Higher plants contain nutrients that bacteria can access through openings like stomata. Gram-negative bacteria like Pseudomonads and Enterobacteria specialize in colonizing plant tissues. These apoplastic colonizers are often pathogens that cause diseases. Plants have evolved two lines of defense: pattern-triggered immunity in response to microbe-associated molecular patterns, and effector-triggered immunity in response to bacterial effectors through resistance proteins. Bacteria deliver effectors into plant cells through type III secretion systems like Hrp, and plants recognize specific effectors through corresponding resistance genes in a gene-for-gene interaction.

2. Higher plants contain potentially vast sources of nutrients for the bacterial
species in their environment, and most bacteria are small enough to pass through
stomata and other natural openings into the apoplast.
Mostly Gram negative bacteria in the Pseudomonadaceae and
Enterobacteriaceae, specialize in colonizing the apoplast.
It is the apoplastic colonizers that are the common pathogens that produce the
rots, spots, wilts, cankers, and blights.
Their ability to multiply and then sooner or later to kill plant cells depends on
secreted enzymes that degrade the wall or on molecules that pass through it.

3. The first line of defence is triggered upon the recognition of general elicitors,
known as microbe-associated (or pathogen-associated) molecular patterns
(MAMPs/PAMPs) by plant transmembrane pattern-recognition
receptors(PRR).
This recognition results in the PAMP-triggered immunity(PTI) or basal
defence that negatively affects the progress of bacterial infection.
Plants have evolved perception for different bacterial MAMPs like flagellin,
lipopolysacchride, elongation factor Tu (Ef-Tu), cold shock protein (CSP) or
peptidoglycan.
MAMP perception triggers several plant responses. Early reactions include
ion-flux across the plasma membrane, increased intracellular Ca2+
concentration, oxidative burst, MAP kinase activation, cell wall thickening
leading to papillae formation and altered expression of pathogenesis-related
(PR) genes and major transcriptional changes.

4. In addition to PTI, plants have evolved a second line of defence acting largely
inside the cell known as effector-triggered immunity (ETI) in which nucleotide-
binding site- LRR resistance proteins (R proteins) guard specific microbial
effector-mediated perturbations of host cell functions.
ETI results in hypersensitive cell death response (HR) at the infection site.
P. syringae effectors AvrPto, AvrRpt2, and AvrRpm1 inhibit defense responses
elicited by PAMP recognition.
Effector proteins such as the members of the Xanthomonas AvrBs3 effector
family have also been implicated in activating plant
, HopPtoK, and AvrPphE have been shown to modulate host signaling via
salicylic acid (SA), jasmonic acid (JA), and ethylene, all of which have a role in
plant defense transcription.
In response to this threat, plants have evolved R proteins which typically are
intracellular receptor proteins of the NBS-LRR type that recognize effectors in the
cytoplasm thereby activating the so-called effector-triggered immunity (ETI).

5. Zig zag model as described by Jones and Dangl (2006)

6. Typically, the ability to trigger ETI is pathogen strain or race-specific and is
associated with programmed cell death, a response which is referred to as
the hypersensitive response (HR), and systemic acquired resistance (SAR)
in the host.
Some of the best characterized R proteins are RPS2, RPM1, and RPS5.
These Arabidopsis R proteins confer resistance to P. syringae carrying the
bacterial effectors AvrRpt2, AvrRpm1/AvrB and AvrPphB respectively.

7. Salicylic acid is a phenolic metabolite that plays key roles in plant disease
resistance. This molecule is involved in both the localized HR and SAR and
can also interfere directly with several aspects of the infection process of
pathogenic bacteria.
Plant perception of avirulent pathogens leads to oxidative burst followed by
massive production of ROS
Functions of ROS – direct killing of pathogens, involvement in structural
changes to reinforce the cell wall, promotion of the HR.
Concomitant with the production of ROS, the recognition of an avirulent
pathogen by a resistant plant induces the accumulation of nitric oxide (NO).
NO has been shown to trigger HR, activate the expression of several defence
genes, increase the level of ROS in the cell, modulate the synthesis of SA,
jasmonic acid and ethylene.

8. Gram negative bacteria are capable of causing necrosis, but their necrogenic
aggressiveness varies.
Plant pathogenic bacteria in the genera Erwinia, Pseudomonas, Xanthomonas,
and Ralstonia cause diverse, and sometimes devastating, diseases in many
different plants, but they all share three characteristics: they colonize the
intercellular spaces of plants, they are capable of killing plant cells, and they
possess hrp genes.
Many of these pathogens are host specific. In host plants, they produce various
symptoms after several days of multiplication, whereas in non host plants, they
trigger the hypersensitive response (HR), a rapid, defense-associated,
programmed death of plant cells at the site of invasion

10. HR elicitation appears to require contact between plant and bacterial cells that
are both metabolically active and synthesizing new proteins.
High inoculum levels (106 bacterial cells/ml) results in macroscopically
observable death of the entire infiltrated tissue, usually within 24 h
HR elicitation and the resulting limitation in host range can occur if the
pathogen possesses any one of many possible avr (avirulence) genes that interact
with corresponding R (resistance) genes in the host plant.
Such “gene-for-gene” interactions result in recognition of the bacterium and the
triggering of plant defenses.

11. Hrp genes (hypersensitive response and pathogenicity)
The ability of the necrogenic phytopathogens to elicit the HR resides in hrp
genes, which were first found in P. syringae pv syringae and P. syringae pv
phaseolicola by identifying Tn5 transposon mutants that grew normally in
minimal media but failed to elicit the HR in non-host tobacco or cause disease or
multiply in host bean
hrp genes are clustered and are likely to occur within “pathogenicity islands”
containing supporting virulence genes
The nine hrp genes that are broadly conserved in plant and animal pathogens
have been redesignated as hrc gene (hypersensitive response and conserved).
The hrp genes, and particularly the hrc subset, are now considered to be
fundamentally involved in type III protein secretion in phytopathogenic bacteria.

12. A simplified scheme of hypothetical molecular interactions between avr and hrp
genes of a pathogenic bacterium and the R genes of two resistant and one susceptible
plant.

13. The type III secretion pathway is pathways that Gram-negative bacteria use
to secrete proteins across their inner and outer membranes (Salmond and
Reeves, 1993).
Plant pathogens use the Hrp type III pathway to transfer avr effector
proteins to the interior of plant cells.
In plant pathogens, harpin proteins are known to be secreted into the milieu
by the Hrp pathway, and there is evidence that Avr proteins are transferred
into plant cells.
Expression of a single secreted protein, harpin, could confer the ability to
induce the cell death response that is a hallmark of disease resistance, and
harpin was soon made available for commercial disease control (Wei et al.,
1992).

14. 1. Harpins
Harpins are glycine-rich, cysteine-lacking proteins that are secreted in culture
when the Hrp system is expressed and that possess heat-stable HR elicitor
activity when infiltrated at relatively high concentrations (> 0.1 µM) into the
leaves of tobacco and several other plants.
They serve parasitism directly by eliciting alkalinization of the apoplast and
nutrient release. Alternatively, they could act indirectly by assisting the delivery
of other bacterial proteins to plant cells.
These proteins are mostly targeted to the extracellular space of the plant
tissues.

15. 2. Avr proteins
Avr genes control host specificity at the race-cultivar level by triggering the
HR when the host carries a corresponding resistance (R) gene, in accordance
with Flor's gene-for-gene (avr-for-R) hypothesis
Unlike harpins, the Avr proteins (and HrmA) reveal no defining physical
characteristics. Furthermore, they have no effect when infiltrated into plants, no
known biochemical activity (except P s. tomato AvrD), and their sequences do
not suggest any function (except X. c. vesicatoria AvrBs2),they are hrp
dependent.
Thus, in fundamental contrast to the hrp genes, avr genes are
characteristically scattered in their distribution among strains of
phytopathogenic bacteria.
The specific recognition of the pathogen results in the induction of the plants
defense response, often a hypersensitive reaction.
They do not appear to be secreted in culture and are hydrophilic proteins
lacking N-terminal signal peptides or other recognizable secretion signals
In other words, avr genes restrict the host range of a particular pathogen to a
certain spectrum of plant lines.

16. Model of Bacterial Pathogenesis Involving Hrp-Mediated Delivery of Avr-like Proteins into Plant
Cells.
The Hrp secretion system of P. syringae

17. 1. Toxins
The toxins are secondary metabolites (mostly small peptides).
They show no host specificity, typically do not contribute to bacterial
multiplication in plants, and are highly diffusible, often producing characteristic
symptoms spreading well beyond developed lesions.
Bacterial toxins are virulence factors. They may contribute to the production
of certain symptoms but in a manner that is not essential for pathogenesis.
Toxins have been known for a long time to play a central role in parasitism and
pathogenesis of plant by several plant pathogenic bacteria. Pseudomonas
syringae, P. syringae pv. tomato, and P. syringae pv. Maculicola are primarily
associated with production of the phytotoxin coronatine
Coronatine functions primarily by suppressing the induction of defense-related
genes, but, as happens with most bacterial phytotoxins, it does not seem to be
essential for pathogenicity by all strains. The bacterium P. syringae, along with its
pathovars, produces several pathotoxins, including syringomycin.

18. Toxins are produced by some nonpathogenic strains of P. syringae, and
many toxins also have antimicrobial activity and thus may function primarily
to reduce microbial competition during epiphytic or pathogenic colonization.
Albicidins, produced by Xanthomonas albilineans, block the replication of
prokaryotic DNA and the development of plastids, thereby causing chlorosis
in emerging leaves. Albicidins interfere with host defense mechanisms and
thereby the bacteria gain systemic invasion of the host plant.

19. 2. Extracellular Polysaccharides
EPSs, unlike toxins, are produced by most bacteria, including many plant
pathogens, and are secreted as a loose slime or as capsular material.
Extracellular polysaccharides (EPS) play an important role in pathogenesis of
many bacteria by both direct intervention with host cells and by providing
resistance to oxidative stress.
EPSs are thought to protect free-living bacteria from a variety of
environmental stresses and may aid pathogenesis by sustaining water-soaking of
intercellular spaces, altering the accessibility of antimicrobial compounds or
defense-activating signals, and blocking the xylem and thereby producing wilt
symptoms.
EPS is generally a virulence factor in Ralstonia solanacearum and Erwinia
amylovora, contributing to wilt and water-soaking symptoms.

20. In the bacterial wilt of solanaceous crops caused by Ralstonia
solanacearum, EPS1 is the main virulence factor of the disease. EPS1 is a
polymer composed of a trimeric repeat unit consisting of Nacetyl
galactosamine, deoxy-l-galacturonic acid, and trideoxy-d-glucose. At least
12 genes are involved in EPS1 biosynthesis. EPS1 is produced by the
bacterium in massive amounts and makes up more than 90% of the total
polysaccharide. EPS likely causes wilt by occluding the xylem vessels and
by causing them to rupture from the high osmotic pressure.
EPS in the fire blight bacterium Erwinia amylovora is amylovoran, which
is biosynthesized and regulated by several clusters of genes. Disturbance of
production of amylovoran eliminates pathogenicity in the mutant.

21. 3. Cell wall degrading enzymes
Plant cell walls are composed of three major polysaccharides: cellulose,
hemicellulose, and pectins and, in woody and some other plants, lignin. The
number of genes encoding cell wall-degrading enzymes varies greatly in the
different plant pathogenic bacteria.
The enzymes include pectinases, cellulases, proteases, and xylanases.
Pectinases are believed to be the most important factor in pathogenesis in a
broad range of plant diseases caused by necrotrophic pathogens. as they are
responsible for tissue maceration by degrading the pectic substances in the middle
lamella and, indirectly, for cell death.
For the bacterial soft rots caused by the necrotrophic pathogens, E. carotovora
and E. chrysanthemi, pathogenesis is dominated by pectic enzymes that cleave β-
1,4-galacturonsyl linkages in plant cell wall polymers by hydrolysis
(polygalacturonases) or β-elimination (pectate or pectin lyases).

22. Four main types of pectin degrading enzymes are produced, three {pectate
lyase (Pel), pectin lyase (Pnl), and pectin methyl esterase (Pme)} with a
high (~8.0) pH optimum, and one polygalacturonase, with a pH optimum
of ~6. All are present in many forms or isoenzymes, each encoded by
independent genes.
For example, E. chrysanthemi produces five major Pel groups arranged into
two families and at least three minor Pel groups induced preferentially in
plant tissue and arranged into three other families.
In contrast, E. carotovora produces three major Pels, an intercellular Pel,
and several minor plant induced Pels.
Because of the structural importance and unique accessibility of pectic
polymers in the primary cell walls and middle lamellae of dicots and some
monocots, pectic enzymes are the big guns of the brute-force approach to
pathogenesis, and they cause both cell killing and tissue maceration, the
primary symptoms of soft rot disease.

23. Some Xanthomonads, e.g., Xanthomonas campestris pv. campestris, the
cause of black rot of crucifers, have genes for two pectin esterases and
polygalacturonases, four pectate lyases, five xylanases, and nine cellulases.
X. citri has no pectin esterases, one less pectate lyase, and three fewer
cellulases.
Other poor pectinolytic bacteria include A. tumefaciens, which has only
four genes encoding pectinases of any type, and Xylella, which has only
one gene coding for a polygalacturonase.

24. Several other components of the bacterial cell or released by the bacteria
appear to play roles as pathogenicity factors. Lipopolysaccharide (LPS)
components of the outer cell wall of gram-negative bacteria play a role in
the pathogenicity in Erwinia spp.
Catechol and hydroxamate siderophores appear to be virulence determinants
for Erwinias. In the fire blight bacterium E. amylovora, its siderophore
protects the bacteria by interacting with H2O2 and inhibiting the generation
of toxic oxygen species.

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