Phytopathogen Rhodococcus Fascians Research Articles

The C-terminal stretch of glycine-rich proline-rich protein (SbGPRP1) from Sorghum bicolor serves as an antimicrobial peptide by targeting the bacterial outer membrane protein.

The C-terminal stretch in SbGPRP1 (Sorghum glycine-rich proline-rich protein) acts as an antimicrobial peptide in the host innate defense mechanism. Cationic antimicrobial proteins or peptides can either bind to the bacterial membrane or target a specific protein on the bacterial membrane thus leading to membrane perturbation. The 197 amino acid polypeptide of SbGPRP1 showed disordered structure at the N-terminal end and ordered conformation at the C-terminal end. In the present study, the expression of N-SbGPRP1, C-SbGPRP1, and ∆SbGPRP1 followed by antimicrobial assays showed potential antimicrobial property of the C-terminal peptide against gram-positive bacteria Bacillus subtilis and phytopathogen Rhodococcus fascians. The SbGPRP1 protein loses its antimicrobial property when the 23 amino acid sequence (GHGGHGVFGGGYGHGGYGHGYGG) from position 136 to 158 is deleted from the protein. Thus, it can be concluded that the 23 amino acid sequence is vital for the said antimicrobial property. NPN assay, SEM analysis, and electrolyte leakage assays showed potent antimicrobial activity for C-SbGPRP1. Overexpression of the C-SbGPRP1 mutant protein in tobacco followed by infection with Rhodococcus fascians inhibited bacterial growth as shown by SEM analysis. To determine if C-SbGPRP1 might target any protein on the bacterial membrane we isolated the bacterial membrane protein from both Bacillus subtilis and Rhodococcus fascians. Bacterial membrane protein that interacted with the column-bound C-SbGPRP1 was eluted and subjected to LC-MS/MS. LC-MS/MS data analysis showed peptide hit with membrane protein YszA from Bacillus subtilis and a membrane protein from Rhodococcus fascians. Isolated bacterial membrane protein from Bacillus subtilis or Rhodococcus fascians was able to reduce the antimicrobial activity of C-SbGPRP1. Furthermore, BiFC experiments showed interactions between C-SbGPRP1 and YszA protein from Bacillus subtilis leading to the conclusion that bacterial membrane protein was targeted in such membrane perturbation leading to antimicrobial activity.

Isolation and Characterization of Rhodococcus spp. from Pistachio and Almond Rootstocks and Trees in Tunisia

The purpose of this study was to isolate and identify Rhodococcus spp. strains from almond and pistachio rootstocks and trees in Tunisia. Twenty-eight strains were identified through 16S rDNA and vicA genes amplification and sequencing. Pea bioassay was performed to determine the pathogenicity of the strains. Representative 16S rDNA and vicA sequences of eight strains from pistachio and seven strains from almond were closely related (>98% similarity) to Rhodococcus spp. accessions in GenBank. Phylogenetic analysis based on 16S rDNA sequences revealed that the yellow-colored strains clustered with phytopathogenic Rhodococcusfascians. The red and orange-colored strains were separated into a different group with R. kroppenstedtii and R. corynebacteiroides isolates. Eleven strains affected the pea seedlings’ growth and exhibited different levels of virulence. The number of shoots was significantly higher in seedlings inoculated with four Rhodococcus strains, whereas the other three strains caused up to 80% of plant height reduction and reduced root secondary growth compared to non-inoculated pea seedlings. These strains, most of which are epiphytes from asymptomatic hosts, showed strong pathogenicity during pea bioassay and were established endophytically in pea tissues. Ten att and five fas genes were detected in four strains and may represent a novel model of plant pathogenic Rhodococcus virulence. The results of our survey showed that Rhodococcus is present but not prevalent in all visited orchards of almond and pistachio rootstocks and trees. Our surveys complemented the investments being made on ornamental species in Tunisia and unveiled the presence of undocumented plant-associated Rhodococcus spp. on economically important crops.

Glycine rich proline rich protein from Sorghum bicolor serves as an antimicrobial protein implicated in plant defense response.

Sorghum glycine rich proline rich protein (SbGPRP1) exhibit antimicrobial properties and play a crucial role during biotic stress condition. Several proteins in plants build up the innate immune response system in plants which get triggered during the occurrence of biotic stress. Here we report the functional characterization of a glycine-rich proline-rich protein (SbGPRP1) from Sorghum which was previously demonstrated to be involved in abiotic stresses. Expression studies carried out with SbGPRP1 showed induced expression upon application of phytohormones like salicylic acid which might be the key in fine-tuning the expression level. Upon challenging the Sorghum plants with a compatible pathogen the SbGprp1 transcript was found to be upregulated. SbGPRP1 encodes a 197 amino acid polypeptide which was bacterially-expressed and purified for in vitro assays. Gram-positive bacteria like Bacillus and phytopathogen Rhodococcus fascians showed inhibited growth in the presence of the protein. The NPN assay, electrolytic leakage and SEM analysis showed membrane damage in bacterial cells. Ectopic expression of SbGPRP1 in tobacco plants led to enhanced tolerance towards infection caused by R. fascians. Though the N-terminal part of the protein showed disorderness the C-terminal end was quite capable of forming several α-helices which was correlated with CD spectroscopic analysis. Here, we have tried to determine the structural model for the protein and predicted the association of antimicrobial activity with the C-terminal region of the protein.

First Report of Rhodococcus Isolates Causing Pistachio Bushy Top Syndrome on 'UCB-1' Rootstock in California and Arizona.

'UCB-1' (Pistacia atlantica × Pistacia integerrima) rootstock is a hybrid cultivar widely used by the U.S. pistachio industry. In the last three years, a large number of micropropagated UCB-1 pistachio rootstocks planted in California and Arizona orchards exhibited shortened internodes, stunted growth, swollen lateral buds, bushy/bunchy growth, stem galls with multiple buds, and twisted roots with minimal lateral branching. Field T-budding success in affected orchards was reduced to approximately 30% with unusual bark cracking often observed around the bud-union. The percentage of abnormal rootstocks within affected orchards varied from 10 to 90%. We have termed the cumulative symptoms "pistachio bushy top syndrome" (PBTS) to describe these affected trees. Two isolates, both containing virulence factors from the phytopathogen Rhodococcus fascians, were identified on symptomatic trees in field and nursery samples. Micropropagated UCB-1 trees inoculated with the Rhodococcus isolates exhibited stunted growth, shortened internode length, swollen lateral buds, sylleptic branching, and differences in root morphology, compared with control UCB-1 trees. To our knowledge, this is the first report of Rhodococcus isolates, related to Rhodococcus fascians, causing disease on a commercial tree crop and the results presented indicate that this organism is responsible at least in part for PBTS in California and Arizona.

Metabolomic-Based Study of the Leafy Gall, the Ecological Niche of the Phytopathogen Rhodococcus fascians, as a Potential Source of Bioactive Compounds

Leafy gall is a plant hyperplasia induced upon Rhodococcus fascians infection. Previously, by genomic and transcriptomic analysis, it has been reported that, at the early stage of symptom development, both primary and secondary metabolisms are modified. The present study is based on the hypothesis that fully developed leafy gall, could represent a potential source of new bioactive compounds. Therefore, non-targeted metabolomic analysis of aqueous and chloroform extracts of leafy gall and non-infected tobacco was carried out by 1H-NMR coupled to principal component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). Polar metabolite profiling reflects modifications mainly in the primary metabolites and in some polyphenolics. In contrast, main modifications occurring in non-polar metabolites concern secondary metabolites, and gas chromatography and mass spectrometry (GC-MS) evidenced alterations in diterpenoids family. Analysis of crude extracts of leafy galls and non-infected tobacco leaves exhibited a distinct antiproliferative activity against all four tested human cancer cell lines. A bio-guided fractionation of chloroformic crude extract yield to semi-purified fractions, which inhibited proliferation of glioblastoma U373 cells with IC50 between 14.0 and 2.4 μg/mL. Discussion is focused on the consequence of these metabolic changes, with respect to plant defense mechanisms following infection. Considering the promising role of diterpenoid family as bioactive compounds, leafy gall may rather be a propitious source for drug discovery.

Plant‐derived auxin plays an accessory role in symptom development upon Rhodococcus fascians infection

The biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainly through its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation. Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstrated that the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutant tryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis (Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towards R. fascians infection, although bacterial colonization and virulence gene expression were not impaired. These observations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore, the increased auxin production and, possibly, the accumulating bacterial cytokinins in infected plants modified the polar auxin transport so that new auxin maxima were repetitively established and distributed, a process that is imperative for symptom onset and maintenance. Based on these findings, we extend our model of the mode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.

Metabolic Shift in the Phytopathogen Rhodococcus fascians in Response to Cell-Free Extract of Infected Tobacco Plant Tissues

The phytopathogen Rhodococcus fascians induces the development of leafy gall, which is considered to be its ecological niche. To obtain a view of the metabolic changes occurring in R. fascians during this process, an in vitro system was used where bacteria are grown in the presence of a leafy gall extract, a condition mimicking that found by the bacteria in infected plants. Proteins of R. fascians grown for 24 h under these conditions were displayed by two-dimensional polyacrylamide gel electrophoresis. Fifteen polypeptides showing a differential accumulation in response to the inducing conditions were analyzed by mass spectrometry. Two polypeptides potentially linked to the Krebs cycle, a pyruvate dehydrogenase and a fumarate hydratase, were further characterized and shown to be downregulated at the transcriptional level. The identification of these two enzymes suggests that R. fascians may shift its metabolism during the interaction with plants from the Krebs cycle to the glyoxylate shunt.

Biosynthesis of Auxin by the Gram-Positive PhytopathogenRhodococcus fasciansIs 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.

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.

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.

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.