bacterial structure

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Bacterial-Plant Interactions - Pathogenesis
Topics - Plant pathogenic bacteria
1. Bacterial infection, multiplication and the hypersensitive response
2. Virulence factors
Toxins
Extracellular polysaccharide
Degradative enzymes
Quorum sensing
3. Type III secretion and hrp genes
4. R genes and Avirulence
Lecture 1 - Overview of bacterial pathogenesis on plants

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Bacterial pathogens specialize in colonizing the plant apoplast. They have two general features:
1) They parasitize plants and spend their life in close interaction with the wall of plant cells.
2) They are necrogenic - that is, they are able to cause the death of plant cells. This ability is
dependent on bacterial proteins that are secreted (to degrade the plant cell wall) and bacterial
proteins that are injected into plant cells through the wall.
Some of the well-studied gram-negative bacterial pathogens are listed in Table 1. The
necrotrophic bacteria generally kill quickly during infection of host tissue, and the biotrophic
pathogens usually multiply to high numbers before they cause necrosis. There are some gram-
positive plant pathogenic bacteria but we won’t discuss their properties. The other important
gram-negative bacterial pathogen that we have discussed is Agrobacterium tumefaciens.

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Bacterial pathogens of plants - background
1. Compared to the large number of bacterial species, relatively few are capable of
infecting plants. This suggests that specialized properties are needed for bacteria to
interact with plants.
2. The bacteria generally gain access to the apoplast through stomates and other natural
openings, or through wounds (e.g. A. tumefaciens).
3. Table 1 indicates that two types of secretion systems are important for plant pathogenic
bacteria to export proteins that contribute to virulence. If one takes a broader view, there
are actually four types of bacterial secretion systems that are important in plant pathogens.
a. Type I secretion - transmembrane channel that exports proteins without cleavage of an
N-terminal export signal. An example is the export of proteases by Erwinia chrysanthemi.
b. Type II secretion - multiple transport components reside in the inner and outer membranes
and in the perisplasmic space between the membranes. Secreted proteins have an N-
terminal signal sequence that is cleaved during transport. Examples of exported proteins
include the pectinases and cellulase produced by soft rot Erwinia species.
c. Type III secretion - multiple components in the inner and outer membranes form a syringe-
type export system that injects virulence (harpin and avirulence) proteins directly into plant
cells. This system plays an important role in all of the gram-negative pathogens.
d. Type IV secretion - this is the system that Agrobacterium tumefaciens uses to deliver T-
DNA into plant cells.

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Possible outcomes for the plant response to bacterial pathogens and non-pathogens.
Figure 1 shows the reactions for compatible (no recognition or defense response), incompatible
(recognition and defense response) and a nonpathogenic bacterium.

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Things to note about Figure 1
1) Incompatible interaction. The hypersensitive response (HR) is the most dramatic of the possible
responses that a plant can elaborate during the interaction with a potentially pathogenic bacterial
species. The HR is seen in the incompatible interaction. In this case, there is no pathogenicity (P)
or disease because of the strong defense response (DR). Two reactions that occur along with the
hypersensitive response are the generation of active oxygen species ((AOII) such as hydrogen
peroxide) and the Exchange Reaction (XR) which is a K+ efflux/H+ influx that happens for plant
cells. This XR results in a rise in pH for the apoplastic fluid during an infection. The HR results
in a localized plant cell death (self destruction) that limits the growth and spread of the pathogen.
This reaction can also happen in incompatible interactions between fungi or viruses and plants.
2) Compatible interaction. The compatible interaction is a disease situation where the pathogenic
bacteria multiply in plant tissue to high numbers. There is no HR because the plant fails to mount
a rapid resistance response. There is no generation of active oxygen species (AOII), the XR is
gradual and the defense response is delayed and ineffective. It is interesting to note that the
XR may contribute to the disease process because it is associated with the leakage of sucrose
from plant cells and with the multiplication of pathogenic bacteria in the plant.
3) Nonpathogenic interaction. The bacteria essentially cause no reaction in the plant. There is
no HR because the bacteria presumably lack the signals that the plant needs to recognize a
pathogenic attack.

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Figure 2 presents a model for bacterial pathogenesis on plants.
This figure diagrams the role of the hrp system and the delivery of avr proteins into plant cells.
Note that the hrp genes and avr genes are clustered together. This is similar to the clustering of
vir (virulence) genes in Agrobacterium tumefaciens and the clustering of nod (nodulation) genes in
Rhizobium species.
Compatibility results in parasitism with the releas of water and nutrients from plant cells.
Incompatibility results in an HR. The model in Figure 2 represents an important conceptual frame-
work from which to explore the functions of the proteins that are injected into plant cells.
The hrp genes = hypersensitive response and pathogenicity genes
Bacterial pathogenesis depends on the type III secretion system encoded by hrp (pronounced harp)
genes and avirulence gene products delivered to plant cells by the hrp system. A critical decision in
the interaction between bacterial pathogens and plants is whether an HR is triggered.
Mutations in these genes block the ability of the bacteria to cause disease and to trigger a
defense response. The mutants thus behave like non-pathogenic bacteria. The hrp genes are present
in large clusters of genes found in pathogenic bacteria. These genes encode the proteins needed to
assemble a type III secretion system that the bacteria use to inject proteins into plant cells. Similar
clusters of genes encoding type III secretion systems are found in bacterial pathogens of humans
including Yersinia (Bubonic plague bacteria), Shigella (dysentery) and Salmonella (diarrhea disease).
hrp genes are generally only expressed in conditions like those found in the plant apoplastic fluid
(low nutrients). That is, the bacterial pathogens don’t express these genes until they find themselves
in conditions that mimic the plant environment.

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The hrp genes (continued)
Some of the proteins that the hrp type III secretion system injects into plant cells are called harpins.
Purified harpin proteins have been found to cause an HR when injected into plants. The function of
harpins is unclear - one would not expect the bacteria to deliberately inject proteins that would cause a
defense response (HR)! The harpins may act to cause the XR to provoke plant cells to release
nutrient that will allow the bacteria to grow in the plant during a compatible (disease) interaction.
The avr genes = avirulence genes.
The gene-for-gene concept applies to bacterial pathogens
The avr genes in bacterial pathogens control host specificity via the gene-for-gene system. Mutations in
these genes make the bacterium invisible to the host defense recognition system. The plant fails to
develop an HR and the bacterium is able to cause disease. Over 40 bacterial avirulence genes have been
isolated and sequenced.
Very little is known about how avirulence gene products (proteins) function but biochemical acitivities
are starting to be discovered. The activity of avr genes and their products in triggering a defense
response depends on the function of the hrp system for protein secretion. That is, at least some of
the known bacterial avirulence proteins act inside plant cells after delivery by the hrp type III
secretion system. Some avirulence products may be involved in virulence (causing disease in a
compatible interaction) in addition to triggering a defense response in an incompatible interaction.

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Bacterial avr genes
The genes have generally been cloned by transferring cloned genomic DNA from an avirulent
strain to a virulent strain and then screening the transformants for their ability to elicit a
hypersensitive response. The procedure works well for bacteria because their genomes are
relatively small and this means the a managable number of transformants can be screened.
In general, the sequence analysis of the bacterial avr genes has not provided clues about their
functions. Functions are starting to be discovered and we will discuss these functions in later lectures.
One avirulence gene appears to encode an enzyme that makes small molecule elicitors. This gene is
avrD of P. syringae pv. tomato and the elicitors are acyl glycosides call syringolides 1 and 2.

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Other disease factors. These include toxins, extracellular polysaccharide
(EPS), degradative enzymes and quorum sensing.
Toxins
These toxins from P. syringae do not show host specificity and they therefore behave quite differently
from the host-selective toxins from fungal pathogens. The bacterial toxins contribute to the virulence
of the bacteria that produce them but the are note essential for pathogenesis.
EPS
Extracellular polysaccharides are produced by many of the plant pathogenic bacteria and they form a
capsule or slime around the bacteria. The material may protect the bacteria in the environment and
contribute to virulence by blocking the xylem and causing wilt symptoms.

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Degradative enzymes - examples for the Erwinia species
Erwinia chrysanthemi Erwinia caratovora
PelA Pectate lyase PelA Pectate lyase
PelB Pectate lyase PelB Pectate lyase
PelC Pectate lyase PelC Pectate lyase
PelD Pectate lyase
PelE Pectate lyase
ExoPeh Polygalacturonase Peh Polygalacturonase
EGY Cellulase CelS Cellulase
EGZ Cellulase CelV Cellulase
Pem Pectin methylesterase
PrtA Protease Prt1 Protease
PrtB Protease
PrtC Protease
PrtG Protease
PlcA Phospholipase
PnlA Pectin Lyase
These Erwinia species cause soft rot diseases in a wide variety of hosts. The enzymes contribute
to a brute force approach to plant cell killing and tissue maceration.

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Quorum sensing
Some bacteria use quorum sensing to monitor their local population density. They accomplish this
by secreting and monitoring small diffusible signaling molecules. These molecules mediate cell-cell
communication for the bacteria with the result that certain traits are only expressed when the
bacteria are crowded together. The signal molecules accumulate when cells are crowded together
and when the concentration reaches a threshold, receptors signal a change in gene expression.
N-acyl homoserine lactones are the most common types of signaling molecules that bacteria use
for quorum sensing.
Plant pathogenic bacteria use quorum sensing to control pathogenesis and colonization of the host.
Thus quorum sensing is used to control a number of traits such as:
1. Production of extracellular polysaccharides
2. Production of degradative enzymes
3. Production of siderophores (low molecular weight iron binding factors)
4. Expression of type III secretion apparatus (hrp genes).
5. Transfer of the Ti plasmid between strains of Agrobacterium tumefaciens.
Examples of quorum sensing in plant pathogenic bacteria

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Agrobacterium tumefaciens
Acyl-homoserine lactones (AHLs) and some opines serve as quorum sensing signals for A. tumefaciens.
These molecules trigger the conjugative transfer of the Ti plasmid from plasmid carrying strains to
other strains that don’t carry the plasmid but happen to reside in the same gall. It is possible that
the bacteria lose the Ti plasmid and grow better during the early stages of crown gall disease. Galls
are known to contain a significant population of bacteria that are missing the Ti plasmid. At later
stages of gall formation, nutrients may be limiting and the bacterial cell density may be sufficiently
high to trigger quorum sensing to result in Ti plasmid transfer to more cells in the galls. These cells
would then be capable of initiating new infections. Cells that obtain the Ti plasmid would also be able
to use opines as a carbon and nitrogen source.
Genes encoding functions needed for
quorum sensing, conjugal transfer and
replication of the Ti plasmid

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Erwinia stewartii
This bacterium causes Stewart’s wilt of corn, a disease transmitted by the corn flea beetle. The
beetle feeds on corn seedlings and introduces the bacterium into the xylem where it can multiply
to cause a wilt disease. The bacterium secretes large amounts of extracellular polysaccharide to
occlude xylem vessels.
A two component regulatory system (EsaI/EsaR) mediates quorum sensing such that the bacterium
does not express EPS until the cell density reaches 108 cells per ml. It appears that the absence of
EPS at low cell densities may help the bacterium attach to plant cells (it is known that EPS interferes
with attachment). The EsaI/EsaR system that senses AHLs also regulates the expression of hrp
genes that the bacterium uses to generate additional symptoms (e.g. water soaked lesions).
Soft Rot Erwinias (E. chrysanthemi and E. caratovora).
A quorum sensing system controls the expression of extracellular degradative enzymes, the hrp
secretion system and the production of an antibiotic (carbapenem) in these bacteria. The quorum
sensing mechanism for the soft rot Erwinia species has been called the “mob” attack mechanism of
pathogenesis. The bacteria avoid producing pectic enzymes to degrade plant tissue during the early
stages of infection possibly to avoid triggering a defense response. It is known that pectic enzymes
and the products of their action can trigger a defense response. The pathogen might withhold
a pectolytic attack (wall degradation) until a sufficiently large population of bacteria has accumulated
to overwhelm the plant defenses.

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Ralstonia solanacearum
This pathogen causes vascular wilt in many different host species including tomato, tobacco, and
potato. The bacteria infects the xylem and can grow to a density of 1010 cells per cm length of
stem tissue. A volatile quorum sensing signal 3-hydroxy palmitic methyl ester is used to sense
population density and to regulate the production of EPS, degradative enzymes such as pectin
metylesterase, endoglucanase, polygalacturonase.
Is there a host plant response to quorum sensing?
Plants may be able to destroy quorum sensing molecules as a way to interfere with bacterial
pathogenesis. Alternatively, plants may be able to sense the signals as a means of triggering a
defense response. There are no reports that plants have these abilities but soil bacteria such as
Bacillus cereus do make lactonase enzymes that destroy the AHL molecules. The transgenic
expression of a lactonase in tobacco and potato caused the plants to be resistant to infection
by Erwinia caratovora.