A Closer Look at Pseudomonas syringae
as a Leaf Colonist
The pathogen P. syringae thrives on healthy plants by employing
quorum sensing, virulence factors, and other traits
Glenn Dulla, Maria Marco, Beatriz Quiñones, and Steven Lindow
acteria, yeasts, and fungi colonize the
aboveground parts of plants—some
within plant tissues, but many more
on the surfaces of healthy plants. This
habitat is called the phyllosphere and
its inhabitants, epiphytes. Bacteria are by far the
most numerous colonists of leaves, often averaging 106 to 107 cells/cm2 and up to 108 cells/g
of leaf. These leaf-associated microbiota differ
substantially from those associated with plant
roots.
The pathogenic bacterium Pseudomonas syringae includes diverse pathovars that differ in
their pathogenicity to one or more of more than
100 plant species. When studying plants in nature, it is quite common to find P. syringae on
healthy host plant tissue. These P. syringae serve
as reservoirs of inoculum for subsequent dis-
B
• Many varieties of Pseudomonas syringae species live on leaves of healthy plants, frequently act
as pathogens, and also can catalyze formation of
ice crystals that damage plants.
• Quorum sensing controls expression of several
traits that contribute to P. syringae epiphytic fitness and virulence, particularly when these bacterial cells occupy nutrient-poor leaf surfaces.
• Signals that interfere with ordinary quorum
sensing activity of P. syringae and other microorganisms on plant surfaces may offer novel means
for reducing disease and freeze damage.
• Studies of habitat-inducible rescue-of-survival
mutants of P. syringae are providing insights into
the wide range of plant-inducible genes that these
bacteria deploy during their complex interactions
with hosts.
ease, whereby the likelihood of which can be
predicted from their epiphytic population sizes.
Many strains of P. syringae also actively catalyze ice formation at temperatures as warm as
–2°C by producing an outer membrane protein
that orients water molecules into an ice-like
structure. Because many frost-sensitive plant
species can supercool to as low as –5° C in the
absence of ice-nucleating bacteria, species such
as P. syringae are important determinants of
frost damage to plants. Because the risks of disease
and frost are both population size dependent,
there is considerable interest in understanding
how P. syringae colonize plants. Moreover, because environmentally damaging bactericides,
including copper compounds and antibiotics,
are widely used to reduce the risk of plant disease and frost, researchers also are studying
plant surface-microbe interactions in the
hope of developing alterative means for reducing those risks.
Leaves Are a Hostile
Niche for Microbes
Because leaf surfaces are exposed to rapidly
fluctuating temperatures, relative humidity,
moisture, and UV irradiation, they provide
a hostile environment for bacterial colonists. Although other habitats also offer extremes of desiccation and temperature,
most are not subject to such rapid and extreme fluctuations. The scarcity of carboncontaining nutrients on leaves also limits
epiphytic colonization. Sugars such as glucose, fructose, and sucrose are the main
carbon source, apparently leaching from
plant interiors at levels of 1 to 10 ␮g/bean
leaf. Molecular biosensors suggest that nutri-
Glenn Dulla is a
graduate student
and Steven Lindow
is a Professor of
Plant Pathology in
the Dept. Plant and
Microbial Biology,
University of California, Berkeley;
Maria Marco is a
scientist at the
Wageningen Centre
for Food Sciences
and NIZO Food Research, Ede, the
Netherlands; and
Beatriz Quiñones is
a Microbiologist in
the USDA ARS,
Western Regional
Research Center,
Albany, Calif.
Volume 71, Number 10, 2005 / ASM News Y 469
ent availability on leaves is highly spatially heterogeneous.
For instance, sucrose-responsive biosensors,
consisting of Erwinia herbicola cells with a
green fluorescence reporter (GFP) reporter gene
fused to the fructose-responsive fruB promoter,
show that nearly all fructose bioreporter cells
consume fructose within 1 hour after inoculation onto plants, according to Johan H. Leveau,
who worked with our group at the University of
California, Berkeley. However, this level
dropped to less than 1% within 24 hours, suggesting a highly heterogeneous availability of
nutrients to individual cells. Furthermore, when
he examined those bioreporter cells directly, he
found that they continued to consume sugars at
particular sites that are not randomly dispersed
across the leaf (Fig. 1).
Microscopy by Jean-Michel Monier, who
worked in our group, and by Cindy Morris and
collaborators at INRA, Avignon, France, provides additional evidence of this nutritional
“landscape.” Bacterial colonies are highly aggregated on plant leaves, according to Morris.
Indeed, within a few days after leaves are uniformly inoculated with P. syringae, those cells
are primarily found in aggregates containing at
least 100 cells. Such aggregates probably arise
from single immigrant cells that find nutrients in
“oases” on leaves.
Cell-Density-Dependent
Behavior of P. syringae
These aggregates of epiphytic P. syringae cells
affect leaf-associated behavior and success of individual cells in colonizing the leaf surface. For instance, after several cycles of wet and dry conditions, the primary survivors among P. syringae
are cells from relatively large aggregates (⬎100
cells), according to Monier (Fig. 1). Because cells
within such aggregates on leaves appear to be
more tolerant to various environmental stresses than
more solitary cells due to their being physiologically
different from such cells, we examined the possibility
that cell density-dependent genes expression influences the ability of P. syringae to adapt to the
phyllosphere environment.
Cell-density-dependent quorum sensing regulates a variety of bacterial behaviors. In this
process, diffusible signal molecules accumulate
as population densities increase. P. syringae and
several other gram-negative bacteria use partic-
470 Y ASM News / Volume 71, Number 10, 2005
ular N-acyl homoserine lactones (AHL) as signal molecules. When such molecules accumulate, the cells express virulence factors and
secondary metabolites that mediate colonization of a host.
The cell-density dependent signal of P. syringae is 3-oxo-hexanoyl-homoserine lactone (3oxo-C6-HSL) synthesized via an AHL synthase,
AhlI, and the AHL regulator, AhlR, according
to Beatriz Quiñones and Catherine Pujol, who
worked in our group. This AhlI-AhlR quorumsensing system is further modulated by AefR,
which positively regulates ahlI and subsequent
AHL production. Another positive regulator,
GacA, is required for AHL production; however, AefR and GacA appear to regulate the
AhlI-AhlR quorum sensing system via separate
pathways.
Quorum sensing controls expression of several traits that contribute to P. syringae epiphytic fitness and virulence, according to Quiñones and Glenn Dulla in our group. For
example, the epiphytic population sizes of an
aefR- mutant and an ahlI- ahlR- double mutant,
both deficient in AHL production, are substantially reduced 1 or 2 days after desiccation stress
on leaves. Both AHL mutants are also greatly
impaired in producing the exopolysaccharide
alginate and are more susceptible than usual to
hydrogen peroxide. This reduced tolerance of an
aefR- mutant and an ahlI- ahlR- double mutant
to environmental stresses appears to be due in
part to their reduced coat of alginate that ordinarily protects against such stresses. Because
tissue maceration is attenuated in both these
mutants, quorum sensing apparently also contributes to virulence.
Moreover, quorum sensing regulates motility
in P. syringae. For instance, on moist bean
leaves, wild-type cells can move on leaf surfaces
from a single inoculation point, presumably by
flagellum-mediated swimming and swarming.
An ahlI- ahlR- double mutant is hypermotile,
moving by swarming at a faster speed than do
wild-type cells on semisolid agar plates. Impressively, an aefR- mutant moves at the same higher
speed as an ahlI- ahlR- double mutant and with
less delay. When placed on moist bean leaves,
quorum sensing mutants enter the interior of
leaves much more rapidly and extensively than
does the wild-type strain. Consistent with leaf
invasion being a prerequisite for subsequent infections, quorum sensing mutants incite a much
Long Fascinated by Plants, Lindow Focuses on Plant-Associated Microbes
Steven Lindow cared about plants
since childhood. He grew up on a
farm southwest of Portland, Ore.,
doing all the home gardening
while also renting land and equipment from his father to earn extra
money by raising several acres of
strawberries and boysenberries.
For his high school science class,
he did an independent project
in his own greenhouse studying
the uptake of mineral nutrients
through the leaves of plants he
grew hydroponically.
Thus, it seemed logical for him
to study botany as an undergraduate student at Oregon State University. What was less predictable,
however, was his burgeoning interest in microbiology. “In my own
experiences as a strawberry and
boysenberry farmer, I had encountered several bad diseases
such as crown gall of boysenberry
canes, Botrytis fruit rot of strawberry, as well as frost damage to
strawberry flowers,” he recalls. “I
could relate to the problem-solving nature of a discipline such as
plant pathology, and the ability to
merge fundamental science with
problem-solving in plant pathology really appealed to me.”
His undergraduate advisor encouraged him to take plant pathology – a practical merger of
plant science and microbiology –
and he did. During one session in
plant disease epidemiology, he encountered an unexpected, yet eminently familiar image. “I recall
vividly one lecture when Bob
Powelson, the instructor, showed
a picture of a wheat field devastated with the fungal disease
‘take-all,’ and a grower standing
with him among the dead plants,”
he says. “The grower was my
Dad! Then I remembered that several years earlier Powelson had
used my Dad’s fields for some of
his field experimentation. . ..”
Lindow graduated in 1973, and
continued to study plant pathology at the University of Wisconsin, where his Ph.D. research focused on leaf surface bacterial ice
nuclei and its role in causing frost
damage to corn and other plants.
Today Lindow, 54, is a professor
in the department of plant and
microbial biology at the University of California, Berkeley, where
he has been since 1978. His current research focuses on the microbial ecology of plant-associated bacteria.
Lindow’s wife of nine years,
Betsy, similarly combines biology
with her primary interest, energy
resources. She is the manager of
energy efficiency programs for a
California utility, Pacific Gas and
Electric Co. Away from the lab,
Lindow spends much of his free
time woodworking— but these
days makes furniture only for
friends and family, keeping none
for himself. “I have no room left
in my own house for the things I
like to build,” he says.
In the 1980s, Lindow was
among the first scientists who
were authorized to release recombinant microorganisms into the
environment, and he participated
in an early field test in which potatoes were sprayed with a product called “Ice-minus” in an attempt to prevent frost damage.
The work grew out of his extensive study of bacterial ice nucleation. Similar studies were soon
done on strawberries.
“We cloned ice nucleation
genes in 1981 and have done quite
a bit of work on the biochemistry
of bacterial ice nucleation proteins,” he says. “Our earlier work
had shown that frost control was
possible by applying non-ice nucleation-active bacteria to plants
at the right time to preempt colonization of plants by ice nucleation-active bacteria that would
cause freezing and frost damage
if temperatures dropped below
freezing.”
The work was met with considerable protest and opposition. “I
received more than my share of
hate mail and death threats at the
time and had, with other members of my lab and some Environmental Protection Agency scientists, to serve as security guards
over the field plot at night after
ecoterrorists had vandalized the
plot,” he recalls. “Many of the
same concerns about use of recombinant technologies for crop
improvement that we hear today
were voiced 20 years earlier. . ..
While it is now easier to obtain
permits for the types of field experiments that we performed, it is
still a significant hurdle, and I am
concerned that it has dissuaded
many . . . from pursuing such research.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
Volume 71, Number 10, 2005 / ASM News Y 471
FIGURE 1
In broth cultures and agar surfaces,
quorums are achieved when cells reach
high densities because AHL signals
freely diffuse in such media. In ordinary
environmental settings, however, absorption, adsorption, destruction, and
diffusion affect such signal molecules—
meaning large numbers of cells may be
required to achieve a quorum where
there is liquid flow. In contrast, on a dry
leaf, an aggregate of only a few cells
might accumulate enough signal to activate quorum sensing. Because a leaf
surface is in a constant flux of wetness,
it offers an opportunity to study how
diffusion and cell number affect quorum sensing.
Altering Quorum Sensing: a New
Paradigm in Biocontrol
Altering the chemistry of plants can affect cell-cell signaling in P. syringae to
achieve disease and frost control. For
instance, Rupert Fray at the University
of Nottingham, United Kingdom, engineered tobacco plants to produce
3-oxo-C6-HSL. Then Quiñones and
Dulla in our group tested whether this
change in the host affected the virulence
of P. syringae pv. tabaci, which has a
quorum-sensing system that is similar
to P. syringae.
When they inoculated leaf surfaces or
infiltrated these bacteria into leaves of
Processes and leaf and environmental features leading to colonization of leaves by
tobacco plants, disease severity was deP. syringae and subsequent entry into plants.
creased in the AHL-producing plants.
However, these reductions were observed only when low concentrations of
larger number of leaf infections after long, moist
inoculum
were infiltrated. Although expression
incubations. An aefR mutant is both the most
of
AHL
in
transgenic tobacco alters this disease,
motile and the most virulent of our quorum
further
work
will be needed before this control
sensing mutants.
process
can
be
applied to other pathovars of P.
Due to the heterogeneity of the leaf surface,
syringae.
the likelihood of P. syringae cells landing near
This pattern is similar to that observed when
abundant nutrients is low. Thus, cells presumE. Tapio Palva at the University of Helsinki,
ably move about on leaves to nutrient-rich oases
Finland, and his collaborators inoculated Eror sites where invasion occurs. Solitary cells are
winia carotovora into transgenic AHL-producnot in a “quorum-sensing state” and, hence,
ing plants. They conjectured that the presence of
would not be repressed for motility (Fig. 2). A
plant-derived AHL induces the premature excell that finds such a site would proliferate and
pression of bacterial virulence traits, even when
form an aggregate. However, upon reaching a
the pathogen is at low population sizes, leading
quorum in such an aggregate, motility would be
to expression of virulence genes and damage
suppressed, preventing detrimental movement
into the surrounding nutritional desert.
followed by a host defense response.
472 Y ASM News / Volume 71, Number 10, 2005
The leaf is a niche accommodating
FIGURE 2
diverse fungal, bacterial, and protist
residents that may also affect quorum
sensing. The leaf itself is also a reactive
component of the community, further
increasing the possibilities of crosstalk
among different organisms. The group
of Steffen Kjelleberg of the University
of New South Wales in Sydney, Australia, has shown that the red alga Delisea
pulchra, for example, produces furanones that inhibit bacterial quorum
sensing and prevent biofouling. Algae
and roots of pea and other plants
secrete a variety of diffusible compounds capable of activating or inhibiting quorum sensing, according to
Proposed behavior of P. syringae on leaf microsites differing in nutrient content. The
topogical complexity of the leaf is depicted in the Scanning Electron Micrograph of a bean
Dietz Bauer at Ohio State University in
leaf surface (from G. Beattie, Iowa State University, Iowa City). Sites of abundant nutrient
Columbus and his collaborators (see
leakage are shown as red fountains where bacterial cells multiply, thereby accumulating
ASM News, March 2005, p. 129). SimAHL signal molecules, which in turn induces EPS production and represses motility at
such sites. Away from such oases, cells sense little AHL and hence motility is enhanced,
ilarly, soil bacteria both stimulate
enabling them to fully explore the leaf.
and inhibit quorum sensing in Pseudomonas aureofaciens and thereby affect
its control over take-all disease of wheat, acProbing Factors That Affect How
cording to Leland Pierson and associates at the
P. syringae Adapts to Leaf Environments
University of Arizona in Tucson.
The prominence of P. syringae on leaves is preAs many as 18% of culturable bacteria from
sumably due to traits that confer a high level of
leaves produce small diffusible molecules that
ecological fitness. Aside from quorum sensing,
can interfere with quorum sensing in P. syrinonly a few individual phenotypes, such as flagelgae, according to Dulla in our group. About 7%
lar motility, UV-mediated mutagenic repair, alof these bacterial epiphytes produce the same
ginate, Type IV pili, and the Type III secretion
3-oxo-C6-AHL, often in amounts far higher
system, are known to contribute to epiphytic
than in P. syringae. Because quorum sensing
fitness. Surprisingly, other traits predicted to
controls both epiphytic fitness and virulence of
function in the phyllosphere, such as antibiosis,
P. syringae, its confusion by interference with
iron sequestration by siderophores, pigment
signals by other community members may lead
production, and ice nucleation, provide little or
to a new paradigm in biological control of plant
no fitness advantage.
pathogens. Thus, compared to plants inoculated
This uncertainty over which phenotypes conwith P. syringae alone, fewer lesions formed on
tribute to epiphytic function was underscored
plants coinoculated with P. syringae and other
when Gwyn Beattie of our group used random
AHL-producing strains, whereas coinoculating
insertional mutagenesis to identify phenotypes
P. syringae with quorum-sensing-inhibiting
required for epiphytic fitness of P. syringae B728a.
strains increased the numbers of lesions.
Most of these 82 epiphytic fitness mutants were
Although AHL-producing transgenic plants
only slightly altered in their growth and stress
or use of AHL-interfering bacterial strains show
tolerance levels on bean leaves, and they disgreat promise in controlling plant pathogens, we
played in vitro phenotypes indistinguishable from
need to study how altering AHL affects other
those of wild-type cells. This result highlights
members of these leaf-based microbial commuthe complexity of leaf surface habitats whereby
nities, including bacteria such as P. aureofaciens
single traits typically do not determine the overand Rhizobia spp., in which quorum sensing
all fitness of P. syringae and expression of many
plays significant roles in antibiotic production
of these traits is apparently limited to conditions
and plant symbiosis.
that cannot be readily reproduced in vitro.
Volume 71, Number 10, 2005 / ASM News Y 473
FIGURE 3
HIRS procedure for selection of plant-inducible genes. Random DNA fragments from P. syringae are cloned upstream of a promoterless
metXW operon and introduced into a P. syringae metXW deletion mutant. The P. syringae transformants are then incubated first under moist
and then dry conditions on plants. Only the clones with active promoters directing the expression of metXW and hence a Met⫹ phenotype
are able to survive desiccation stress upon drying of the leaf surfaces. . These cells can then be screened for methionine auxotrophy in
culture medium to identify those clones harboring pig promoters.
To get a better understanding of what P.
syringae traits are expressed only on plants,
Maria Marco in our group designed a genetic
screen, called habitat-inducible rescue of survival (HIRS; Fig. 3). This variant of in vivo
expression technology (IVET) screens, which
typically are used to identify induced bacterial
virulence genes, is based on expression of a
selectable phenotype that is conditionally required for survival. The HIRS system took advantage of the fact that, when exposed to moist
conditions on leaves, methionine auxotrophs of
P. syringae B728a grow identically to wild-type
cells but suffer large declines in population size
and subsequently are unable to resume growth
on dry leaves (Fig. 3). Promoters contained in
random P. syringae genomic DNA could then
be“trapped” by HIRS as a result of their ability
to induce the expression of metXW required for
methionine biosynthesis and, hence, restore
growth to Met- P. syringae on plants but not
during their growth in minimal medium.
Several Mutants Provide Insights
into Epiphytic Lifestyle
HIRS has revealed several surprising responses
of P. syringae during its first stages of leaf colonization, including nutrition, virulence, trans-
474 Y ASM News / Volume 71, Number 10, 2005
port, and transcription changes that are encoded
among plant-inducible genes (PIGs). Differential expression of these genes in response to
changes in leaf wetness indicates that P. syringae
also appears to modulate its responses depending on environmental cues in the phyllosphere.
For example, PIGs with highest expression levels on moist plants include genes involved in the
export and biosynthesis of the cyclic peptides
syringolin and syringomycin, an operon encoding the multifunctional porin OprF, and genes
related to organosulfur metabolism. While
Pseudomonas species grow on various sulfonates, expression of this capacity during growth
on plant leaves was not observed previously.
Moreover, mutants disrupted in ssuE, a PIG that
is also a member of the sulfate starvation regulon, are not discernibly impaired in epiphytic
fitness. Although applying sulfate to leaves inhibits expression of ssuE, the population size of
wild-type P. syringae does not increase. Therefore, it is unlikely that sulfur is a limiting nutrient for P. syringae growth on leaf surfaces, and
genes involved in organosulfur metabolism encode only one mechanism by which sulfur is
incorporated into the cell.
On plants exposed to low moisture, the highest levels of expression were found for several
PIGs, including greA, a transcription elongation
factor and stress response protein, xerD, a sitespecific recombinase, and orf6, a gene with unassigned function conserved among P. syringae
plant pathogens and located in the conserved
effector locus of the P. syringae hrp/hrc pathogencity island. The orf6 locus was among the
most highly expressed genes identified by HIRS.
Most of the other PIGs identified by the
metXW HIRS approach were characterized by
low absolute levels of expression, both in culture
medium and under inducing conditions on
plants. This result suggests that the metXW
HIRS system is exquisitely sensitive for detecting gene promoters with such low levels of activity as to be “off” in culture medium but
expressed at somewhat higher levels on leaves.
The HIRS screen resulted in our isolating many
genes with low levels of activity in vitro and that
are less likely to have been studied previously.
These factors may help to explain why more
than half of all these PIGs encode proteins with
unknown functions.
Prominent among these plant-inducible loci
are several whose transcripts are initiated from
the antisense strand of annotated P. syringae
genes. While several of these transcriptional
units still need to be confirmed, this phenomenon is apparently not limited to epiphytic P.
syringae. For example, Paul Rainey and associates at Oxford University in Oxford, United
Kingdom, found in vivo-inducible antisense
transcripts in rhizosphere-associated bacteria,
opening the possibility that bacterial genomes
encode many more functional or regulatory
traits than previously thought.
Meanwhile, Rainey and Barbara Kunkel at
Washington University in St. Louis, Mo., and
their respective collaborators are using IVET
screening to study plant-inducible activities of
Pseudomonas species that live on roots or are
invading plant tissue. These screens reveal little
overlap between traits involved in the colonization of subterranean plant tissue, the phyllosphere, and during infection. A remarkable exception is that several P. syringae PIGs induced
during epiphytic growth are also induced during
disease when P. syringae pv. tomato is invading
tomato leaves. This overlap between leaf colonization and pathogenesis suggests that P. syringae is establishing intimate contact with the
plant host while an epiphyte.
The recent sequencing of the genomes of both
P. syringae pv. syringae strain B728a and P.
syringae pv. tomato strain DC3000 will further
accelerate studies of these important microbial
colonizers of plants. Genomic approaches enable directed studies of previously cryptic traits
that might control epiphytic fitness and virulence and will also facilitate studies of single
epiphytic cells. In combination, these studies
will help us to understand plant-mediated, environmentally controlled, and cell density-dependent patterns of gene expression of P. syringae
in its natural environment.
SUGGESTED READING
Boch, J., V. Joardar, L. Gao, T. L. Robertson, M. Lim, and B. N. Kunkel. 2002. Identification of Pseudomonas syringae pv.
tomato genes induced during infection of Arabidopsis thaliana. Mol. Microbiol. 44:73– 88.
Hirano, S. S., and C. D. Upper. 2000. Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae—a pathogen, ice
nucleus, and epiphyte. Microbiol. Mol. Biol. Rev. 64:624 – 653.
Leveau, J. H. J., and S. E. Lindow. 2001. Appetite of an epiphyte: quantitative monitoring of bacterial sugar consumption in
the phyllosphere. Proc. Natl. Acad. Sci. USA 98:3446 –3453.
Lindow, S. E., and M. T. Brandl. 2003. Microbiology of the phyllosphere. Appl. Environ. Microbiol. 69:1875–1883.
Marco, M. L., J. Legac, and S. E. Lindow. 2003 Conditional survival as a selection strategy to identify plant-inducible genes
of Pseudomonas syringae. Appl. Environ. Microbiol. 69:5793–5801.
Marco, M. L., J. Legac, and S. E. Lindow. 2005 Pseudomonas syringae genes induced during colonization of leaf surfaces.
Environ. Microbiol., in press.
Monier, J.-M., and S. E. Lindow. 2003. Differential survival of solitary and aggregated bacterial cells promotes aggregate
formation on leaf surfaces. Proc. Natl. Acad. Sci. 100:15977–15982.
Quiñones, B., C. J. Pujol, and S. E. Lindow. 2004. Regulation of AHL production and its contribution to epiphytic fitness in
Pseudomonas syringae. Mol. Plant-Microbe Interact. 17:521–531.
Quiñones, B., G. Dulla, and S. E. Lindow. 2005. Quorum sensing regulates exopolysaccharide production, motility, and
virulence in Pseudomonas syringae. Mol. Plant-Microbe Interact. 18:682– 693
Von Bodman, S. B., W. D. Bauer, and D. L. Coplin. 2003. Quorum sensing in plant-pathogenic bacteria. Annu. Rev.
Phytopathol. 41:455– 482.
Volume 71, Number 10, 2005 / ASM News Y 475