Chapter_ 6 Plant pathogenic Bacteria_Pathogencity.ppt

Bacterial pathogenesis genes and virulence factors
 Gram-negative pathogenic bacteria : Evolved sophisticated strategies to
exploit the attractive nutritional menu provided by plants and animals.
 The majority of bacterial pathogens are highly specialized for a limited
number of eukaryotic host organisms.
 However, some bacterial strains are capable of infecting a wide range of
diverse hosts that includes both plants and animals
 Pathogenicity genes are genes that make a particular (micro) organism
a pathogen
 Bacterial invasion and colonization of eukaryotic tissues involves a
variety of extracellular factors,polysaccharides, adhesins,toxins
degradative enzymes and bacterial effector proteins

Bacterial pathogenesis genes and virulence factors…continued
 Pathogenesis and disease resistance are closely related= subjects treat host-
pathogen interactions from different point of view.
 Pathogenesis treat from the side of compatible interactions and resistance from
the compatible one.
 Genetic analysis of the ability of plant pathogenic bacteria to induce pathogenic as
well as resistant reactions on plant is one of the most rapidly developing fields in
bacterial plant pathology.

 The pathogenecity of plant pathogenic bacteria can be expressed through several
infection stages
 invasion (or ingress)
 recognition
 multiplication of bacteria,
 production of virulence factors, and
 symptom development. These stages often occur in continuity and are difficult to
recognize as independent phenomena

 Invasion of plant pathogenic bacteria through portals of entry such
as natural openings and wound is usually a passive phenomena.
 The genetic functions found in the infection process of most fungal
pathogens are not necessary.
 Chemotaxis is sometimes, referred to as the active response of
bacteria to invasion.

 When plant tissues are infected by compatible bacteria, partial
degeneration of cell membrane takes place, activating the K+ efflux –
H+ influx change.
 The potassium ion released into intercellular space increase the pH of
intercellular fluid from 5.5, to 7.0-7.5, and this change further induces
efflux of sucrose, amino acids, and inorganic ions without causing
structural damage to plasma membrane.
 As bacterial growth progresses in the intercellular space, water-soaking
becomes visible to the naked eye, and creates conditions for
accelerated multiplication of bacteria.

Production of virulence factors:
 Some plant pathogenic bacteria growing in the intercellular
spaces produce virulence factors

 Five main types of bacterial pathogenicity factors are known:
I. Effector Proteins: These can be secreted into the extracellular environment
or directly into the host cell, often via theType three secreetion system( TTSS) .
Some effectors are known to suppress host defense processes.
II. Phytohormones: for example Agrobacterium changes the level of Axuxin to
cause tumours.
III. Cell wall degrading enzymes - used to break down the plant cell wall in order
to release the nutrients inside. Used by pathogens such as Erwina to
cause soft rot.
IV. Toxins: These can be non-host specific, and damage all plants, or host
specific and only cause damage on a host plant.
V. Exopolysaccharides:- these are produced by bacteria and block xylem
vessels, often leading to the death of the plant.

Initial events at the host–pathogen interface
 Physical contact between the bacterium and the host cell
 Bacterial attachment to the host cell surface is mediated by surface proteins,
termed adhesins
 Adhesins assembled into pilus-like structures (fimbrial adhesins) or anchored
in the outer membrane (afimbrial adhesins)
 In animal pathogenic bacteria, adhesins bind to specific host-cell receptors,
thus allowing a tight contact between the pathogen and the host cell
 In plant pathogenic bacteria, however, the role of adhesins in the interaction
with the cell wall, a natural barrier that surrounds plant but not animal cells, is
less clear
 Bacteria control the production of Pathogenicity factor via quorum sensing.

Bacteria control the production of Pathogenicity factor via quorum sensing.
 The production of small molecular weight signals as a mechanism of cell–cell
communication among bacteria is well recognized.
 Regulation of bacterial gene expression in response to N-acyl-homoserine lactone
(AHL) signals is the best characterized example of such a system. This form of
communication has been referred to as ‘quorum sensing’ as the level of signal required
for gene induction occurs only when the appropriate density or ‘quorum’ of signal
producers is present.
 The basic model for AHL-mediated gene regulation involves a transcriptional regulator
(i.e. an R protein) and an AHL synthase (i.e. an I protein) (Figure 1).

A simplified scheme illustrating some key control features of AHL-mediated autoinduction by I and R proteins.
Bacterial cells (shown in blue) contain an I protein that is responsible for the synthesis of freely diffusible signals (green
ovals).
At high cell density, the signal accumulates intracellularly and interacts with the R protein.
This interaction induces a conformational change in the R protein, which alters the affinity of the R protein for specific
DNA sequences, known as ‘lux’ boxes, that are located within the promoters of the AHL-regulated genes.

 This model is based upon studies in which the bacterial population size determined the
pattern of AHL-mediated gene expression. The R protein can recognize specific
promoter sequences and stimulate gene expression only when complexed with an AHL
signal.
 Typically, the AHL synthase is expressed at low levels and, at low cell densities,
insufficient intracellular AHL exists to activate the R protein.
 As the bacterial population increases, AHLs accumulate until sufficient intracellular AHL
is present to ensure that some of it binds to the R protein, resulting in the binding of the
R protein to its target promoter sequences

How the bacterial plant pathogen Xanthomonas campestris pv. Vesicatoria conquers the host
 Xanthomonas campestris pv. Vesicatoria (Xcv) is the causal agent of bacterial spot disease on pepper
and tomato.
 In natural infections, the bacteria enter the plant through stomata or wounds to reach the intercellular
spaces of the tissue where they establish an intimate relationship with the plant cell
 Pathogenicity on susceptible plants and the induction of the hypersensitive reaction (HR) on resistant
plants requires a number of genes, designated hrp , most of which are clustered in a 23-kb chromosomal
region.
 Nine hrp genes encode components of a type III protein secretion apparatus that is conserved in Gram
negative plant and animal pathogenic bacteria.
 Recently has been demonstrated that Xcv secretes proteins into the culture medium in a hrp-dependent
manner.
 Substrates of the Hrp secretion machinery are pathogenicity factors and avirulence proteins, e.g.
AvrBs3.
 The AvrBs3 protein governs recognition, i.e. HR induction, when bacteria infect pepper plants carrying
the corresponding resistance gene Bs3 . Intriguingly, the AvrBs3 protein contains eukaryotic signatures
such as nuclear localization signals (NLS), and has been shown to act inside the plant cell.
 Finally being postulate that AvrBs3 is transferred into the plant cell via the Hrp type III pathway and that
recognition of AvrBs3 takes place in the plant cell nucleus.

Fig….. Model for the interaction of X. campestris pv. vesicatoria with susceptible and resistant host plants.

Bacterial Secretion Systems
 Secretion systems are essential pathogenicity tools
 They make possible the translocation of bacterial proteins and other molecules into host plant
cells
 Once the bacteria are close to a host cell, they start to inject effector proteins into the cytosol of
the eukaryotic cell
 The delivery of effector proteins is mediated by the TTS system, which spans both bacterial
membranes and is associated with an extracellular appendage


 TTS systems are present not only in many Gram-negative pathogenic
bacteria but also in some plant symbionts, such as Rhizobium spp., in wh
they presumably influence the host range

 The structure and function of TTS systems differences among the TTS
systems of plant and animal pathogenic bacteria reside in the extracellula
part of the secretion machinery.

 Type I-SS: present in almost all plant pathogenic bacteria
 carries out the secretion of toxins such as hemolysins, cyclolysin,
and rhizobiocin
 They consist of ATP-binding cassette (ABC) proteins
 involved in the export and import of a variety of compounds
through energy provided by the hydrolysis of ATP.
 Type II-SS is common in gram-negative bacteria and is involved
in the export of various proteins, enzymes, toxins, and virulence

 Type III-SS is the most important in terms of pathogenicity of the
bacteria in the genera Pseudomonas, Xanthomonas, and Ralstonia.
 The primary function of type III-SS: transport of effector proteins
across the bacterial membrane and into the plant cell

 The TTS system of animal pathogens is associated with a needle,
which is essential for the delivery of effector proteins into the host
cell
 In plant pathogenic bacteria, the TTS system is connected to a pilus
structure, which is up to 200 nm in length and can potentially cross
the plant cell wall
 The pilus serves as a conduit for secreted proteins and.

Figure 1. Enzymatic activities of bacterial effector proteins. Known
enzymatic activities of effector proteins from animal pathogenic bacteria
(blue circle) and their major effects on infected host cells
(yellow circle).
Homologous effector proteins that have been identified in plant
pathogenic bacteria are indicated in the green area. An enzymatic activity
has not yet been demonstrated for members of the YopJ/AvrRxv family or
for effector proteins from plant pathogenic bacteria

Figure 2 Simplified model of the interaction between Gram-negative plant pathogenic bacteria and the plant cell. It is
suggested that bacterial effector proteins are secreted (injected?) into the plant cytoplasm via the Hrp type III secretion
system. Once in the plant cell, the effectors interfere with host metabolism for their own benefit, leading to disease. In case
of specific recognition of an effector protein by the plant resistance gene (R gene) mediated surveillance system, plant
defence reactions are induced resulting in resistance

Figure 3. Model describing the role of TTS systems in bacterial interactions with plants
and animals.

(
a). The TTS system of plant pathogenic bacteria is associated with the Hrp pilus, which
presumably spans the plant cell wall (200 nm thick; not drawn to scale) and serves as a
conduit for secreted proteins. Among the secreted proteins are harpins (yellow) that
presumably act at the plant cell surface and effector proteins (dark green). The translocation
of effector proteins into the host cell cytosol is mediated by the putative TTS translocon, a
bacterial protein complex in the host plasma membrane (PM)].
(b) The TTS system of animal pathogenic bacteria is associated with a needle structure
that is significantly shorter than the Hrp pilus. The translocation of effector proteins into
the host cell cytosol is mediated by a putative channel formed by the TTS translocon.
Several animal pathogenic bacteria (e.g. species of Salmonella and Shigella) are able to induce
their uptake into non-phagocytic cells].

Figure 4 Proposed virulence functions of type-III effector proteins from plant and animal pathogenic bacteria. The
TTS system of plant and animal pathogenic bacteria delivers effector proteins into the host cell cytosol where they
interfere with specific host target proteins.

(a). Some effector proteins from plant pathogenic bacteria presumably
localize to the plant cell nucleus and modulate host gene expression, as
has been shown for AvrBs3
The molecular activities of effector proteins inside the plant cell lead to
a suppression of host defense responses.
Furthermore, effector proteins probably cause the release of water and
nutrients into the extracellular medium.
(b) In animal host cells, effector proteins trigger a variety of cellular
responses. Nuclear localization and modulation of host gene expression
has been demonstrated for the effector protein YopM (see text for
details). PM, plasma membrane.

Fig.6. Model for the interaction of X. campestris pv. vesicatoria with susceptible and resistant host plants.

Model for the interaction of X. campestris pv. vesicatoria
 The bacterial TTS system spans both bacterial membranes and is
associated with an extracellular pilus that presumably crosses the plant cell
wall (200 nm thick, not drawn to scale).
 Upon contact with the host cell, effector proteins are delivered into the host
cell cytosol with the help of the predicted TTS translocon, a bacterial protein
complex that contains HrpF and inserts into the plant cell membrane.
 In susceptible plant cells, effectors interfere with host cellular pathways,
ultimately leading to the formation of disease symptoms. In resistant plants,
however, effector proteins are recognized by matching plant resistance (R)
proteins and induce the HR.
 Photographs of plant phenotypes were taken 3 days after inoculation of a
high-density bacterial culture into leaves of susceptible and resistant pepper
plants. PM, plasma membrane; TTS, type III secretion.

Figure . Basic interactions of pathogen avirulence (A)/ virulence (a) genes with host resistance
(R)/susceptibility (r) genes in a gene-for-gene relationship and final outcomes of the
interactions

Figure Basic events in an incompatible host–pathogen interaction: Elicitors from pathogen interact with plant cell
receptors. Signal transductions activate hypersensitive (host defense) responses that lead to programmed cell death
and systemic acquired resistance

Pathogenicity of Bacterial Enzymes That Degrade
Cell Walls
 Plant cell walls compositon: 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:
 Softrotting erwinias produce a wider range of enzymes able to degrade plant cell wall
components than any other plant pathogenic bacteria.
 The enzymes include pectinases, cellulases, proteases, and xylanases.
 Pectinases are believed to be the most important in pathogenesis, as they are responsible
for tissue maceration by degrading the pectic substances in the middle lamella and,
indirectly, for cell death.

 Four main types of pectindegrading 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 byindependent 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
plantinduced
 Pels.

PATHOGENICITY AND VIRULENCE FACTORS in Pseudomonas
TTSS and effectors
 The type III protein secretion system (TTSS) is key to the plant parasitism of P.
syringae pathovars and has been found in all of the P. syringae strains
examined.
 Most of the hrp (hypersensitive response and pathogenicity) and hrc (hrp
conserved) genes encoding the TTSS system are essential for pathogenicity,
which indicates the collective importance of the ffector proteins that are injected
into plant cells by the system.
 Type III effectors are believed to contribute to pathogenesis in two ways: by
eliciting the release of water and/or nutrients from the host cell in the apoplastic
space; and by suppressing and/or evading plant host defense responses

TTSS and effectors…continued
 The type III effectors produced and secreted by the pathogen interact with
plant molecules known as virulence targets.
 In resistant plants, effectors function as avirulence determinants that activate
the hypersensitive response (HR), a primary defense response triggered by
recognition of the effector-virulence target complex by plant resistance genes.
 In susceptible plants, effectors avoid specific recognition by the plant host
surveillance mechanisms and function as virulence determinants that facilitate
pathogenesis and modulate host defense responses and physiology to the
benefit of the pathogen.

Phytotoxins
 Pseudomonas spp. produce a wide spectrum of phytotoxic compounds.
 Among the most well-characterized bacterial phytotoxins are those
produced by Pseudomonas syringae
 The toxins produced by P. syringae include monocyclic lactam (tabtoxin),
sulfodiaminophosphinyl peptide (phaseolotoxin), lipodepsipeptide
(syringomcins, syringopeptins) and polyketide (coronatine) structures.

Phytotoxins ..continued
 Although phytotoxins are not required for pathogenicity in P. syringae, they
generally function as virulence factors and their production results in
increased disease severity.
 P. syringae phytotoxins can contribute to systemic movement of bacteria
in planta, lesion size, and multiplication of the pathogen in the host.
 Tagetitoxin is a cyclic hemithioketal molecule that is only
 produced by strains of P. syringae pv. tagetis.
 The toxin interferes with RNA polymerase in protein biosynthesis of
chloroplasts.
 The toxin can rapidly be detected by its ability to elicit apical chlorosis in
plant tissues.

Phytotoxins ..continued
Toxins produced by other Pseudomonas species include the
lipodepsipeptides corpeptin, fuscopeptin, tolaasin and viscosin
produced by P. corrugata, P. fuscovaginae, P. tolaasii and P.
fluorescens (marginalis), respectively.
 The best studied phytotoxins are coronatine, syringomycin, tabtoxin
and phaseolotoxin.

Tabtoxin
 Tabtoxin is a monocyclic β-lactam produced by P. syringae pv. tabaci,
coronafaciens, and garcae, which cause wildfire on tobacco, and halo blight of
oats and coffee, respectively.
 P. syringae pv. striafaciens, the causal agent of bacterial stripe of oats, is
tabtoxin-deficient, but further indistinguishable from P.syringae pv.
coronafaciens and pv. garcae.
 Recent evidence suggests that P.syringae pv. coronafaciens, garcae and
striafaciens are likely the same pathovar.

Tabtoxin..,continued
 Introduction of the tabtoxin biosynthetic region in P. syringae pv. Striafaciens
resulted in the production of lesions on oat leaves there were indistinguishable from
those caused by P. syringae pv. coronafaciens (Barta and Willis, 2005).
 Tabtoxin contains tabtoxin-β-lactam linked by a peptide bond to threonine.
 The chlorosis-inducing activity occurs only after hydrolysis of the peptide bond by
aminopeptidases of plant or bacterial origin.
 Cleavage of the peptide bond releases tabtoxin-β-lactam, the toxic moiety.
 Tabtoxin-β-lactam irreversibly inhibits glutamine synthetase.
 This enzyme is the only way to efficiently detoxify ammonia.

Phaseolotoxin
 Phaseolotoxin is produced by P. syringae pv. phaseolicola and P. syringae pv.
actinidiae, which cause halo blight on beans and bacterial canker on kiwifruit,
respectively.
 Phaseolotoxin consists of a sulfodiaminophosphinyl moiety linked to a
tripeptide.
 Phaseolotoxin competitively inhibits ornithine carbamoyl transferase
(OCTase), a critical enzyme in the urea cycle, which converts ornithine and
carbamoyl phosphate to citrulline.
 Phaseolotoxin is hydrolysed in planta by peptidases to produce octicidine.
 Octicidine is an irreversible inhibitor of OCTase and the predominant form of
the toxin in infected tissues.
 Inhibition of OCTase causes an accumulation of ornithine and a deficiency in
intracellular pools of arginine, leading to chlorosis.

Auxin production
 Auxin production by pathovars of P. syringae and related species.
 Most of the analysed strains produced IAA, especially in the presence of tryptophan.
 The strains P. syringae pv. syringae 1392 and P. syringae pv. aceris 2339
(genomospecies 1); P. savastanoi pv. savastanoi 1670, P. syringae
 pv. myricae 2897 and P. syringae pv. photiniae 2899 (genomospecies 2); P.
 syringae pv. maculicola 1657 and P. syringae pv. ribicola 10971t (genomospecies
 3); and P. syringae pv. cannabina 2341 (genomospecies 9)
 synthesized IAA at concentrations over 2 μg/ml when grown in modified King B
medium without tryptophan and produced high amounts of IAA in the presence of
tryptophan.
 These strains harbor genes homologous to the iaaM/iaaH genes of P. savastanoi.
 The involvement of IAA in pathogenicity has been unambiguously demonstrated for P.
savastanoi pv. savastanoi

Auxin production…continued
 P. syringae pv. myricae also induce proliferation of plant tissues and also
harbor the iaaM/iaaH genes.
 IAA production has also been associated with epiphytic survival or with toxin
production as demonstrated for P. syringae pv. Syringae strains on
Phaseolus vulgaris.
 There are also indications that IAA may inhibit plant
 defense mechanisms

Ethylene production
 Ethylene production has been demonstrated in various pathovars of P. syringae,including
pvs. glycinea, pisi
 In addition strains of P. syringae pv. Phaseolicola isolated from kudzu (Pueraria lobata)
also produce ethylene unlike P.s. pv. phaseolicola strains isolated from bean
 The efe gene encoding the ethylene-forming enzyme appears to be plasmid encoded
 Study on role ethylene production for Virulence of P. syringae pv. phaseolicola was not
affected by disruption of the efe gene, while efe mutants of P. syringae pv. glycinea were
significantly reduced in their ability to grow in planta

Exopolysaccharides
 The production of exopolysaccharide polymers by phytopathogenic bacteria has been implicated in several
symptoms, including wilting induced by vascular pathogens and the water soaking associated with foliar
pathogens.
 P. syringae pathovars generally produce two EPS molecules: levan, a fructofuranan polymer, and alginate,
a co-polymer of O-acetylated β-1,4-linked D-mannuric acid and L-guluronic acid
 When grown on media with excess sucrose, many P. syringae pathovars produce levan P. syringae pv.
ciccaronei, which causes leaf spots on carob plants produces a mannan exopolysaccharide.
 The pure polysaccharide showed phytotoxic effects,i.e., chlorosis and necrosis on tobacco leaves

Pectinolytic enzymes
 Soft-rotting Pseudomonas fluorescens (marginalis) strains are capable of degrading pectic
components of plant cell walls by producing a wide variety of pectolytic enzymes, including pectin
methyl esterase, pectin lyase, polygalacturonase and two pectate lyase isozymes
 P. viridiflava produces a single pectate lyase (PelV), which has a very alkaline PI, like the major Pel
enzyme of P. fluorescens (marginalis)
 At least some of the P. syringae pathovars also produce pectic enzymes and pel gene sequences are
available in the database for P. syringae pv. lachrymans, P. syringae pv. phaseolicola, P. syringae pv.
tabaci, and P. syringae pv. glycinea. P. syringae pv. glycinea produces two alkaline ectate lyase
isozymes with pIs of 9.0 and 9.5 and an alkaline polygalacturonase
 P. syringae pv. Lachrymans produces a single pectate lyase enzyme with a pH optimum between 8.0
and 8.5 which is encoded by the pelS gene

Chapter_ 6 Plant pathogenic Bacteria_Pathogencity.ppt

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