoculated R and S plants were compared, postulating that genes preferentially expressed in the R line may be part of the network sustaining resistance to TYLCV. Further, we assumed that silencing genes located at important nodes of the network would lead to collapse of resistance. Approximately 70 different cDNAs representing genes preferentially expressed in R plants were isolated and their genes identified by comparison with public databases. A Permease I-like protein gene encoding a transmembranal transporter was further studied: it was preferentially expressed in R plants and its expression was enhanced several-fold following TYLCV inoculation. Silencing of the Permease gene of R plants using Tobacco rattle virus-induced gene silencing led to loss of resistance, expressed as development of disease symptoms typical of infected susceptible plants and accumulation of large amounts of virus. Silencing of another membrane protein gene preferentially expressed in R plants, Pectin methylesterase, previously shown to be involved in Tobacco mosaic virus translocation, did not lead to collapse of resistance of R plants. Thus, silencing of a single gene can lead to collapse of resistance, but not every gene preferentially expressed in the R line has the same effect, upon silencing, on resistance.     

Stimulation of ALK by the growth factor midkine renders glioma cells resistant to autophagy-mediated cell death

Δ?-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.     

Stimulation of ALK by the growth factor midkine renders glioma cells resistant to autophagy-mediated cell death

Δ⁹-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.     

An alternatively-spliced exon in the 5'-UTR of human ALAS1 mRNA inhibits translation and renders it resistant to haem-mediated decay

Haem controls its own synthesis in non-erythroid cells primarily by regulation of ALAS1 mRNA stability. Alternative splicing of human ALAS1 generates two mRNAs with different 5'-UTRs: a major one, where exon 1B is omitted, and a minor form containing exon 1B. We show that, unlike the major ALAS1 mRNA, the minor form was resistant to haem-mediated decay. Furthermore, we demonstrate that the ALAS1 5'-UTR alone did not confer haem-mediated decay upon a heterologous mRNA and the inclusion of exon 1B inhibited translation. These data suggest that translation of ALAS1 mRNA itself might be required for destabilisation in response to haem.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.