Koen Goethals

Broad Host Range and Promoter Selection Vectors for Bacteria that Interact with Plants

A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed. Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571. They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E. coli (Y. Itoh, J.M. Watson, D. Haas, and T. Leisinger, Plasmid 11:206-220, 1984). Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene. These vectors facilitate the study of gene expression in bacteria under particular environmental conditions. This is illustrated by the expression of the gusA gene under the control of a nod promoter in A. caulinodans nodulating stem-located infection sites on Sesbania rostrata.

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

ABSTRACT 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.

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: nucleotide sequence and plant-inducible expression

SummaryAzorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium mehloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nod[ gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible β-galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.

The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications.

Abstract.Rhodococcus fascians is a Gram-positive bacterium that infects dicotyledonous and monocotyledonous plants, leading to an alteration in the normal growth process of the host. The disease results from the modulation of the plant hormone balances, and cytokinins are thought to play an important role in the induction of symptoms. Generally, on the aerial parts of the plants, existing meristems were found to be most sensitive to the action of R. fascians, but, depending on the infection procedure, differentiated tissues as well gave rise to shoots. Similarly, in roots not only actively dividing cells, but also cells with a high competence to divide were strongly affected by R. fascians. The observed symptoms, together with the determined hormone levels in infected plant tissue, suggest that auxins and molecules of bacterial origin are also involved in leafy gall formation. The complexity of symptom development is furthermore illustrated by the necessary and continuous presence of the bacteria for symptom persistence. Indeed, elimination of the bacteria from a leafy gall results in the further development of the multiple embryonic buds of which it consists. This interesting characteristic offers novel biotechnological applications: a leafy gall can be used for germplasm storage and for plant propagation. The presented procedure proves to be routinely applicable to a very wide range of plants, encompassing several recalcitrant species.

FUCOSYLATION AND ARABINOSYLATION OF NOD FACTORS IN AZORHIZOBIUM CAULINODANS : INVOLVEMENT OF NOLK, NODZ AS WELL AS NOEC AND/OR DOWNSTREAM GENES

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild‐type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D‐arabinosyl and/or an L‐fucosyl branch. Nod factors produced by Tn5‐insertion mutants in nodZnoeC, and the separate nolK locus, were analysed by thin‐layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK. Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP‐fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ. In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans. Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP‐fucose.

Leafy Gall Formation Is Controlled by fasR, an AraC-Type Regulatory Gene in Rhodococcus fascians

Rhodococcus fascians can interact with many plant species and induce the formation of either leafy galls or fasciations. To provoke symptoms, R. fascians strain D188 requires pathogenicity genes that are located on a linear plasmid, pFiD188. The fas genes are essential for virulence and constitute an operon that encodes, among other functions, a cytokinin synthase gene. Expression of the fas genes is induced by extracts of infected plant tissue only. We have isolated an AraC-type regulatory gene, fasR, located on pFiD188, which is indispensable for pathogenesis and for fas gene expression. The combined results of our experiments show that in vitro expression of the fas genes in a defined medium is strictly regulated and that several environmental factors (pH, carbon and nitrogen sources, phosphate and oxygen content, and cell density) and regulatory proteins are involved. We further show that expression of the fas genes is controlled at both the transcriptional and the translational levels. The complex expression pattern probably reflects the necessity of integrating a multitude of signals and underlines the importance of the fas operon in the pathogenicity of R. fascians.

The plant pathogen Rhodococcus fascians colonizes the exterior and interior of the aerial parts of plants.

Rhodococcus fascians is a plant-pathogenic bacterium that causes malformations on aerial plant parts, whereby leafy galls occur at axillary meristems. The colonization behavior on Nicotiana tabacum and Arabidopsis thaliana plants was examined. Independent of the infection methods, R. fascians extensively colonized the plant surface where the bacteria were surrounded by a slime layer. R. fascians caused the collapse of epidermal cells and penetrated intercellularly into the plant tissues. The onset of symptom development preceded the extensive colonization of the interior. The meristematic regions induced by pathogenic strain D188 were surrounded by bacteria. The nonpathogenic strain, D188-5, colonized the exterior of the plant equally well, but the linear plasmid (pFiD188) seemed to be involved in the penetration efficiency and colonization of tobacco tissues.

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.

Patterns of phenolic compounds in leafy galls of tobacco

The chemical composition of ethanolic and aqueous extracts from leafy galls produced after infection of Nicotiana tabacum L. plants with Rhodococcus fascians was drastically changed compared to uninfected controls. Chlorogenic acid was abundant both in uninfected and infected plants, but caffeic acid and another cinnamoyl analogue were new in leafy galls. The most pronounced product induced in leafy galls was identified as 7-O-methyl-6-O-β-D-glucopyranosyl coumarin (7-methyl esculin). This is the first report of the presence of this coumarin derivative in tobacco. Interestingly, 7-methyl esculin did not accumulate in the presence of avirulent R. fascians strains nor was it found in leafy galls on other plant species. However, it did appear in crown galls induced by Agrobacterium tumefaciens on tobacco plants. Intriguingly, none of the phenolics known to accumulate in Solanaceae under pathogen attack were found in leafy galls. 7-Methyl esculin barely affected growth of R. fascians nor was it catabolized. Microscopical analysis showed that autofluorescent compounds were located mainly in the abundant meristematic regions of the leafy galls. We postulate that 7-methyl esculin might locally influence plant cell division.

The att locus of Rhodococcus fascians strain D188 is essential for full virulence on tobacco through the production of an autoregulatory compound

The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified. The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence. To gain insight into the function of the att locus, sequence and expression analyses were performed. The att locus contains nine open reading frames homologous to arginine and β‐lactam biosynthetic genes. att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR‐type transcriptional regulator. The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts. Because the induction of the fas locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R. fascians.