Download what is galls

WHAT IS GALLS ?
Galls or plant galls are proliferations and modifications of plant cells and can
be caused by various parasites, from fungi and bacteria, to insects and mites.
Galls are often very organised structures and because of this, the cause of
the gall can often be determined without the actual agent being identified. This
applies particularly to some insect and mite galls
The Bacterial galls are caused by several genera of bacteria some of them
are shown in table 1
Agrobacterim tumifaciens
Rhodococcus fascians
Pseudomonas syringae
Crown gall
Leafy gall
oleander and olive knot
Erwinia herbicola
Various gall-forming bacteria employ some common elements during infection
and symptom development, different bacteria use distinctive systems to
produce galls. Except Rhodococcus (corynebacterium) fascians and leafy
galls are no exception to this rule ***
First, ipt genes were described in Agrobacterium and P. savastanoi and later
in E. herbicola, in which they are involved in virulence (65,89,90,118).
Although ipt genes contribute to the type or degree of symptom development
in these bacteria, they are not necessary for virulence. Mutants in the T-DNAencoded ipt gene of Agrobacterium sp. still induce crown galls, although the
tumors have an altered morphology with the formation of multiple roots (1, 9,
17). In P savastanoi, ipt mutants produce galls that are smaller and have a
changed anatomy (71), and in E. herbicola, mutation of an ipt gene essential
for cytokinin production caused a reduction in gall size (89,90). These
observations show that cytokinin production by these bacteria contributes to,
but is not indispensable for, gall formation.
In P. savastanoi, auxin is the major pathogenicity factor, stimulating cambium
cell division and the proliferation of disorganized tissue (71). Furthermore, the
relation between different P. savastanoi strains and particular plant hosts of
the Oleaceae family can in part be correlated with different levels of auxins
produced by the pathogen (54,70,124,132).
In E. herbicola, auxins are also involved in pathogenicity but, as for cytokinins,
they are not indispensable for gall formation (24, 89,90). Instead, an hrp gene
cluster has been identified in this bacterium that is essential for gall formation
(99,106,107,142). The hrp genes have been described in biotrophic,
Gramnegative bacteria in which they are implicated in the formation of a type
III delivery system that injects protein virulence factors directly into the host
cells (27, 50). Perhaps both auxin and cytokinin produced by E. herbicola
boost a gall induction process that is initiated by one or more proteinaceous
factors delivered to the host cells.
These data are in contrast with the absolute requirement of then.fascians D
188 ipt gene in leafy gall formation. The significance of this gene for
pathogenicity has been confirmed by a strict correlation with virulence in
different R. fascians isolates (134). *****
Anatomy of galls:
1- Crown gall:
The crown is the point at the soil line where the main root joins the stem
where is galls typically form at . but the galls may also develop on secondary
or lateral roots and on the main stem and branches above the soil line ***
The study of the development of crown gall disease in plants is important, not
only because the disease affects a wide range of dicotyledonous plants
(especially those in the rose family, including fruit trees and raspberries as
well as roses), but also because of the nature of the developmental changes
that occur. Understanding tumorigenesis and crown gall development could
provide important insights into plant hormone signaling pathways, carbon and
nitrogen metabolism, and source-sink relationships.*******
Furthermore, The virulence mechanism of the pathogene turns out to be
unique among interactions between prokaryotic pathogens and eukaryotic
hosts(1-2)
For example, Gall diseases on the roots of tobacco plants were first
found in a field of Kawai-mura, Iwate Prefecture, Japan in 1995. Since then,
the disease has continued to occur in the same field. A. tumefaciens was
isolated from the galls of the tobacco plants, and the Agrobacterium tobacco
strains were fully characterized by pathogenicity tests, reinoculation of
healthy tobacco plants, growth on selective medium, and biochemical
properties in this study. This report is the first of crown gall of tobacco caused
by A. tumefaciens in the field.
Several changes have been proposed related to the no-menclature and
taxonomy of Agrobacterium (Bouzar 1994;Ophel and Kerr 1990; Sawada et
al. 1993; Young et al.Introduction 2001) ****
Symptoms and signs
Crown gall is identified by overgrowths appearing as galls on roots and at the
base or "crown" of woody plants such as pome (e.g., apple, pear) and stone
(e.g., cherry, apricot) fruit and nut (e.g., almond, walnut) trees (Figure 1).
Crown galls are also formed on ornamental woody crops such as roses,
Marguerite daisies, and Chrysanthemum spp. as well as on vines and canes
such as grapevines (Figure 2) and raspberries. Marguerite daisies,
chrysanthemums and grapevines can become infected systemically.
Occasionally, galls have been observed on field crops such as cotton, sugar
beets, tomatoes, beans (Figure 3) and alfalfa (Figure 4), but the disease does
not impact such crops economically. Crown gall is caused by Agrobacterium
tumefaciens, a Gram-negative, bacilliform bacterium that is normally
associated with the roots of many different plants in the field. This bacterium
can survive in the free-living state in many soils with good aeration such as
sandy loams where crown gall diseased plants have grown. The bacterium
can also survive on the surface of roots (rhizoplane) of many orchard weeds.
Plants representing over 93 plant families are susceptible to crown gall as
judged by experimental inoculations. Owing to their high susceptibility to
crown gall, plants such as Jimson weed (Datura stramonium) and sunflower
(Helianthus annuus) are used as assay hosts for testing the degree of
virulence of A. tumefaciens. Also, Kalanchoë daigmontiana (also known as
Bryophyllum) is used for assaying A. tumefaciens, but the plant is less
sensitive than Datura.
Pathogen Biology
Agrobacterium tumefaciens is a Gram-negative rod-shaped bacterium that is
commonly found in the rhizosphere of many plants, where it survives on root
exudates. It will infect a plant only through a wound site (which often occurs in
nursery stock through transplanting and grafting and in vineyards through
pruning).
Agrobaterium is widely recognized for its ability to transfer foreign DNA into
plant cells, whereby T-DNA becomes integrated into the plant genome.
Certain phenolic compounds produced by the plant (including acetosyringone)
cause the induction of agrobacterial virulence genes encoding, among other
proteins, an endonuclease that excises T-DNA from the bacterial tumorinducing plasmid. The T-DNA then becomes integrated into the plant genome,
and T-DNA genes are expressed via the plants normal transcriptional and
translational machinery. Some of the salient features of crown gall disease
were reviewed by Nester et al. (1984), and a review concerning T-DNA
transfer was presented by Gelvin (2003(.*******
So Agrobacterium tumefacience is one of the transgenic organism witch
contains genes foreign to its own genome (Griffiths 1999). Such organisms
can be used for research or for specialized commercial applications(1-4),
There are multiple ways to construct transgenic organisms. Expression
plasmids are widely used tools, especially in the genetic modification of
plants. The expression plasmid is a circular piece of DNA that contains an
origin of replication, the appropriate regulatory regions located 5’ to the site in
which the gene of interest is inserted, and a criterion for selection. The origin
of replication is important to assure the replication of the plasmid within the
host bacterium (Darnell 1990). Containing the correct regulatory regions,
such as promoters, allows for the transcription and translation of the inserted
gene, ultimately resulting in the expression of the gene. Having a mechanism
for selection enables for the discrimination between self-ligated (which do not
contain the gene of interest) and recombinant plasmids (which do contain the
gene of interest). Selectable bacterial genes, such as antibiotic resistance,
are often used for the selection process. Expression is finally achieved when
the plasmid is reinserted into bacteria, which have replication, transcription,
and translation machinery (Griffiths 1999). (1-4)
Agrobabacterium tumefaciens genom and plasmid:
Most of the work on Agrobacterium tumefaciens, since its identification as the
causal agent in crown gall disease of dicotyledonous plants at the turn of the
century, has rightfully focused on the mechanism of tumor induction (52; for
recent reviews of all aspects of the disease, see references 2, 10, 23, 39, and
58).. Since most of the virulence genes lie on the Ti plasmid, the
chromosomal complement of A. tumefaciens has been relatively understudied
Initial chromosomal maps for A. tumefaciens, based on chromosome mobilization and
recombination of genetic markers, suggested a single circular chromosome .
However, recent physical mapping data strongly suggests that A. tumefaciens has two
chromosomes, one circular chromosome of ~3 Mbp and one linear chromosome of
~2.1 Mbp This chromosome organization appears to be a conserved trait throughout
the genus. While multiple chromosomes have been found in some other eubacteria
(1-2)
Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel
electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and
2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by PacI or
SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons,
used as probes, hybridize with different restriction fragments, showing that these
replicons are two independent genetic entities. A 16S rRNA probe and genes
encoding functions essential to the metabolism of the organism were found to
hybridize with both replicons, suggesting their chromosomal nature. In PFGE,
megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome always
generated an intense band, while the 3-Mb band was barely visible. After linearization
of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of
the 2.1-Mb remained constant. This suggests that the 3-Mb chromosome is circular
and that the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomic DNA,
trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA
present. The plug was then recovered, and the remaining DNA was digested with
either PacI or SwaI and then separated by PFGE. The fragments corresponding to the
small chromosome were found to be absent, while those corresponding to the circular
replicon remained, further proof of the linear nature of the 2.1-Mb chromosome.(1-3)
Agrobacterium tumefaciens biotypes and biovars
Based on some distinct phenotypic differences, A. tumefaciens isolates were
originally classified into three biotypes or biovars (biotype I, II and III; biovar 1,
2, and 3). Biotype I or biovar 1 strains produce 3-ketosugars and usually have
wide host ranges. Biotype II or biovar 2 strains mainly classify as the hairy
root-forming organism, A. rhizogenes. Biotype III or biovar 3 isolates are
mainly confined to grapevines, prefer L-tartaric acid over glucose and produce
polygalacturonase. Because grapevine isolates formed a distinct group
verified by DNA homology studies and were frequently limited in host-range to
grapevines, biovar 3 strains have been reclassified into one species, A. vitis.
Agrobacterium rubi strains infect canes of the genus Rubus, representing
blackberry and raspberry
Leafy gall:
In 1927, Brown (1) 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 gallforming 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 (139) 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 Lacey (83) 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 (38), 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 (84),
Corynebacteriae (62,159), and the Rhodococci (58,59). The controversy
ended when Corynebacterium fascians was reclassified as Rhodococcus
fascians, based on chemical, genetic, and phenotypic data (57).
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
(45). Analysis of the SS and 16S rRNA sequences (91,121) and a study of the
rRNA operon structure (116) confirmed this taxonomic position. Rhodococcus,
together with the genera Gordona, Tsukamurella, Mycobacterium, and
Nocardia, is now classified into the family Mycobacteriaceae (23).
R.fascians is aerobic and pleiomorphic and can form branched hyphae that
soon break up in rods and cocci (nocardioform) (87). 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 (14, 43, 44, 57,
139). Within the genus Rhodococcus, R.fascians is the only known
phytopathogen, although animal pathogens and symbionts have been
described (11).
After the bacterium had been isolated from sweet pea, in which it severely
reduced flower setting (139), it was later found in "cauliflower" strawberry
plants, which carry a large number of dwarfed buds, and in carnation,
chrysanthemum, and asparagus (81,82). Since then, the effects of R. fascians
have been noticed primarily in ornamental plants because infection usually
results in decreased flower production (98, 110, 143-145) or deformed flowers
(160). The disease is spread by infected plant material and infested seeds or
soils (7, 83, 110, 139).
Dicotyledonous as well as monocotyledonous plants respond to R. fascians
infection and the host range encompasses 39 plant families and 86 genera
(134, 150; our own observations). 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 (19, 51,52).
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 (46), the bacterial strain (40), and the bacterial
growth conditions (46) 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 (150).
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 (126), but soon growth of
the terminal bud is blocked, and the plants become dwarfed and produce
fewer blossoms (7, 16, 82, 139). 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 (126,127). 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 (81,125). R. fasciansinduced fasciations are formed when several
hypertrophied shoots coalesce, carrying malformed small leaves with
thickened petioles and veins (125).
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 required for the multiplication of the shoots and also for gall persistence
(126, 150). 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 (55, 125, 126, 150). 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 (7, 83, 88, 125) until leafy galls were found to also
originate from leaf margins and veins (150). 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 (46,81,125,139). Inoculation of germinating seedlings leads
to a complete inhibition of root growth, a thickening of the hypocotyl, and
seedling growth inhibition (150). 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
(150).
Analysis of the phenolic and amino acid content shows that leafy galls are
distinct from surrounding plant tissues (42, 149, 152). 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 (152).
Leafy galls are clearly distinct in origin and formation from the knots, blisters,
and wart-like formations induced by P. savastanoi (51, 52) and the galls
produced by E. herbicola that consist of parenchymatous cells carrying
necrotic centers (25,49). 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 (150). 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 (77, 111), and exogenous cytokinins have a similar,
albeit less pronounced, effect as R.fascians on shoot meristems and cell cycle
gene expression (92,152).
The primary source of cytokinins released by R. fascians has been proposed
to be the tRNA (95, 103), but the increased quantity of non hydroxylated
cytokinins in virulent strains when compared to nonpathogenic strains was
interpreted as involving an alternative biosynthetic pathway (103). Although
cytokinin residues in tRNA may serve as precursors of free cytokinin, tRNAmediated cytokinin release is probably not biologically significant in any
organism (20, 100).
Although Balazs & Sziraki (8) 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 (41,92,152).
The nature of the R.fascians signal molecules implicated in leafy gall
formation is unknown, but evidence points to the involvement of a cytokininlike compound. Crespi et al (30) showed the presence of an isopentenyl
transferase (ipt) gene in R. 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 (20).
For the R.fascians DI 88 ipt gene, the enzymatic activity of its gene product
has been demonstrated, but at relatively low efficiency (30). The low overall
sequence identity of the enzyme with isopentenyl transferases of P.
savastanoi, E. herbicola, and Agrobacterium species (ranging from 20% to
26%) (30,135) 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 (135). 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 (75,125) that
can, in combination with the simultaneous secretion of cytokinins, rapidly
increase the cytokinin/auxin ratio leading to symptom development (8). 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 (92, 152). 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 (15,94).
Other Plant Hormones
In peas, gibberellic acid was reported to counteract the effects of R. fascians
infection (126), 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 (61, 119), results in
symptoms on Aracea that are very similar to R. fascians-induced phenotypes
(155). 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 (156).
Nevertheless, gibberellin treatments of R. fascians-infected tobacco and
Arabidopsis wild-type and gibberellic acid mutants do not significantly alter
gall formation (135), 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.fasciansproduced 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 (60, 103) but was later contested due
to the observation that all R.fascians strains carry circular plasmids,
irrespective of their pathogenicity (86).
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 (72). 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 (34), a
transformation protocol and shuttle vectors (35), and a random mutagenesis
procedure (33) were developed. Reporter genes useful for the molecular
study of R. fascians were characterized (36).
Crespi et al (30) 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 (28). 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 (34).
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 (134), 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 (134).
Linear replicons have been described in Streptomyces, Rhodococcus, and
Mycobacterium where they are linked with diverse processes (67,113). The
chromosome of Streptomyces strains is also linear (21). In Actinomycetes,
linear plasmids carry termini that are covalently bound by proteins at the 5'
end (67). 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 (30), later data have
proven the genome to be circular (115).
Random mutagenesis of R. fascians D 188 resulted in the isolation of different
virulence mutants that were mapped on pFID188 (30). 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 (30,31,55, 135). 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 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
(68, 101, 135). Cytokinin oxidases irreversibly inactivate cytokinins by
catalyzing the cleavage of the N^sup 6^ side chain (157). 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 transferaseproduced 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 (39,134,154). Whereas the
functions of most bacterial GSTs remain to be established (154), eukaryotic
GSTs are involved in the detoxification of exogenous substances and
endogenous reactive products of cellular metabolism (26). 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 (31,109, 128).
No homologies with ferredoxin reductase, the third component of these oxidoreduction systems, could be detected. However, the ORF2 product carries a
carboxyl-terminal domain, which resembles the (alpha) subunit of pyruvate
dehydrogenase (18,64, 134). Pyruvate dehydrogenases catalyze the oxidative
decarboxylation of pyruvate, requiring thiamine pyrophosphate (TPP) as a
cofactor. The proteins are composed of two (alpha) and two (beta) subunits
(12,105) and, interestingly, ORF3 encodes a protein similar to the (beta)
subunit. Regions involved in TPP binding (102) 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 (80,114). These enzymes can use the high reducing power of
pyruvate for reactions requiring strong reductants (76). 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 (108). The fas-encoded cytochrome P450
system could be unique in its ability to use pyruvate or another a-keto acid as
an electron donor. The highenergy electrons, generated from the oxidative
decarboxylation of these molecules by the action of the ORF2- and ORF3encoded proteins, could be delivered directly to cytochrome P450 via the
ferredoxin-like domain of the ORF2 product (55, 135).
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 (74,96). In R. fascians, nonpolar mutants in ORF1 are
completely nonvirulent, 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 (141). 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 (140).
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 adeninegenerated by the isopentenyl transferase-is oxidized to trans- or cis-zeatin.
These latter cytokinins have been detected in R. fascians supernatants (2,41,
77, 129), but have not been correlated with virulence. Considering the
proposed role of the ORF5and ORF6-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 (31). 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 (136). Furthermore, gene
expression in culture has been detected in the presence of gall extracts, but
not of extracts of noninfected plants (30, 136). In other pathogens, virulence
gene expression is often correlated with the pH conditions met in the host (93,
120, 133).
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 noninfected parts of the plant and the
surrounding medium, suggesting the involvement of diffusible factors (149).
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, attproduced 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 (136,153). Histidine levels remain unchanged in leafy galls
and the levels necessary 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 (136). 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 (48,118). However, the production of an active IAA pool is
controlled by the endogenous tryptophan concentration (79), feedback
inhibition of the biosynthesis (69), and the formation of IAA-lysine conjugates
(53, 112, 124). 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 (94). The
cytokinin biosynthetic genes are constitutively expressed, although some
regulation in function of the growth cycle of the bacterium has been observed
(89,90).
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 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 (135). Other
highly virulent strains of R. fascians also secrete low amounts of isopentenyl
adenine (24 ng/ml in kinetin equivalents) (103) or zeatin (2 ng/ml) (2). 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 (135). 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) (117), E. herbicola
(200-300 ng/ml) (89,90), and A. tumefaciens C58 (500 ng/ml) (2).
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 (135). 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
(135).
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 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 fasderived 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 (30). 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 (135, 153). These proteins are important in the regulation of
mRNA expression and translation initiation (131). 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 (30). 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 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
(153), 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 (28). 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
att-encoded 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 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 (30). 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 (28) and exhibit strongly reduced pathogenicity
(149,151,153). To understand how a gene of the glyoxylate shunt (78) 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 sources or to attack a host, stimulating it to release nutrients, the
strategy of necrogenic plant pathogens (3). 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 (137), certain flavonoids (63), proline (73), L-homoserine,
and betaines (13, 56) 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 (122).
Only one type of rhizopine has been identified to date, but it has been
predicted that other types with similar functions are produced analogously
(104).
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
gall-inducing bacteria that carry the respective catabolic genes (37, 130). An
appealing hypothesis on the origin of the opine concept has been formulated
by Vaudequin-Dransart et al (147, 148). 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 T-DNA 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 (22). 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. Deoxyfructosylglutamine 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.
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.