BACTERIAL GALLS AND CANKERS
LEAFY GALL
LEAFY GALL
In 1927, Brown described fasciations on the crown of sweet pea ( Lathyrus
odoratus) as bunches of short, fleshy stems carrying misshaped and aborted leaves
and she proposed that this was a form of crown gall, indused by a weak or
specialized form of Agrobacterium tumefaciens . Currently , the bacterium
responsible for these fasciation is known as Rhodococcus fascians , a Gram –
psitive bacterium that infects diverse plant families and genera and provokes as
variety of disease symptoms, the most dramatic being the formation of leafy galls
as centers of abundant shoot meristem amplificatin.
Starting from its originally alleged connection with crown galls, leafy gall
formation was considered as another plant disease caused by bacterially produced
phytohormones. As parallelism with crown gall formation had been ruled out,
research was oriented toward the role of plant hormones in the galling syndrome.
Molecular analysis of the pathogenicity determinants of R. fascians indicates that
leafy gall formation is based on some elements common to Gram-negative galling
bacteria and on more specific elements that may relate to the highly particular
nature of leafy galls.
In this sense, R.fascians should not be regarded as a bacterium that acquired its
tumorigenic properties from well-described, Gram-negative bacteria. Molecular
studies of different gall-inducing bacteria show no recurring strategies for plant
gall formation. It would be erroneous to assume that all galls are alike, an idea
refuted by the existing literature. Although various gall-forming bacteria employ
some common elements during infection and symptom development, different
bacteria use distinctive systems to produce galls. R.fascians and leafy galls are no
exception to this rule.
THE CAUSAL AGENT OF LEAFY GALL FORMATION
Ten years after Brown's proposition, research by Tilford on the causal agent of the
sweet pea fasciations led to the isolation of the pathogen: a Gram-positive
bacterium that was named Phytomonasfascians. Although there was an agreement
on the distinct nature of the fasciating bacterium at that time, just three years later
Rhodococcus fascians
17
BACTERIAL GALLS AND CANKERS
LEAFY GALL
Lacey renamed the bacterium Bacterium fascians. The unclear taxonomic status
was resolved by classifying the pathogen as an Actinomycete, coryneform
bacterium, and the species name Corynebacterium fascians was proposed, a name
that was retained until 1984.
This proposal was not without controversy, because from the early 1940s until the
early 1980s discussion on the exact taxonomic position within the Actinomycetes
tossed the bacterium between the Nocardiae, Corynebacteriae , and the
Rhodococci. The controversy ended when Corynebacterium fascians was
reclassified as Rhodococcus fascians, based on chemical, genetic, and phenotypic
data.
Based on cell wall composition, DNA base composition (61-67% GC), and the
presence of tuberculostearic and mycolic acids, R. fascians belongs to the
Actinomycetes and is classified within the mycolic acid-containing genera.
Analysis of the SS and 16S rRNA sequences and a study of the rRNA operon
structure confirmed this taxonomic position. Rhodococcus, together with the
genera Gordona, Tsukamurella, Mycobacterium, and Nocardia, is now classified
into the family Mycobacteriaceae.
R.fascians is aerobic and pleiomorphic and can form branched hyphae that soon
break up in rods and cocci (nocardioform). It is non motile, non spore-forming and
produces moist cream to orange colonies that can be smooth or rough, a feature
that does not affect pathogenicity. Within the genus Rhodococcus, R.fascians is
the only known phytopathogen, although animal pathogens and symbionts have
been described.
After the bacterium had been isolated from sweet pea, in which it severely reduced
flower setting, it was later found in "cauliflower" strawberry plants, which carry a
large number of dwarfed buds, and in carnation, chrysanthemum, and asparagus.
Since then, the effects of R. fascians have been noticed primarily in ornamental
plants because infection usually results in decreased flower production or
deformed flowers. The disease is spread by infected plant material and infested
seeds or soils.
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
Dicotyledonous as well as monocotyledonous plants respond to R. fascians
infection and the host range encompasses 39 plant families and 86 genera. In its
broad host range, the bacterium resembles Agrobacterium and differs from other
gall-forming bacteria, such as Pseudomonas savastanoi and Erwinia herbicola,
which have a very restricted host range.
SYMPTOM DEVELOPMENT
Depending on different parameters, the morphological changes induced upon R.
fascians infection range from leaf deformation, witches' broom formation, and
fasciation to the initiation of leafy galls. In addition to the plant species or cultivar,
the age of the plant, the bacterial strain, and the bacterial growth conditions affect
symptom development.
Leaf deformation in tobacco is accompanied by a wrinkling of the lamina and a
swelling of the petioles and veins, resulting from enlarged parenchyma cells and
secondary growth of the vascular tissues. Patches of "green islands" occur on the
lamina, possibly representing sites with increased chloroplast content.
A witches' broom is characterized by a bunch of fleshy stems with misshapen and
aborted leaves that develop on the crown of the host plant. In peas, this is
accompanied by initial stimulation of epicotyl growth, but soon growth of the
terminal bud is blocked, and the plants become dwarfed and produce fewer
blossoms. This inhibition of the main shoot is not a consequence of trophic
competition with the lateral shoots but is endogenous and directly caused by the
bacteria. The hypertrophied shoots of witches' brooms contain regions with
actively dividing, meristematic cells, enlarged cortical parenchyma cells, and
multiple vascular bundles located at the center). R. fascians induced fasciations are
formed when several hypertrophied shoots coalesce, carrying malformed small
leaves with thickened petioles and veins.
Leafy galls represent the most severe symptom and originate from a local
amplification of multiple buds that are inhibited for outgrowth. They form
exclusively at the site of bacterial inoculation and no secondary leafy gall
formation at noninfected sites has been described. The presence of bacteria is
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
required for the multiplication of the shoots and also for gall persistence. The
inhibition of shoot outgrowth has been interpreted as an extreme form of apical
dominance whereby the shoot primordia mutually exclude their further
development provoking the formation of the densely packed gall. By inhibiting the
bacterial activity in isolated leafy galls, shoot outgrowth is activated. Eventually,
these shoots develop roots and grow into normal, fertile plants. Thus, in contrast to
crown galls that rely on the transgenic production of phytohormones, leafy galls
are non autonomous structures, and gall maintenance depends on the presence
activity of R. fascians.
At first, buds and meristematic tissue seemed to be the only sites of the plant
affected by R.fascians until leafy galls were found to also originate from leaf
margins and veins. Local infection of tobacco shoot axils revealed that R.fascians
induces cell division and de novo meristem formation in subepidermal cells. The
reactivation of cortical cell division was correlated with induction of the CYCB1;l
promoter, a mitotic marker, and with increased transcription of the CYCD3;2
gene, involved in the regulation of the Gl-to-S transition of the cell cycle (92).
These data show that R.fascians can reprogram cells to re-enter the cell cycle in a
controlled way because the outcome is the formation of shoot meristems. The
results demonstrate that leafy galls consist of shoot primordia that originate both
from amplification of existing meristems and from the formation of adventitious
meristems from differentiated tissues.
Although the root system is generally not affected by R. fascians, severe infection
of plants can result in a thickened main root and the absence of secondary roots.
Inoculation of germinating seedlings leads to a complete inhibition of root growth,
a thickening of the hypocotyl, and seedling growth inhibition. By following the
expression of cell cycle genes that are correlated with active cell division and/or
competence to divide, a significant decrease of the meristematic activity of
Arabidopsis thaliana roots upon R. fascians infection is observed, accompanied by
a diminished root number and length. Interestingly, such infections also stimulate
lateral root initiation at the pericycle; however, growth of these root initials is
arrested.
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
Analysis of the phenolic and amino acid content shows that leafy galls are distinct
from surrounding plant tissues. In tobacco leafy galls, the relative concentrations
of phenolics differ from those in noninfected plant parts, but no phenolics that are
characteristic for a defense response have been detected.
Leafy galls are clearly distinct in origin and formation from the knots, blisters, and
wart-like formations induced by P. savastanoi and the galls produced by E.
herbicola that consist of parenchymatous cells carrying necrotic centers. The leafy
galls are active centers of ongoing shoot meristem production and growth
repression. They do not become necrotic, but remain green long after the mother
plant is senescent. This characteristic has been interpreted as a result of delayed
senescence.
Cytokinins
Several disease symptoms, such as wrinkling of leaves, formation of green islands,
shoot proliferation, delayed senescence, and root inhibition, are reminiscent of
cytokinin effects. Some symptoms can be mimicked by the addition of cytokinins ,
and exogenous cytokinins have a similar, albeit less pronounced, effect as
R.fascians on shoot meristems and cell cycle gene expression.
The primary source of cytokinins released by R. fascians has been proposed to be
the tRNA, but the increased quantity of non hydroxylated cytokinins in virulent
strains when compared to nonpathogenic strains was interpreted as involving an
alternative biosynthetic pathway. Although cytokinin residues in tRNA may serve
as precursors of free cytokinin, tRNA-mediated cytokinin release is probably not
biologically significant in any organism.
Although Balazs & Sziraki showed that the cytokinin activity in R. fasciansinfected Pelargonium zonale plants was considerably higher than that of healthy
plants, similar observations could not be made by others.
The nature of the R.fascians signal molecules implicated in leafy gall formation is
unknown, but evidence points to the involvement of a cytokinin-like compound.
Crespi et al (30) showed the presence of an isopentenyl transferase (ipt) gene in R.
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
fascians strain D188 that is absolutely required for virulence. Isopentenyl
transferases catalyze the first dedicated step of cytokinin biosynthesis, the
formation of isopentenyl adenine from 5' AMP and dimethylallyl pyrophosphate,
and have been isolated from plants and other organisms.
For the R.fascians DI 88 ipt gene, the enzymatic activity of its gene product has
been demonstrated, but at relatively low efficiency. The low overall sequence
identity of the enzyme with isopentenyl transferases of P. savastanoi, E. herbicola,
and Agrobacterium species (ranging from 20% to 26%) may reflect the acquisition
of different enzymatic specificities. Cytokinin activity as detected in the
supernatant of R. fascians D 188 cultures using a standard cytokinin bioassay
could not be correlated with the expression of the ipt gene and an ipt mutant could
not be complemented by the exogenous application of isopentenyl adenine. These
data suggest that the function of the R.fascians ipt gene is distinct from the
production of classical cytokinins
Auxins
An auxin-degrading capacity has been reported for R. fascians that can, in
combination with the simultaneous secretion of cytokinins, rapidly increase the
cytokinin/auxin ratio leading to symptom development. On the other hand, cell
swelling, secondary differentiation of vascular tissue, and induction of lateral root
initiation are typical auxin effects. In plants carrying a leafy gall, a significantly
higher in the indole-3-acetic acid (IAA) level than in normal tissues has been
detected. Recent experiments show that R. fascians can produce IAA in culture (M
Jaziri, unpublished results). In addition to a possible direct role of auxin in the
initiation of leafy galls, a function in the epiphytic survival of the bacterium, as
shown for E. herbicola, should be considered.
Other Plant Hormones
In peas, gibberellic acid was reported to counteract the effects of R. fascians
infection, suggesting a direct involvement of gibberellins in fasciation.
Interestingly, a combined treatment with cytokinins and imidazole-type fungicides
that block the activity of P450 cytochromes, results in symptoms on Aracea that
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
are very similar to R. fascians-induced phenotypes. This effect can be counteracted
by the addition of gibberellin, indicating that a combined activity of cytokinins and
a decrease in gibberellin content phenocopy leafy gall induction.
Nevertheless, gibberellin treatments of R. fascians-infected tobacco and
Arabidopsis wild-type and gibberellic acid mutants do not significantly alter gall
formation, indicating a more complex mechanism than the mere alteration of
endogenous gibberellin concentrations.
An array of A. thaliana mutants that are defective in ethylene, cytokinin, auxin,
gibberellin, and abscisic acid production and sensitivity have been infected with
R.fascians D188 without significant differences in symptom production. Similarly,
treatment of R.fascians-infected Arabidopsis with different hormones or hormone
inhibitors does not affect the disease symptoms (our unpublished observations).
These results suggest that endogenous hormone levels do not play a central role in
leafy gall formation, but that R.fascians-produced signals act autonomously from
the existing hormone landscape in the host during initiation and development of
leafy galls.
MOLECULAR GENETICS OF R. FASCIANS VIRULENCE GENES
Plasmids often are associated with virulence and phytohormone biosynthesis in
hyperplasia-inducing bacteria. The involvement of plasmids in gall formation by
R. fascians was proposed but was later contested due to the observation that all
R.fascians strains carry circular plasmids, irrespective of their pathogenicity.
Before the 1990s, reports on the genetics of the interaction were restricted to a
publication on the effects of pigment and auxotrophic mutants on pathogenicity.
Studies on the underlying mechanisms of the interaction were mainly hampered by
the absence of molecular tools to study the bacterium.
In the early 1990s, plasmid isolation and conjugation methods, a transformation
protocol and shuttle vectors, and a random mutagenesis procedure were
developed. Reporter genes useful for the molecular study of R. fascians were
characterized.
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
Crespi et al showed the presence of a large (180 kb) conjugative linear plasmid
pFID188 that is essential for pathogenesis in R. fascians strain D188. Although a
linear plasmid-free strain can still penetrate plants, the efficiency of endophytic
colonization is lower than that of the wild-type bacteria. In addition to pFID188,
R.fascians strain D188 also carries a circular plasmid that is involved in cadmium
resistance but plays no role in pathogenesis.
The occurrence of a linear virulence plasmid explains the earlier contesting results
on a linkage between pathogenicity and plasmids, because linear plasmids behave
differently during gel electrophoresis when compared to circular replicons. The
presence of a linear plasmid as a major determinant of pathogenicity has been
expanded to other R.fascians strains, and a strict correlation has been shown
between the presence of linear plasmids and fasciation, with only one R. fascians
strain carrying virulence genes on a circular replicon.
Linear replicons have been described in Streptomyces, Rhodococcus, and
Mycobacterium where they are linked with diverse processes. The chromosome of
Streptomyces strains is also linear. In Actinomycetes, linear plasmids carry termini
that are covalently bound by proteins at the 5' end. The structure of the pFID188
ends complies with the typical actinomycete linear plasmids (our unpublished
results). Although the chromosome of R. fascians was thought to be linear, later
data have proven the genome to be circular.
Random mutagenesis of R. fascians D 188 resulted in the isolation of different
virulence mutants that were mapped on pFID188. Three types of mutants were
described: a nonpathogenic fas mutant, an att mutant showing attenuated
virulence, and a hyp mutant with a hypervirulent phenotype.
Sequence of the fas Locus
The fas locus carries a large operon with six open reading frames (ORFs)
including the ipt homologue (ORF4). Mutants in ORFI and ORF4 are nonvirulent .
At least one ORF downstream of ipt (ORFS or ORF6) is not required for full
virulence on seedlings, but is involved in repression of shoot outgrowths in leafy
galls. This observation suggests that the fas-encoded proteins are required for the
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
production of a signal molecule that mediates both shoot initiation and growth
inhibition and that ORES and/or ORF6 are important for the shoot inhibition
process.
ORES is homologous to cytokinin oxidase genes from maize and A. thaliana.
Cytokinin oxidases irreversibly inactivate cytokinins by catalyzing the cleavage of
the N^sup 6^ side chain. The presence of a cytokinin oxidase gene downstream of
ipt, involved in cytokinin biosynthesis, raises the question as to why a bacterium
would spend energy to produce cytokinin that is degraded subsequently. Cytokinin
oxidases act via the formation of an intermediate with an activated N^sup 6^ atom
and hydrolysis of this intermediate releases the N^sup 6^ side chain. When another
molecule reacts with the activated N^sup 6^ atom, the reaction product would be
an adenine carrying another side chain at the N6 position. Alternatively, the
isopentenyl transferase-produced side chain would not be cleaved, but an adenine
derivative carrying two side chains would be formed. In either case, the molecule
may be more active than the initial isopentenyl transferase-produced cytokinin,
allowing fasciation of both young and older plants.
The ORF6 gene product shows similarity to glutathione S-transferases (GSTs) of
the 0 class with conservation of particular amino acids involved in glutathione-binding and catalytic activity. Whereas the functions of most bacterial GSTs
remain to be established, eukaryotic GSTs are involved in the detoxification of
exogenous substances and endogenous reactive products of cellular metabolism.
The similarity of the ORF6 protein to such enzymes suggests its involvement in
transfer of glutathione, or another peptide residue, to the N6 atom activated by the
ORFS gene product.
Two ORFs that are located upstream of the ipt gene are homologous with P450
cytochromes (ORF1) and the ancillary ferredoxins (ORF2) found in
biodegradation and biosynthesis pathways of Actinomycetes. No homologies with
ferredoxin reductase, the third component of these oxido-reduction systems, could
be detected. However, the ORF2 product carries a carboxyl-terminal domain,
which resembles the (alpha) subunit of pyruvate dehydrogenase. Pyruvate
dehydrogenases catalyze the oxidative decarboxylation of pyruvate, requiring
Rhodococcus fascians
25
BACTERIAL GALLS AND CANKERS
LEAFY GALL
thiamine pyrophosphate (TPP) as a cofactor. The proteins are composed of two
(alpha) and two (beta) subunits and, interestingly, ORF3 encodes a protein similar
to the (beta) subunit. Regions involved in TPP binding are also found in ORF2 and
ORF3. The fusion between a ferredoxin-like domain and a TPP-binding domain in
the ORF2-encoded protein is reminiscent of the pyruvate:ferredoxin
oxidoreductases that are responsible for the oxidation of pyruvate in several
bacteria. These enzymes can use the high reducing power of pyruvate for reactions
requiring strong reductants. The presence of such an enzyme in the fas locus may
be relevant to the action of the cytochrome P450 encoded by ORF1. In most
bacterial cytochrome P450 systems, the reducing power is generated by the
oxidation of NAD(P)H through the action of a ferredoxin reductase. The electrons
are subsequently delivered to the cytochrome P450 by a ferredoxin. The fasencoded cytochrome P450 system could be unique in its ability to use pyruvate or
another a-keto acid as an electron donor. The high energy electrons, generated
from the oxidative decarboxylation of these molecules by the action of the ORF2and ORF3-encoded proteins, could be delivered directly to cytochrome P450 via
the ferredoxin-like domain of the ORF2 product.
Cytochromes P450 have been described in other plant-pathogenic microorganisms
such as A. tumefaciens and the fungus Nectria haematococca, where they enhance
virulence by detoxification of phytoalexins. In R. fascians, nonpolar mutants in
ORF1 are completely non virulent, and a role for the gene product in phytoalexin
degradation is not very probable. Like the fas locus of R. fascians, the cytochrome
P450 gene cluster from Bradyrhizobium japonicum also comprises a ferredoxin
gene and a prenyltransferase. This operon is thought to encode a pathway for the
synthesis of gibberellic acid in bacteroids, but no role in symbiotic properties has
been assigned.
The linked presence of P450 monooxygenase, an ancillary electron transfer
system, and an isopentenyl transferase in the fas locus could indicate a functional
relationship between these enzymes by which isopentenyl adenine-generated by
the isopentenyl transferase-is oxidized to trans- or cis-zeatin. These latter
cytokinins have been detected in R. fascians supernatants, but have not been
correlated with virulence. Considering the proposed role of the ORF5and ORF6-
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
encoded proteins in the attachment of another side chain at the activated N6
position, hydroxylation of this moiety by cytochrome P450 is equally possible.
However, a function in modifying the side chain added by the ORF6 product is not
consistent with the observed phenotype of the ORF1 and ORFS mutants. If the
essential role of the P450 monooxygenase was to act on this side chain, the ORFS
mutant in which the completed fas product cannot be formed, should be
nonvirulent. In contrast, the ORFS mutant has been shown to be virulent. The role
of cytochrome P450 remains to be established. Possibly it is involved in the
hydroxylation at an unorthodox site of a cytokinin-like substrate with properties
that differ from those of well-known cytokinins.
Regulation of fas Genes
The expression of the fas genes is high in plants. Optimal expression in cultures
requires a defined medium and pH 5. Furthermore, gene expression in culture has
been detected in the presence of gall extracts, but not of extracts of noninfected
plants. In other pathogens, virulence gene expression is often correlated with the
pH conditions met in the host.
The nature of the compound inducing fas gene expression is unknown, but
inducing activity could already be detected 24 hours after infiltration of tobacco
plants by R. fascians, long before symptoms are visible. Inducing activity has been
detected in non infected parts of the plant and the surrounding medium, suggesting
the involvement of diffusible factors. There is now evidence that the factor is
produced by the bacterium through the action of the att locus (our unpublished
data). In a working model, att-produced compounds build up during plant
colonization and act as autoregulatory molecules inducing the expression of both
the att and fas genes. Initial induction of the att genes requires no plant-derived
signal molecules; instead confinement of the bacteria under specific conditions
during plant infection creates the settings necessary to build up a critical amount of
attderived inducer, resulting-via a positive feedback loop-in inducer quantities that
meet the demands for fas gene expression.
Interestingly, histidine has been shown to induce fas gene expression in a defined
medium. Histidine levels remain unchanged in leafy galls and the levels necessary
Rhodococcus fascians
27
BACTERIAL GALLS AND CANKERS
LEAFY GALL
to induce gene expression are relatively high (2-5 mM). It can be hypothesized
that such levels in the media somehow mimic the conditions in and on the plant
that are compatible with an initial att gene induction. Alternatively, histidine could
resemble the native inducer.
The regulatory gene, fasR, which belongs to the AraC family of transcriptional
regulators and is essential for both fas gene expression and leafy gall formation, is
located upstream of the fas locus. Curiously, fas gene expression is regulated at the
posttranscriptional level. A posttranscriptional regulator has been proposed to be
encoded by the linear plasmid and its transcription to be regulated by the fasR
product. The induction of the fas genes would then be mediated through an
interaction of an inducer with the posttranscriptional regulatory protein or with the
fasR product.
The complex and tight regulation of the R.fascians fas gene expression probably
reflects the importance of a controlled release of signal molecules. An
uncontrolled production of signals might lead to unfavorable side effects on the
plant host that could counteract a successful interaction or interfere with a
stringent control of shoot primordium induction.
In P. savastanoi, expression of the auxin and cytokinin biosynthetic genes is
constitutive. However, the production of an active IAA pool is controlled by the
endogenous tryptophan concentration, feedback inhibition of the biosynthesis, and
the formation of IAA-lysine conjugates. The interpretation of these results is that a
controlled production of IAA is important for optimal colonization and symptom
development in the host.
For E. herbicola, expression of the IAA biosynthetic genes is relatively low in
cultures and on the plant surface but is highly induced in plants. The cytokinin
biosynthetic genes are constitutively expressed, although some regulation in
function of the growth cycle of the bacterium has been observed.
The regulatory requirements of cytokinin production by P. savastanoi and E.
herbicola seem less relevant for controlled pathogenicity, in contrast to the tightly
controlled expression to which the fas locus is subjected. This suggests a
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
distinctive role of the fas locus in the synthesis of an active signal that differs both
in activity and structure from the classical cytokinins described in relation to
hyperplasia formation.
Function of the fas Genes
Culture supernatant extracts from R. fascians strain D 188 display a very low
cytokinin activity of approximately 5 ng/ml in kinetin equivalents. Other highly
virulent strains of R. fascians also secrete low amounts of isopentenyl adenine (24
ng/ml in kinetin equivalents) or zeatin (2 ng/ml). The cytokinins detected in
R.fascians culture media must be of chromosomal origin because they are also
present in extracts derived from a plasmid-free strain. They may support cell
division and/or render the plant cells more sensitive to the activity of the fas
molecule.
High amounts of trans-zeatin and isopentenyl adenine are found in cultures of
several P. savastanoi strains (400 ng/ml for EW 1006), E. herbicola (200-300
ng/ml) (89,90), and A. tumefaciens C58 (500 ng/ml). Nevertheless, infection of
plants with these phytopathogens never leads to the formation of leafy galls,
indicating that virulence of R. fascians is not due to the secretion of typical
cytokinins alone.
Partly purified supernatant or bacterial extracts of D188 cultures induced for fas
expression do not contain additional cytokinin activity as measured using an
Amaranthus caudatus betacyanin bioassay, implying that fas-derived molecules
have only weak activity, if any, in this assay. Such extracts reduce the internode
length and the leaf size when applied to plants, but formation of leafy galls has
never been observed. Similarly, leaf and root development of seedlings are
seriously reduced by the extracts. Such effects are not seen when extracts of
noninduced or fas mutant cells are applied.
BY-2 tobacco cell cultures synchronized for cell division were treated with R.
fascians, preinduced for fas gene expression (135a). In this assay, cytokinins
increase the proportion of cells in mitosis. In contrast, preinduced R. fascians cells
partly delay the prophase of mitosis of BY-2 cells. This effect depends on an intact
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
and induced fas locus and has been shown to be dominant over the effects exerted
by cytokinins. Furthermore, partly purified extracts provoke the same effects, also
relying on an intact and induced fas locus. Whatever the biological relevance of
these observations might be, they suggest that fas-derived signals have a different
action on the plant cell than do cytokinins.
These results indicate that the fas locus is involved in the synthesis of signals that
affect the growth and development of plants in a particular way. The fas genes
have been proposed to be involved in the synthesis of a cytokinin analogue,
employing the basic biochemistry for cytokinin biosynthesis but with particular
substrates and modifications.
The hyp and the att Loci
Upon infection of decapitated plants, mutants in the hyp locus cause the
development of larger leafy galls. The hypervirulent phenotype suggests the
involvement of the hyp locus in the reduction of symptoms by modifying the
bacterial signals to a less active form or by repressing the expression of virulence
genes.
Sequence analysis of the hyp locus showed the presence of an ORF whose
deduced amino acid sequence is homologous to the "D-E-A-D box" family of
RNA helicases. These proteins are important in the regulation of mRNA
expression and translation initiation. The hyp locus could encode a regulatory
protein involved in the posttranscriptional control of the fas gene expression.
However, the evidence is still scanty because it currently relies on the sequence
analysis only.
The att locus is closely linked to the fas locus on pFID188. Infection of plants with
att mutant bacteria results in a range of phenotypes. Most plants develop less
compact and smaller leafy galls, and seedlings infected with the att mutant grow to
an intermediate height. The att gene products) is unknown, but judging from the
attenuated phenotype provoked by the mutant, several modes of action can be
hypothesized (153; our unpublished data). The intermediary phenotype could be
the result of impaired attachment or growth of the mutant, but this is contradicted
Rhodococcus fascians
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
by the mutant's capacity to successfully colonize Arabidopsis thaliana plants (our
unpublished data). Another function could be the production of one or more
phytotoxins that weaken the plant's surface barrier, facilitating infection by R.
fascians. The fact that wounding, although not necessary for virulence of R.
fascians, greatly enhances symptom development is in agreement with this
hypothesis. Furthermore, wounding is necessary for symptom development by att
mutant bacteria on detached tobacco leaves (our unpublished data). However,
leafy gall and surrounding plant tissues never show signs of chlorosis or
deterioration. If a toxin is involved, it does not cause symptoms typically
associated with phytotoxins.
The att product could directly interact with the fas product by enhancing the plant's
sensitivity to cytokinins or, alternatively, both products could act in concert and
give rise to full symptom development. However, coinfection of plants with att
and fas mutant bacteria does not result in complementation, but instead gives rise
to typical att mutant symptoms (our unpublished data).
Expression of the att genes is induced by a gall-derived factor and histidine, and in
galls incited by att mutants, the production of such a factor is absent (our
unpublished data). Therefore, the att locus may form an autoregulatory compound
that also induces expression of the fas locus. The attenuated phenotype could be
explained by decreased production of the fas product because of an inefficient
expression due to lack of an att-encoded inducer. Such a function does not exclude
the possibility that att serves other functions in gall formation.
Recently, the expression of fas and att genes has been evaluated during infection
of tobacco plants. While expression of the att genes gradually decreases during
infection and only occurs in bacteria present at the plant surface, the fas genes are
expressed both at the surface and the interior of the plant. Furthermore, fas
expression remains high over a longer time period than att expression.
These data suggest that fas gene regulation is very complex and involves more
than the mere presence of att-encoded inducer molecules. Possibly, the attencoded inducer initiates fas gene expression, resulting in the onset of leafy gall
formation. At later stages, when the bacteria penetrate the plant's surface, the role
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
of att-derived inducer may be secondary or even superfluous and other regulatory
elements may abound, precluding att gene expression and allowing further fas
gene expression. Because fas and att mutant bacteria fail to complement one
another, other cell-contained factors that depend on att and/or fas activity must
play a role in disease development.
Although important, the loci affected by the insertions in the fas and att mutants
are not sufficient for pathogenicity because a pFID 188-cured strain carrying a
cosmid spanning these loci is nonvirulent. More recently, another pFID 188 locus
has been isolated that is necessary for full virulence (our unpublished data). This
locus could be involved in penetration, regulation of gene expression in planta, or
the synthesis of accessory signals or compounds that affect colonization of the
plant's interior. Whatever their activities, functions associated with additional
pathogenicity genes can be predicted to be integrated into a regulatory and
functional web involving both the fas and att genes.
METABOLIC COLONIZATION: THE FATE OF A LEAFY GALL
An R. fascians D 188 chromosomal mutant has been isolated that carries an
insertion in a malate synthase homolog. The mutant cannot grow on acetate and
grows less well than wild type on leafy gall extracts and accumulates glyoxylate.
The mutant bacteria also show a reduced endophytic colonization of symptomatic
tissues and exhibit strongly reduced pathogenicity. To understand how a gene of
the glyoxylate shunt is involved in virulence, it was hypothesized that the locus is
involved in the metabolism of plant components that are used as nutrients during
the interaction. The catabolism of these compounds would be mediated through
glyoxylate, requiring the action of a malate synthase. During in planta growth, the
malate synthase mutant is poisoned by accumulating glyoxylate and becomes less
virulent.
Nutritional requirements push soil bacteria to interact more or less intimately with
plants. From an evolutionary point of view, this intimacy for nutritional
sequestering probably occurred gradually. The most primitive way for a soil
bacterium to profit from the vast amount of nutritional sources that plants
represent is to colonize the rhizosphere and utilize the exuded nitrogen and carbon
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
sources or to attack a host, stimulating it to release nutrients, the strategy of
necrogenic plant pathogens. These approaches give little selective advantage
because common compounds are readily utilized by all soilborne microorganisms.
A more advanced strategy is to specialize in the metabolism of specific exudates
from the plant roots that are catabolized by a limited amount of soil bacteria.
Calystegin, certain flavonoids, proline , L-homoserine, and betaines are produced
by many plants, but are rather specifically used by certain Rhizobium species.
Another approach is to divert photosynthate of the plant to compounds that are
specifically used by the inciting bacteria. Approximately 10% of all Rhizobium
meliloti and R. leguminosarum pv viciae strains are known to use this strategy in
which modified bacteria, bacteroids, produce rhizopines derived from plant
metabolites that are used by the free-living rhizobia. Only one type of rhizopine
has been identified to date, but it has been predicted that other types with similar
functions are produced analogously.
The ultimate step in specialization for the use of nutritional mediators is
represented by Agrobacterium strains. Genetic colonization of the host leads to a
free production of opines that are almost exclusively used by the crown gallinducing bacteria that carry the respective catabolic genes. An appealing
hypothesis on the origin of the opine concept has been formulated by VaudequinDransart et al. Primitive agrobacteria evolved to acquire genes for the catabolism
of an ancestral opine produced by uninfected plants at only a certain stage in their
development. These catabolic genes were the first to be incorporated into the TDNA and upon integration into the plant cell nucleus, their activity shifted toward
the biosynthesis of the ancestral opine. Later, other opine biosynthetic genes
would have evolved from these ancestors. Deoxy-fructosyl-glutamine is an opine
of the chrysopine family. It is produced in uninfected plants upon ageing, can be
used by a wide range of agrobacteria, irrespective of their pathogenicity, and is an
intermediate in the biosynthesis of several other opines. Deoxyfructosyl-glutamine
is therefore a good candidate for being an ancestral opine, but it is not
unreasonable to assume that other plant metabolites could fit this profile.
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BACTERIAL GALLS AND CANKERS
LEAFY GALL
In this context, R. fascians has been proposed to use plant carbon sources that are
only present at low concentrations in the plant. However, because of the
proliferation of specific plant tissues-meristems and shoots-during leafy gall
formation, the concentration of these compounds would increase to levels that
could serve as specialized nutrients for the bacteria. As such, a leafy gall would
represent a specific niche for R.fascians in which the bacteria experience a
selective advantage over other plant-inhabiting bacteria. The occurrence of a
nutrient pool inaccessible to others is the driving force for such biotrophic
pathogens. We feel that this concept of "metabolic habitat modification" may fit
many, if not all, plant-galling bacteria. (Goethals et al 2001)
Rhodococcus fascians
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