Rhodococcus fascians
Encyclopedia
Rhodococcus fascians is a Gram positive bacteria
l phytopathogen
that causes leafy gall disease
. R. fascians is the only phytopathogenic member of the Rhodococcus
genus; its host range includes both dicotyledonous and monocotyledonous hosts. Because it commonly afflicts tobacco
(Nicotiana
) plants, it is an agriculturally significant pathogen.
, pleiomorphic, actinomycete that is nonmotile and does not form spores. When grown on the surface of an agar plate, colonies are orange in color and appear both smooth or rough.
, which development depends on the plant's cultivar
, plant's age, and the bacterial strain.
Leaf deformation consists of widening of parenchyma
and growth of vascular system, resulting in wrinkling of laminae and widening of veins. Leafy gall is a gall originated from a bud
which would not develop under normal conditions.
All effects coming from the infection of R. fascians do not depend 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 a junction or cavity of a 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.
in R. fascians is controlled by genes on a plasmid
(strains lacking that plasmid are 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.
The 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
, for fas a main role in virulence was proposed . Gene fasR is an araC-like transcriptional regulator. Its transcription can be induced in vitro in cultures containing certain carbon
sources (such as glucose
, sucrose
, arabinose
, glycerol
, pyruvate, mannitol
, mannose
) or nitrogen
sources (such as histidine
), and is influenced by culture pH
and optical density. Also, fasR can be induced by gall extract created by virulent strain.
The operon codifies for genes involved in cytokinin
synthesis and degradation (orf 4,5,6), in particular for an isopentenyl transferase, a cytokinin oxidase and a glutation-s transferase.
The orf1,2,3 transcribe for a cytochrome
450, a ferridoxine containing also a pyruvate dehydrogenase
alfa-like domain and a pyruvate dehydrogenase beta subunit. It was supposed that the first three genes supply energy for the synthesis and degradation of cytokinin, performed by the last three genes of the operon: R. fascians can actually produce and degradate zeatin
and isopentenil adenine
.
The compound cytokin oxidase(orf4) can also create adenine with a reactive nitrogen in position 6, which can react with other lateral chains, to form cytokininn-like compounds, more efficient in inducing plant tissue growth.
The att is an operon composed of nine genes: attR, a transcriptional regulator, attX, a gene including domains for transmembrane localization (perhaps needed for exportation of compounds made by other att genes), and several genes attA-attH. Many point and Δatt mutants show an attenuated virulence.
Gene attR is a transcriptional factor including a helix-turn-helix
motif. Its transcription is regulated by the same factors that regulate fasR transcription, but with a higher intensity, suggesting, with the attenuation of virulence in att mutants, that att may regulate fas transcription. Transcription of att operon is regulated with a quorum-sensing
mechanism: indeed, density of cultures can influence transcription of attR, and leafy gall extracts coming from galls made by att mutant strains are less effective on transcription of attR.
Genes attA-attH may be involved in synthesis of compounds needed for transcription of attR and attX. In fact, attA, attD & attH are involved in betalactamase synthesis, but no traces of those compounds were found in culture supernatants.
The hyp codifies for an RNA-helicase; mutants for this gene are hypervirulent. Also, hyp is involved in post transcriptional control of virulence-related genes, maybe on fas products.
Operon vic is an operon made of five genes, located on the bacterial chromosome. The only known gene is vicA, the fourth gene in the operon, whose product is a Mas homologue, a protein needed for the switch from citric acid cycle
to glyoxylate cycle
, both for metabolic reasons and to avoid glyoxylate accumulation, which is toxic for the bacteria. Mutations in vicA reduce virulence due to incapacity of R. fascians to resist glyoxylate accumulation.
, infection of R. fascians leads to hyperexpression of a cytochrome P450, homologue to a gene involved in inactivation of abscisic acid
in Arabidopsis thaliana
, of a gibberellic acid
oxidase, which inactivates this hormone and its precursors, a proline
dehydrogenase, which has its transcription induced by cytokinin and turns proline into glutamic acid
, and a factor involved in molybdenum
cofactor, needed for sulfur
, carbon and nitrogen metabolism control and for abscisic acid synthesis.
and cytokinin, such as: formation of green islands on leaves, wrinkling of laminae, bud proliferation, delay of senescence, and inhibition of lateral roots. In fact, R. fascians can produce itself cytokinin, or cytokinin-like compounds: using orf4 and orf5 in the fas operon, it can stimulate infected plants to produce cytokinin, and it can produce indole-3-acetic acid
itself, using a pathway starting from tryptophan and passing through production of 3-indol-piruvic acid and 3-indol-acetaldeid. R.fascians can also degradate cytokinin to influence the cytokinin/auxin ratio.
Beside cytokinin and auxin, R. fascians acts on other hormones: in particular, it can block abscisic acid
and gibberellic acid
synthesis in infected plants. Abscisic acid represses growth, so a block of production is needed to allow proliferation of cells in leafy galls. Gibberellic acid controls cellular differentiation
, so its block is needed for maintenance of meristem
atic cells and for their proliferation.
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
l phytopathogen
Phytopathology
Plant pathology is the scientific study of plant diseases caused by pathogens and environmental conditions . Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants...
that causes leafy gall disease
Gall
Galls or cecidia are outgrowths on the surface of lifeforms caused by invasion by other lifeforms, such as parasites or bacterial infection. Plant galls are abnormal outgrowths of plant tissues and can be caused by various parasites, from fungi and bacteria, to insects and mites...
. R. fascians is the only phytopathogenic member of the Rhodococcus
Rhodococcus
Rhodococcus is a genus of aerobic, nonsporulating, nonmotile Gram-positive bacteria closely related to Mycobacteria and Corynebacteria. While a few species are pathogenic, most are benign and have been found to thrive in a broad range of environments, including soil, water, and eukaryotic cells...
genus; its host range includes both dicotyledonous and monocotyledonous hosts. Because it commonly afflicts tobacco
Tobacco
Tobacco is an agricultural product processed from the leaves of plants in the genus Nicotiana. It can be consumed, used as a pesticide and, in the form of nicotine tartrate, used in some medicines...
(Nicotiana
Nicotiana
Nicotiana is a genus of herbs and shrubs of the nightshade family indigenous to North and South America, Australia, south west Africa and the South Pacific. Various Nicotiana species, commonly referred to as tobacco plants, are cultivated and grown to produce tobacco. Of all Nicotiana species,...
) plants, it is an agriculturally significant pathogen.
Physiology and morphology
R. fascians is an aerobicAerobic organism
An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.Faculitative anaerobes grow and survive in an oxygenated environment and so do aerotolerant anaerobes.-Glucose:...
, pleiomorphic, actinomycete that is nonmotile and does not form spores. When grown on the surface of an agar plate, colonies are orange in color and appear both smooth or rough.
Virulence
R. fascians can be a pathogen of plants, both angiosperm or gymnosperm. Infected plants show typical symptoms, such as leaf deformation, witches broom and leaf gallGall
Galls or cecidia are outgrowths on the surface of lifeforms caused by invasion by other lifeforms, such as parasites or bacterial infection. Plant galls are abnormal outgrowths of plant tissues and can be caused by various parasites, from fungi and bacteria, to insects and mites...
, which development depends on the plant's cultivar
Cultivar
A cultivar'Cultivar has two meanings as explained under Formal definition. When used in reference to a taxon, the word does not apply to an individual plant but to all those plants sharing the unique characteristics that define the cultivar. is a plant or group of plants selected for desirable...
, plant's age, and the bacterial strain.
Leaf deformation consists of widening of parenchyma
Parenchyma
Parenchyma is a term used to describe a bulk of a substance. It is used in different ways in animals and in plants.The term is New Latin, f. Greek παρέγχυμα - parenkhuma, "visceral flesh", f. παρεγχεῖν - parenkhein, "to pour in" f. para-, "beside" + en-, "in" + khein, "to pour"...
and growth of vascular system, resulting in wrinkling of laminae and widening of veins. Leafy gall is a gall originated from a bud
Bud
In botany, a bud is an undeveloped or embryonic shoot and normally occurs in the axil of a leaf or at the tip of the stem. Once formed, a bud may remain for some time in a dormant condition, or it may form a shoot immediately. Buds may be specialized to develop flowers or short shoots, or may have...
which would not develop under normal conditions.
All effects coming from the infection of R. fascians do not depend on plant cells transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
(as for Agrobacterium tumefaciens
Agrobacterium tumefaciens
Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of dicot. It is a rod shaped, Gram negative soil bacterium...
or Agrobacterium rhizogenes
Agrobacterium rhizogenes
Agrobacterium rhizogenes is a Gram negative soil bacterium that produces hairy root disease in dicotyledonous plants.A...
), but on expression of virulence-related genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
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 a junction or cavity of a 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
VirulenceVirulence
Virulence is by MeSH definition the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism - its ability to cause disease - is determined by its...
in R. fascians is controlled by genes on a plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
(strains lacking that plasmid are not virulent) and on the chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
. Using deletion mutations, it was possible to identify three loci
Locus (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
on the plasmid: fas, att, and hyp, and one locus on the chromosome, vic.
The fas is an operon
Operon
In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create...
made of six genes (orf 1-6) and a regulatory gene, fasR. Because deletions of some fas genes give a non-virulent phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
, for fas a main role in virulence was proposed . Gene fasR is an araC-like transcriptional regulator. Its transcription can be induced in vitro in cultures containing certain carbon
Carbon
Carbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
sources (such as glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
, sucrose
Sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
, arabinose
Arabinose
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde functional group.For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or structurally analogous to D-glyceraldehyde.For sugars, the D/L...
, glycerol
Glycerol
Glycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
, pyruvate, mannitol
Mannitol
Mannitol is a white, crystalline organic compound with the formula . This polyol is used as an osmotic diuretic agent and a weak renal vasodilator...
, mannose
Mannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. Mannose is a C-2 epimer of glucose. It is not part of human metabolism, but is a component of microbial cell walls, and is therefore a target of the immune system and also of antibiotics....
) or nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
sources (such as histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
), and is influenced by culture pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
and optical density. Also, fasR can be induced by gall extract created by virulent strain.
The operon codifies for genes involved in cytokinin
Cytokinin
Cytokinins are a class of plant growth substances that promote cell division, or cytokinesis, in plant roots and shoots. They are involved primarily in cell growth and differentiation, but also affect apical dominance, axillary bud growth, and leaf senescence...
synthesis and degradation (orf 4,5,6), in particular for an isopentenyl transferase, a cytokinin oxidase and a glutation-s transferase.
The orf1,2,3 transcribe for a cytochrome
Cytochrome
Cytochromes are, in general, membrane-bound hemoproteins that contain heme groups and carry out electron transport.They are found either as monomeric proteins or as subunits of bigger enzymatic complexes that catalyze redox reactions....
450, a ferridoxine containing also a pyruvate dehydrogenase
Pyruvate dehydrogenase
Pyruvate dehydrogenase complex is a complex of three enzymes that transform pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the...
alfa-like domain and a pyruvate dehydrogenase beta subunit. It was supposed that the first three genes supply energy for the synthesis and degradation of cytokinin, performed by the last three genes of the operon: R. fascians can actually produce and degradate zeatin
Zeatin
Zeatin is a plant hormone derived from the purine adenine. It is a member of the plant growth hormone family known as cytokinins. Zeatin was first discovered in immature corn kernels from the genus Zea...
and isopentenil adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
.
The compound cytokin oxidase(orf4) can also create adenine with a reactive nitrogen in position 6, which can react with other lateral chains, to form cytokininn-like compounds, more efficient in inducing plant tissue growth.
The att is an operon composed of nine genes: attR, a transcriptional regulator, attX, a gene including domains for transmembrane localization (perhaps needed for exportation of compounds made by other att genes), and several genes attA-attH. Many point and Δatt mutants show an attenuated virulence.
Gene attR is a transcriptional factor including a helix-turn-helix
Helix-turn-helix
In proteins, the helix-turn-helix is a major structural motif capable of binding DNA. It is composed of two α helices joined by a short strand of amino acids and is found in many proteins that regulate gene expression...
motif. Its transcription is regulated by the same factors that regulate fasR transcription, but with a higher intensity, suggesting, with the attenuation of virulence in att mutants, that att may regulate fas transcription. Transcription of att operon is regulated with a quorum-sensing
Quorum sensing
Quorum sensing is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population. In similar fashion, some social insects use quorum sensing to determine where to nest...
mechanism: indeed, density of cultures can influence transcription of attR, and leafy gall extracts coming from galls made by att mutant strains are less effective on transcription of attR.
Genes attA-attH may be involved in synthesis of compounds needed for transcription of attR and attX. In fact, attA, attD & attH are involved in betalactamase synthesis, but no traces of those compounds were found in culture supernatants.
The hyp codifies for an RNA-helicase; mutants for this gene are hypervirulent. Also, hyp is involved in post transcriptional control of virulence-related genes, maybe on fas products.
Operon vic is an operon made of five genes, located on the bacterial chromosome. The only known gene is vicA, the fourth gene in the operon, whose product is a Mas homologue, a protein needed for the switch from citric acid cycle
Citric acid cycle
The citric acid cycle — also known as the tricarboxylic acid cycle , the Krebs cycle, or the Szent-Györgyi-Krebs cycle — is a series of chemical reactions which is used by all aerobic living organisms to generate energy through the oxidization of acetate derived from carbohydrates, fats and...
to glyoxylate cycle
Glyoxylate cycle
The glyoxylate cycle, a variation of the Tricarboxylic Acid Cycle, is an anabolic metabolic pathway occurring in plants, bacteria, protists, fungi and several microorganisms, such as E. coli and yeast. The glyoxylate cycle centers on the conversion of acetyl-CoA to succinate for the synthesis of...
, both for metabolic reasons and to avoid 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 transcription in infected plant
In tobaccoTobacco
Tobacco is an agricultural product processed from the leaves of plants in the genus Nicotiana. It can be consumed, used as a pesticide and, in the form of nicotine tartrate, used in some medicines...
, infection of R. fascians leads to hyperexpression of a cytochrome P450, homologue to a gene involved in inactivation of abscisic acid
Abscisic acid
Abscisic acid , also known as abscisin II and dormin, is a plant hormone. ABA functions in many plant developmental processes, including bud dormancy. It is degraded by the enzyme -abscisic acid 8'-hydroxylase.-Function:...
in Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
, of a gibberellic acid
Gibberellic acid
Gibberellic acid Gibberellic acid Gibberellic acid (also called Gibberellin A3, GA, and (GA3) is a hormone found in plants. Its chemical formula is C19H22O6. When purified, it is a white-to-pale-yellow solid....
oxidase, which inactivates this hormone and its precursors, a proline
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...
dehydrogenase, which has its transcription induced by cytokinin and turns proline into glutamic acid
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
, and a factor involved in molybdenum
Molybdenum
Molybdenum , is a Group 6 chemical element with the symbol Mo and atomic number 42. The name is from Neo-Latin Molybdaenum, from Ancient Greek , meaning lead, itself proposed as a loanword from Anatolian Luvian and Lydian languages, since its ores were confused with lead ores...
cofactor, needed for sulfur
Sulfur
Sulfur or sulphur is the chemical element with atomic number 16. In the periodic table it is represented by the symbol S. It is an abundant, multivalent non-metal. Under normal conditions, sulfur atoms form cyclic octatomic molecules with chemical formula S8. Elemental sulfur is a bright yellow...
, carbon and nitrogen metabolism control and for abscisic acid synthesis.
Role of phytohormones during infection
All the effects of R. fascians infection can be attributed to hormone hyperdosage. In particular, most of the effects are connected to auxinAuxin
Auxins are a class of plant hormones with some morphogen-like characteristics. Auxins have a cardinal role in coordination of many growth and behavioral processes in the plant's life cycle and are essential for plant body development. Auxins and their role in plant growth were first described by...
and cytokinin, such as: formation of green islands on leaves, wrinkling of laminae, bud proliferation, delay of senescence, and inhibition of lateral roots. In fact, R. fascians can produce itself cytokinin, or cytokinin-like compounds: using orf4 and orf5 in the fas operon, it can stimulate infected plants to produce cytokinin, and it can produce indole-3-acetic acid
Indole-3-acetic acid
Indole-3-acetic acid, also known as IAA, is a heterocyclic compound that is a phytohormone called auxin. This colourless solid is native plant compound, potent and the most important auxin...
itself, using a pathway starting from tryptophan and passing through production of 3-indol-piruvic acid and 3-indol-acetaldeid. R.fascians can also degradate cytokinin to influence the cytokinin/auxin ratio.
Beside cytokinin and auxin, R. fascians acts on other hormones: in particular, it can block abscisic acid
Abscisic acid
Abscisic acid , also known as abscisin II and dormin, is a plant hormone. ABA functions in many plant developmental processes, including bud dormancy. It is degraded by the enzyme -abscisic acid 8'-hydroxylase.-Function:...
and gibberellic acid
Gibberellic acid
Gibberellic acid Gibberellic acid Gibberellic acid (also called Gibberellin A3, GA, and (GA3) is a hormone found in plants. Its chemical formula is C19H22O6. When purified, it is a white-to-pale-yellow solid....
synthesis in infected plants. Abscisic acid represses growth, so a block of production is needed to allow proliferation of cells in leafy galls. Gibberellic acid controls cellular differentiation
Cellular differentiation
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...
, so its block is needed for maintenance of meristem
Meristem
A meristem is the tissue in most plants consisting of undifferentiated cells , found in zones of the plant where growth can take place....
atic cells and for their proliferation.