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.     

Chromosomal Locus That Affects Pathogenicity of Rhodococcus fascians

The gram-positive plant pathogen Rhodococcus fascians provokes leafy gall formation on a wide range of plants through secretion of signal molecules that interfere with the hormone balance of the host. Crucial virulence genes are located on a linear plasmid, and their expression is tightly controlled. A mutant with a mutation in a chromosomal locus that affected virulence was isolated. The mutation was located in gene vicA, which encodes a malate synthase and is functional in the glyoxylate shunt of the Krebs cycle. VicA is required for efficient in planta growth in symptomatic, but not in normal, plant tissue, indicating that the metabolic requirement of the bacteria or the nutritional environment in plants or both change during the interaction. We propose that induced hyperplasia on plants represents specific niches for the causative organisms as a result of physiological alterations in the symptomatic tissue. Hence, such interaction could be referred to as metabolic habitat modification.     

红球菌线型质粒pNSL1的克隆、测序和复制区的鉴定

从红球菌NS1中检测到两个线型质粒pNSL1和pNSL2。【目的】克隆、测序和分析pNSL1,并鉴定质粒的复制区。【方法】利用脉冲电泳方法从凝胶中回收大量的质粒DNA,进行鸟枪法克隆、测序和拼接,通过生物信息学分析和实验证明质粒的自主复制区。【结果】克隆、测序和拼接获得pNSL1全长为117252bp的序列,包括在红球菌中保守的1282bp端粒的序列。序列预测含有103个蛋白编码区,包括质粒的复制、分配、转移等功能基因。将pNSL1中一个与分枝杆菌质粒的复制基因同源的pNSL1.038及其上游的767bp非编码序列克隆到大肠杆菌质粒,电击转化珊瑚诺卡氏菌4.1037,获得了抗性转化子。【结论】克隆、测序了全长的线型质粒pNSL1,鉴定了质粒的复制区。     

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10⁵/μg of DNA to 10⁷/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.     

pH control of limonin debittering with entrapped Rhodococcus fascians cells

Summary Rhodococcus fascians cells were immobilized by entrapment in -carrageenan. The ability of the system to continuously degrade limonin was tested against pH. A burst of activity was observed when changing from pH 4.5 to 5.0, and a small increase could be seen above the latter value. Such behaviour was not only a response of the metabolic activity of the cells to changes in the medium pH, but to selectivity towards the chemical form of the limonin substrate, which also depends on pH. Additionally, the immobilized cells showed increased resistance against pH changes, since the system recovered almost full activity when the pH was restored to 7.0 after being operated for long periods at pH 4.0. The decrease in limonin-degrading capability of the immobilized cells at low pH values could be overcome by choosing an appropriate dilution rate.Offprint requests to: J. L. Iborra     

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N⁶-(∆²-isopentenyl)adenine and dimethylated N⁶-(∆²-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N⁶-(∆²-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N⁶-(∆²-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.The balance of phytohormones, such as cytokinins (CKs) and auxins, is finely controlled to maintain proper plant growth and development but is often disturbed following pathogen infection (Robert-Seilaniantz et al., 2007; Pieterse et al., 2012). As a virulence strategy, many phytopathogens synthesize phytohormones that cause aberrant organogenesis and modulate primary carbon metabolism that ultimately aids disease establishment (Jameson, 2000; Robert-Seilaniantz et al., 2007). For several pathogens, CK production is essential for virulence, and they carry genes for CK biosynthesis in a harbored plasmid (Jameson, 2000). Fungal pathogens employ CKs to form green islands with delayed senescence, whereas bacterial pathogens develop gall structures (Sakakibara et al., 2005; Walters et al., 2008; Giron et al., 2013). Rhodococcus fascians is a gram-positive actinomycete that causes symptoms ranging from leaf deformation to differentiated shooty outgrowths known as leafy galls in more than 150 different plant species (Goethals et al., 2001; Stes et al., 2011). In ornamental plants, such infections reduce their value and contribute to economic losses worldwide (Putnam and Miller, 2007). Leafy gall symptoms are reminiscent of CK overproduction and can be partially induced by exogenous application of CKs (Thimann and Sachs, 1966; Eason et al., 1996). Although several CKs have been isolated from R. fascians culture filtrates, a clear correlation with pathogenesis is lacking partially owing to the low concentration of bacterial CKs (Eason et al., 1996). A synergistic action by a mixture of bacterially produced CKs has been proposed, leading to persistent accumulation of CKs locally (Pertry et al., 2009). Nevertheless, to date, no virulence-associated CK analogs have been identified that could contribute to the infection symptoms.Naturally occurring CKs are adenine derivatives with different side chains at the N6 position. Major plant CKs are N⁶-prenylated adenine derivatives such as N⁶-(Δ²-isopentenyl)adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin, collectively known as isoprenoid CKs (Sakakibara, 2006). Among them, iP and tZ are the major CKs in Arabidopsis (Arabidopsis thaliana). iP is synthesized by adenosine phosphate-isopentenyl transferase (IPT) using dimethylallyl diphosphate (DMAPP) and adenosine phosphate as substrates (Sakakibara, 2006). tZ is formed by hydroxylation of the trans-end of the prenyl side chain of the iP nucleotide. CK homeostasis is governed by both biosynthesis and catabolism and has an important regulatory role in plant growth (Sakakibara, 2006; Werner et al., 2006). CK oxidase/dehydrogenase (CKX) is responsible for an irreversible reaction cleaving the unsaturated isoprenoid side chain that results in the formation of adenine and the corresponding aldehyde (Werner et al., 2006). In Arabidopsis, CKs are perceived by a subset of sensory His kinases, ARABIDOPSIS HIS KINASE2 (AHK2) to AHK4, which undergo a His-Asp phosphorelay leading to induction of direct target genes including type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes (Kieber and Schaller, 2010). This two-component signaling system has been implicated in mediating basal and pathogen-induced plant immunity (Choi et al., 2010; Argueso et al., 2012). For instance, infection of Arabidopsis plants by R. fascians reportedly activates type-A ARR5 expression with increased expression of AHK3 and AHK4, resulting in mitotic cell divisions that arrest the infected leaves in a meristematic state to establish a nutrient-rich niche (Depuydt et al., 2008, 2009; Pertry et al., 2010; Stes et al., 2011). As the infection progresses, IPT genes are switched off, whereas the expression of all CKX genes are strongly induced in symptomatic tissues (Depuydt et al., 2008).The virulence determinant of R. fascians is located within the fasciation (fas) locus, an operon encoding several genes involved in CK metabolism, indicating that CKs are essential for this plant-pathogen interaction (Stes et al., 2011). fas4 encodes IPT that catalyzes the rate-limiting step of CK biosynthesis and is vital for virulence (Stes et al., 2013). Interestingly, two methyltransferase-like genes are present upstream of the fas gene, whose functions have been unknown. Despite the presence of the fas genes in R. fascians, fewer known CKs have been detected compared with other gall-causing pathogens such as Pantoea agglomerans, Agrobacterium tumefaciens, and Pseudomonas savastanoi (Goethals et al., 2001). Further, the leafy gall phenotype is unique, not invoked by any of the above-mentioned pathogens, implying that the virulence of R. fascians might not be due to typical CKs alone (Goethals et al., 2001). R. fascians has long been hypothesized to produce CK analogs using similar or modified substrates (Goethals et al., 2001; Galis et al., 2005; Stes et al., 2011), but no such molecules have been discovered so far. Here, we report the identification and mode of biosynthesis for methylated cytokinins (MeCKs) as hormone mimics from R. fascians. These compounds are synthesized by two methyltransferases and FAS4. Their CK-like activity and higher in planta stability suggest a role for the methylated analogs as CK mimics that foster efficient pathogenesis.     

Purification and characterization of limonoate dehydrogenase from Rhodococcus fascians.

Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.     

Biosurfactant Production by Halotolerant Rhodococcus fascians from Casey Station, Wilkes Land, Antarctica

Isolate A-3 from Antarctic soil in Casey Station, Wilkes Land, was characterized for growth on hydrocarbons. Use of glucose or kerosene as a sole carbon source in the culture medium favoured biosynthesis of surfactant which, by thin-layer chromatography, indicated the formation of a rhamnose-containing glycolipid. This compound lowered the surface tension at the air/water interface to 27 mN/m as well as inhibited the growth of B. subtilis ATCC 6633 and exhibited hemolytic activity. A highly hydrophobic surface of the cells suggests that uptake occurs via a direct cell–hydrocarbon substrate contact. Strain A-3 is Gram-positive, halotolerant, catalase positive, urease negative and has rod–coccus shape. Its cell walls contained meso-diaminopimelic acid. Phylogenetic analysis based on comparative analysis of 16S rRNA gene sequences revealed that strain A-3 is closely related to Rhodococcus fascians with which it shares 100% sequence similarity. This is the first report on rhamnose-containing biosurfactant production by Rhodococcus fascians isolated from Antarctic soil.     

pH influence on the consumption of limonin species by Rhodococcus fascians cells

Summary The ability of Rhodococcus fascians cells to degrade limonin and limonin species (limonoate, limonoate-D-ring lactone and limonoate-A-ring lactone) was checked against pH. These studies showed a marked effect of pH on cell growth mainly due to substrate availability (limonin species). Evolution of limonin and its species within the medium were followed at different pH values. The best substrate for Rhodococcus fascians at pH 7.0 was limonoate whereas at pH 4.0 to 5.5 it appeared to be limonin. Results suggest that the citrus juice debittering process start only once the natural precursor of limonin (limonoate A ring lactone) has been transformed into limonin, the equilibrium displacement being governed by the citrus juice pH.     

Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains.

The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.     

De novo cortical cell division triggered by the phytopathogen Rhodococcus fascians in tobacco

Plant growth, development, and morphology can be affected by several environmental stimuli and by specific interactions with phytopathogens. In many cases, plants respond to pathogenic stimuli by adapting their hormone levels. Here, the interaction between the phytopathogen Rhodococcus fascians and one of its host plants, tobacco, was analyzed phenotypically and molecularly. To elucidate the basis of the cell division modulation and shoot primordia initiation caused by R. fascians, tobacco plants were infected at leaf axils and shoot apices. Adventitious meristems that gave rise to multiple-shoot primordia (leafy galls) were formed. The use of a transgenic line carrying the mitotic CycB1 promoter fused to the reporter gene coding for beta-glucuronidase from Escherichia coli (uidA), revealed that stem cortical cells were stimulated to divide in an initial phase of the leafy gall ontogenesis. Local cytokinin and auxin levels throughout the infection process as well as modulation of expression of the cell cycle regulator gene Nicta;CycD3;2 are discussed.     

Characterization of the 450-kb linear plasmid in a polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1

A strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 x 10(-5) transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.     

The fas locus of the phytopathogen Rhodococcus fascians affects mitosis of tobacco BY-2 cells

The effect of Rhodococcus fascians, the causal agent of leafy gall disease, on the mitotic behavior of synchronized tobacco Bright Yellow-2 (BY-2) cells was investigated. Incubation of aphidicolin-synchronized BY-2 cells with R. fascians cells specifically resulted in a broader mitotic index peak, an effect that was linked to an intact and expressed fas virulence locus. The obtained results pointed towards an effect of R. fascians on the prophase of mitosis. The relevance of these results to the virulence of the bacterium is discussed.     

Specificity of Antisera Against Rhodococcus fascians (Tilford) Goodfellow in Indirect Immunofluorescence

Abstract The possibility to obtain specific antisera to detect Rhodococcus fascians in imported lily bulbs is taken into consideration for not allowing the admission of this pathogen in Italy. For the production of specific antisera and in order to avoid the occurtence of cross-reactions between R. fascians and some of the most widespread soilborne, plant decay bacteria and with Erwinia carotovora subsp. carotovora a preliminary work for singling out the best immunogens, scheduleof immunization and antisera dilution for using in indirect immunofluorescence has been carried out. Living cells and heattreated cells were proved to be good immunogens and long-term immunization provided higher titers than short-term immunization. Toobtain a satisfactory specificity the dilution of the antisera is required. The 1: 800 dilution is quite effective in overcoming cross-reactions whereas undiluted antisera and antisera used at 1: 100 and 1: 200 dilution did not provide specificity.     

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.