Mechanism of wilt (ralstonia solanacearum) development

2. • Kingdom : Prokaryota ( Bacteria)
• Phylum : Proteobacteria
• Class : Betaproteobacteria
• Order : Burkholderiales
• Family : Burkholderiaceae
• Genus : Ralstonia
• Species : solanacearum
Considered as one of the most important bacterial diseases of plants,
bacterial wilt was firstly described by E.F. Smith in potato, tomato and
eggplant in 1896 and subsequently in tobacco in 1908.
The disease has a worldwide distribution.
According to host range, R. solanacearum strains have been classified into
five races

3. Ralstonia solanacearum is a-
 Gram - negative
 Aerobic
 Non -spore forming
 Soil-borne
 Rod shape about 0.5-0.7 µm×1.5-2.0 µm
 Motile with a polar flagellum (Sneath et al.,1953)
Other name for R. solanacearum
• Bacillus solanacearum (Smith 1896)
• Pseudomonas solanacearum (Smith 1896) (Smith 1914)
• Burkholderia solanacearum (Smith 1896) Yabuuchi and Arakawa (1993)
• Ralstonia solanacearum (Smith 1896) Yabuuchi et al. (1995)
Host plants-
R. solanacearum is pathogenic to more than 200 plant species belonging to over 50
different botanical families. The majority of them mostly belong to the
Solanaceae and Musaceae families. Eg:- Potato, Tomato, Brinjal, Banana,
Geranium, Ginger, Tobacco, Sweet paper, Olive, Soybean and Rose etc.

4. Vascular browning
Diagnostic bacterial streaming in water
Tomato Potato
Wilted plant

5. Scanning electron microscopy of Tomato xylem
Healthy plant Infected plant

6. Bacterial wilt virulence depends on a large consortium of virulence factors
like:
EPS 1 (Exopolysaccharide)
CWDE (cell wall degrading enzyme)
Quorum sensing
Hrp Gene (Hypersensitive reaction and pathogenecity)
Motility (Flagella)

7.  Extracellular polysaccharide (EPS) is a major virulence factor of R.
solanacearum
 Polymer of N-acetyled sugar and a major virulence factor for infection.
 Production of ESP regulated by cell density and it is higher when pathogen
density is greater 107 cells/ml.
 ESP1 production requires nitrogen in the form of nitrate.
 R. solanacearum consumes the available oxygen and was able to use
inorganic N as a energy source with nitrate as a terminal electron acceptor.
 Ability to use nitrate is an important part of R. solanacearum virulence.
 EPS synthesis is regulated by the PhcA quorum sensing system such that it is
produced abundantly at high cell densities in culture or when the bacterium
grows in the confines of host plant xylem vessels
 The bacteria needs EPS to form biofilms on vessel surfaces during disease
development.
 EPS helps R. solanacearum survive desiccation or antibiosis in soil during
periods away from host plants; and
 EPS protects R. solanacearum from plant antimicrobial defenses by cloaking
bacterial surface features that could be recognized by hosts

8.  In heavily colonized vessels, bacterial cells and extracellular polymeric
substances (EPSs) can occlude xylem vessels.
 Vessels can also be blocked by tyloses, a type of plant immunity. Tyloses are
balloon-like out-growths of the living parenchyma cells adjacent to vessels
that extend into and block vessels.
 Gas embolisms- Embolisms could be formed by bacterial damage to xylem
pit membranes or by dinitrogen gas, a byproduct of R. solanacearum
denitrifying respiration. Evaporative transpiration from leaves generates
negative pressure in xylem vessels that draws sap upward, as in a straw. The
resulting negative pressure can suck gases into the vessels and displace the
sap. In response, some plant structures reduce embolisms and limit their
spread between vessels. The pit membrane, a modified primary cell wall that
separates vessels from the xylem apoplast, has microscopic pores that deter
embolism spread by bolstering the surface tension of sap and limiting gas
entry into vessels
 R. solanacearum has several cell-wall-modifying proteins that likely target
primary plant cell walls: two cellulases that degrade cellulose (ChbA and
Egl), one expansin that nonenzymatically loosens cellulose by disrupting
interstrand hydrogen bonds (ExlX), and four enzymes that degrade pectin
(Pme, PehA, PehB, and PehC)
 Cellulose degradation is important for R. solanacearum virulence, and pectin
degradation makes a smaller contribution to virulence

9. Mechanisms that Contribute to Plant Vascular Dysfunction during Bacterial Wilt Disease

10.  Phytopathogenic bacteria have often developed enzymes to hydrolyze plant cell
wall components to obtain nutrients and energy, which are further involved in
early stages of the infective process, favouring the entry and advance of the
pathogenic agent in host tissues.
 R. solanacearum produces several plant cell wall-degrading enzymes, secreted
the type two secretion system (T2SS). These include one β-1,4-
cellobiohydrolase (CbhA) and some pectinases whose activities have been
identified respectively as one β-1,4-endoglucanase (Egl). one
endopolygalacturonase (PehA), two exopolygalacturonases (PehB and PehC)
and one pectin methyl esterase (Pme).
 R. solanacearum Egl is a 43-Kd a protein that has proved to be involved in
pathogenicity.
 Inactivation of egl, pehA or pehB genes revealed that each contribute to R.
solanacearum virulence, and a deficient mutant lacking the six enzymes wilted
host plants more slowly than the wild-type.

11.  Since pectin catabolism does not significantly contribute to bacterial
fitness inside the plant, it seems that cellulase and pectinolytic activities
are preferably required for host colonization than for bacterial nutrition.
Thus, R. solanacearum hydrolytic enzymes are thought to be involved in
pathogenicity in plant.

12.  The recognition between R. solanacearum and the host has long been
thought to implicate an interaction between R. solanacearum LPS and plant
lectins, so involving LPS in the pathogenicity of the bacterium .
 Bacterial LPS is a component of the outer membrane and has three parts
the lipid A, the oligosaccharide core and the O-specific antigen.
 The core structure is composed of rhamnose, glucose, heptose, and 2-
ketodeoxy-octonate, whereas the O-specific antigen is a chain of repeating
rhamnose, N-acetylglucosamine, and xylose in a ratio of 4:1:1.
 Two genes encoding lectins have been characterised in R. solanacearum
presumably with a function in adhesion to plant surfaces, which is
important for R. solanacearum pathogenicity during the early stages of
infection.
 In fact, it was found that these lectins bind L-fucose and interact with the
plant xyloglucan polysaccharide belonging to the hemicellulose fraction of
plant primary cell walls.

13.  Bacterial quorum sensing (QS) is a powerful communication tool that
coordinates behaviors based on cell density
 Bacteria produce and secrete QS signals, which are small diffusible
molecules. As the bacterial population increases, QS signal accumulates
until it reaches a threshold that activates a signal transduction cascade that
transcriptionally and phenotypically reprograms the cells. QS ensures that
public goods such as virulence factors are produced only when beneficial to
the community, and it also enables subpopulations in the community to
quickly adjust to a different lifestyle
 R. solanacearum has LuxI/LuxR homologs (SolI/SolR) that produce and
respond to AHLs
 The lead QS system in R. solanacearum involves the LysR-family
transcriptional regulator PhcA, which is activated by either 3-
hydroxymyristatic methyl ester or 3-hydroxypalmitatic methyl ester
 When sufficient signal accumulates, PhcA is activated, triggering
expression of some virulence traits, such as EPS and cellulases, and
repression of others, such as siderophores and swimming motility

14. Quorum sensing in R. solanacearum

15. • Most of the traits needed for infection and virulence are regulated by the Phc
(phenotype conversion) regulatory system in a population density–dependent
manner.
• PhcA, a LysR-type transcriptional regulator, is at the center of a complex
regulatory hierarchy and its activity is modulated by a unique, volatile signal
molecule, 3-OH palmitic acid methyl ester (3-OH PAME).
• PhcA directly regulates endoglucanase and pectin methyl esterase production
but interfaces with four other response regulators and two sensors, which affect
the remaining Phc-controlled traits. 3-OH PAME is synthesized by the phcB.
• 3-OH PAME then acts as a signal for an atypical two-component regulatory
system that post transcriptionally modulates the activity of PhcA.
• This consists of a membrane-bound sensor-kinase, PhcS, that phosphorylates
PhcR
• QS regulation may be important to R. solanacearum as it makes the transition
from life in the soil to that of a parasite.
• Low levels of 3-OH PAME lead to reduced EPS and exoenzyme synthesis, but
enhanced motility and siderophore production. Conversely, inducing levels of
3-OH PAME promote PhcA activity, resulting in enhanced expression of EPS
and exoenzymes and decreased motility and siderophore synthesis.

16. • R. solanacearum possesses hrp genes (hypersensitive response and
pathogenicity genes) encoding structural constituents of the T3SS
• Bacteria use the T3SS to interact with host plants and to insert virulence
factors—effectors—into the host cytosol
• R. solanacearum hrp cluster consists of a region of DNA about 25 kb long,
which was shown by transposon mutagenesis to contain at least 6 transcription
units
• Several Hrp Proteins are Conserved Among Plant and Animal Pathogenic
Bacteria Sequence analysis of transcription units 1 to 4 and 7 revealed the
presence of 20 genes and nine of the corresponding Hrp proteins have
homologues in every other bacterial hrp gene cluster analysed to date. For this
reason, the corresponding genes were renamed hrc. These are thought to
encode the core of T3SS.
• A protein transiting through the Hrp secretion pathway, PopA, has been
characterised in R. solanacearum. The purified PopA protein is able to provoke
an HR-like necrosis on resistant plants like tobacco or certain genotypes of
petunia;

17. Genetic organisation of the hrp region
• - The expression of hrp genes is strongly activated by (a) plant signal(s)
• - The prhA gene encodes a putative receptor for a plant signal and this
receptor might be involved in the control of host specificity
• - The hrrG gene encodes a new regulatory protein which acts upstream
to HrpB and which might be involved in the plant signal(s) regulatory
cascade

18. Under hrp gene inducing conditions, R. solanacearum produces straight 5-10
nm diameter exo-structures which are located at one pole of the bacterium.
When present in the medium, these so-called pili very often assemble in
bundles which can be very long (up to 10 microns) which might be involved
either in the attachment of bacteria to plant cells or in the injection of proteins
directly into plant cell cytoplasm
-Besides proteins involved in the bio genesis of a type III secretion system, the
hrp gene cluster might encode proteins that transit through this pathway and
interact with plants

19.  R. solanacearum has two forms of active motility: swimming mediated by
flagella and twitching mediated by type IV pili.
 R. solanacearum uses flagellar motility and chemotaxis to locate host roots,
but swimming motility is largely repressed in xylem
 R. solanacearum becomes motile when the populations reaches 109
CFU/ml sap this is also the point when wilt symptoms appear, likely due to
reduced xylem sap flow.
 Bacteria move along surfaces with type IV pili-mediated twitching motility.
Flow orients polarly attached bacterial cells such that the pilus points
upstream; this allows bacteria to move down plant xylem vessels, against
the sap flow

20. • The pathogen moves to the host plant, attaches to the plant roots, infects the
cortex and colonizes the xylem which requires secretion of cell wall-
degrading enzymes and EPS controlled by a regulatory network that uses
PhcA.
• After destroying the host, the bacterium returns to the environment and is
likely to survive in soil, water or reservoir plants
• Within plant tissues, high densities of the pathogen increase expression of
pathogenicity genes, repressed by low bacterial densities in non-host
environments .

21. • In the environment, R. solanacearum senses specific stimuli and moves
towards plants by swimming motility to find more favourable conditions .
• R. solanacearum was actively attracted by chemotaxis to diverse amino
acids and organic acids, and specially to host root exudates, whereas those
from a non-host were less attractive .
• Furthermore, the ability of the pathogen to locate and interact with the host
was dependent on aerotaxis or energy taxis already described for R.
solanacearum . Thus, several aerotaxis-deficient mutants were impaired in
either localizing on host roots or moving up an oxygen gradient .
• Swimming motility, chemotaxis and aerotaxis seem to have a role in the
early stages of host invasion.

22. R. solanacearum host pathogenic interaction: root colonization, cortical
infection and xylem penetration
Root colonization
 After localizing the host roots, R. solanacearum can enter through
physical wounds and/or natural openings and attaches at two precise root
sites root elongation zones and axils of emerging or developed lateral
roots probably due to the fact that the epidermal barrier is usually weaker
in them.
 Moreover, root elongation zones are major sites of plant root exudation. In
the attachment to roots, pili and/or LPS seem to have a role and the
implication of flagella has also been demonstrated.
 Plant root cortical infection- It starts at the sites previously colonized i.e.
root extremities and axils of secondary roots. Due to the R. solanacearum
infection, the root cortex of these zones has the intercellular spaces
invaded and filled with bacteria In the intercellular spaces the bacterium
is likely to obtain nutrients from pectic polymers.

23. Plant root cortical infection
• It starts at the sites previously colonized i.e. root extremities and
axils of secondary roots. Due to the R. solanacearum infection, the
root cortex of these zones has the intercellular spaces invaded and
filled with bacteria.
• In the intercellular spaces the bacterium is likely to obtain nutrients
from pectic polymers of the middle lamella by action of pectinolytic
enzymes and also folate concentration in the spaces seems to
contribute to vigorous proliferation of the pathogen. Infection
proceeds to the inner cortex level of primary roots, with bacteria
forming large intercellular pockets, and cortical cells next to them
displaying features of degeneration .
• As disease progresses, swimming motility may help the invasive
cells go through the cortex.

24. Vascular cylinder infection and xylem penetration
• Bacterial advance from cortex to vascular parenchyma implies crossing
the endodermis, a cell layer with suberized walls and phenolic
compounds, thought to be a barrier to vascular pathogens.
• Therefore, to bypass the endodermis, the bacterium might reach the
vascular cylinder at sites where this barrier is compromised.
• Once in the vascular cylinder, R. solanacearum infects the intercellular
spaces of vascular parenchyma adjacent to xylem vessels.
• The pathogen can then be observed breaking into and filling xylem
vessels, with the surrounding parenchyma cells being highly degraded.
• Cell walls are destroyed by the hydrolytic enzymes secreted by R.
solanacearum Within the xylem vessels, the pathogen moves
throughout the stem to the upper parts of the plant while it is
multiplying, being reported to reach even more than 1010 cells per cm
of stem in tomato plants.

25.  It has been suggested that some R. solanacearum cells might form
biofilms on host xylem vessel walls, which would protect them from
host defenses and could filter nutrients from the flow of xylem fluid .
Although motility could help the pathogen spread out of infected
vessels into adjacent uninfected ones, R. solanacearum is effectively
non-motile in xylem vessels. Extensive multiplication and EPS
production taking place in the water-conducting system lead to wilting
of the host due to clogging of the vessels.

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27. Thank You