Rhodococcus fascians

Rhodococcus fascians (known as Corynebacterium fascians till 1984) is a gram positive, aerobic, pleiomorphic, nonmotile, non-sporeforming. Its colonies looks orange, both smooth or rought. R.fascians is the only phytopathogen specie among Rhodococcus genre, it can be indeed a pathogen for both angiosperm or gymnosperm.

Virulence
R.fascians can be a pathogen of plant, both angiosperm or gymnosperm. Infected plants show typical synthoms, as leaf deformation, witches broom and leaf gall, which development depends on plant's cultivar, plant's age and bacterial strain. Leaf deformation consists of widening of parenchyma and groth of vascular system, resultin in wrinkling of laminae and widening of veins. Leafy gall is a gall originated from a bud which would not develop in normal condition. All effects coming from the infection of R.fascians doesn't depends on plant cells transformation( as for Agrobacterium tumefaciens or Agrobacterium rhizogenes), but on expression of virulence related genes of bacterium and on the production of compounds that can interfere with normal plant growth and development. During the infection, R.fascians usually stays outside vegetal tissues, near junction or cavity of plant's cell walls, maybe to avoid environmental stresses. Presence of R.fascians was also observed in intercellular spaces inside tissues (in leaf or galls) and even inside cell walls. Presence of R.fascians on the infected plant is necessary not only for the initiation of infection but also for its maintenance.

Genes that control virulence
Virulence in R.fascians is controlled by genes on a plasmid (strains missing of that plasmid e not virulent) and on the chromosome. Using deletion mutations, it was possible to identify three loci on the plasmid, fas, att, and hyp, and one locus on the chromosome, vic.

fas is an operon made of six genes (orf 1-6) and a regulatory gene, fasR. Because deletions of some fas genes give a non-virulent phenotype, it was proposed for fas a main role in virulence. fasR is an araC-like transcriptional regulator. Its transcription can be induced in vitro in colture containing certain carbon sources (as glucose, sucrose, arabinose, glycerol, pyruvate, mannitol, mannose) or nitrogen souces (as histidine) and is influenced by colturepH and optical density. fasR can be induced also by gall extract created by virulent strain. The operon codify for genes involved in cytokinin synthesys and degradation (orf 4,5,6), in particular for an isopentenyl transferase, a cytokinin oxidase and a glutation-s transferase. orf1,2,3 transcribe for a cytochrome 450, a ferridoxine containing also a piruvate deidrogenase alfa-like domain and a piruvate deidrogenase beta subunit. It was suppposed that the first three genes supply energy fo the syntesys and degradation of cytokinin performed by the last three genes of the operon: R.fascians can actually produce and degradate zeatin and isopentenil adenin. cytokin oxidase(orf4) can also create adenin with a reactive nitrogen in position 6, which can react with other lateral chain to form cytokininn-like compounds, more efficient in inducing plant tisuess groth.

att is an operon made of nine genes: attR, a trascrptional regulator, attX, a gene including domanins for transmembrane localization, maybe neede fro exportation of compuonds made by other att genes, and genes attA-H. Many att mutants show an attenuated virulence. attR is a transcriptional factor including a helix-turn-helix motif. Its transcription is regulated by the same factors that regulates fasR transcription, but with an higher inensity, suggesting, with the attenuation of virulence in att mutants, that att may regulates fas transcription. Transcription of att operon is regulated with a quorum sensing mechanism: indeed, ottic density of coltures can influence transcription of attR and leafy gall extracts coming from galls made by att mutant strains are less effective on trascription of attR. attA-H may be involved in syntesis of compounds needed for transcription of attR and attX. in fact, attA, D,H are involved in betalactamase syntesis, but no traces of those compounds were found in colture supernatants.

hyp codifys for an RNA-helicase, mutants for this gene are hypervirulent. hyp is involved in post transcriptional control of virulence-related genes, maybe on fas products.

vic is an operon made of five genes, located on the bacterial chromosome. The only known gene is vicA, the forth gene on the operon, whose product is a Mas homologue, a protein needed for the switch from citric acid cycle to glyoxylate cycle, both for metabolic reason and for avioid glyoxylate accumulation, which is toxic for the bacteria. Mutations in vicA reduce virulence due to incapacity of R.fascians to resist glyoxylate accumulation

Induction of trascription in infected plant
In tobacco, infection of R.fascians leads to hyperexpression of a cytochrome P450, homologue to a gene involved in inactivation of abscissic acid in Arabidopsis thaliana, of a gibberellic acid oxidase, which inactivates this hormon and its precursors, a proline dehidrogenase, which has its trascription induced by cytokinin and turns proline into glutamic acid, and a factor involved in molybdenum cofactor, neded for sulfur, carbon and nitrogen metabolisms control and for abscissic acid syntesis.

Role of phytormones during infection
All the effects of R.fascians infection can be attributed to hormones hypredosage. In particular, most of the effects are connected to auxine and cytokinin, as formation of green islands on leafes, wrinkling of laminae, buds proliferation, delay of senescence and inhibition of lateral roots. In fact, R.fascians can produce itself cytokinin, or citokinin-like compounds, using orf4 and orf5 in fas operon, it can stimulate infected plants to produce cytokinin and it can produce itself indole-3-acetic acid, using a pathway starting from tryptophane and passing through production of 3-indol-piruvic acid and 3-indol-acetaldeid. R.fascians can also degradate cytokinin to influence cytokinin/auxin ratio. Beside cytokinin and auxine, R.fascians acts on other hormon: in particular, it can block abscissic acid and gibberellic acid syntesis in infected plants. Abscissic acid represses groth, so a blok of production is needed to allow proliferation of cells in leafy galls. Giberellic acid controls cellular differentiation, so its block is needed for maintenance of meristematics cells and for their proliferation.