Virulence is a pathogen's or microorganism's ability to cause damage to a host. In most, especially in animal systems, virulence refers to the degree of damage caused by a microbe to its host.[1] The pathogenicity of an organism—its ability to cause disease—is determined by its virulence factors.[2][3] In the specific context of gene for gene systems, often in plants, virulence refers to a pathogen's ability to infect a resistant host.

1. UNIVERSITY OF AGRICULTURAL
SCIENCES, BANGALORE
COLLEGE OF AGRICULTURE V. C. FARM
MANDYA
ASSIGNMENT TOPIC :
Bacterial virulence and host-bacterial pathogen interaction
LAVANYA N
PAMM2005
Dept. of Plant Pathology

2. Major diseases of crop plants that are caused by plant pathogens are a
considerable threat to global food security.
The co-evolution of plant pathogens and their hosts has resulted in highly adapted
microbial invasion strategies and plant counter-defence mechanisms.
Phytopathogenic bacteria colonize all plant tissues. Although bacteria can live in
the phyllosphere or rhizosphere without causing any harm to the plant, to become
a fully virulent pathogen, it must incite diseases in plants by penetrating into host
tissues through natural openings, such as hydathodes, stomata, lenticels, stigma,
nectarthodes or through wounds and bacteria are directly deposited by insect
vectors (Benali et al., 2014).

3.  Commonly phytopathogenic bacteria colonize the apoplast (intracellular space of
plants) and from this location outside the walls of plant cells they provoke a range
of diseases in most economical plants.
 Besides the endophytic nature, some bacterial species also have the epiphytic
habitat on plant surfaces (rhizoplane, phylloplane, carpoplane, etc.) (Dangl and
Jones, 2001).
 Once inside plant tissues, various species can inhabit the dead xylem vessels or live
in phloem sieve elements; however, most of pathogenic bacteria are limited to
intercellular space, i.e. apoplast.
 As the plant bacterial pathogens are extracellular, they deploy a delivery of
secreted virulence factors to interfere with host cell processes from outside plant
cells.

4. The terms virulence and pathogenicity, which are often erroneously considered
synonyms. Shurtleff and Averre defined that pathogenicity is the ability of a
pathogen to cause disease, whereas virulence is the degree of pathogenicity of a
given pathogen.
Most phytopathogens must evolve strategies to survive in different environmental
conditions to invade and colonize their host known as virulence factors (Toth et
al., 2003).
Bacteria evade, overcome or suppress antimicrobial plant defences using these
virulence factors, which elicit release of water and nutrients from host cells to
colonize in the apoplast successfully.

5. Virulence factors: The molecules that assists the bacterium
to colonize the host at the cellular level.

6. Virulence
factors
Adhesion of bacteria to
plant surfaces
Secretion system of
bacteria
Production of plant cell
wall degrading enzymes Production of
phytohormones
Production of
extracellular
polysaccharides
Productions of bacterial
toxins

7. Adhesion of bacteria to
plant surfaces
 Adhesins are considered as biomolecules such as proteins and glycoproteins
that mediate the binding of the bacteria to the host cell (Coa et al., 2001).
 The adherence is the first step interaction between the pathogen and the plant
host which lead to the attachment and colonization of foliage or root tissues of
the host plant.
 Bacterial cell adhesion to their host cell depends on the specific binding to
carbohydrates presented at the cell surface which is mediated by adhesive
organelles of bacteria, called fimbria.

8. Secretion system of
bacteria
 Plant pathogenic bacteria have evolved numerous sophisticated strategies for
selective transport of proteins and nucleoproteins involved in the virulence across
the cell membrane.
 Six major classes of systems implicated in the virulence have been identified in
plant pathogenic bacteria from type I to type VI or T1SS to T6SS.
 In plant pathogenic Gram-negative bacteria, two major systems:
* Single step process in which the secretion proteins are exported inner and outer
membrane without periplasmic step.
* The two steps process namely Sec and the Tat secretion system are first exported
in periplasmic and then transported across the external membrane to the exterior of
bacteria cell.

9. Secretion system of bacteria

10. Type 1
secretion
system
 Type I secretion system also known as the ATP binding cassette (ABC) transporters.
 ABC are involved in the export of various molecules from the cytosol to the
external environment without periplasmic step (Delepelaire et al., 2004).
 The type I secretion system have three different proteins that composed of
continuous channel.
 ABC proteins transporters is specific outer membrane known as outer membrane
protein (OMP) and also called as membrane fusion protein (MFP) which is
connected to the inner membrane and spans the periplasmic space and extends to
the outer membrane.
 Many proteins have great importance in pathogenesis are transferred by ABC
secretion system in plant pathogenic bacteria including proteases, lipases or
performing toxins.
E.g. T1SS required bacteria are Erwinia amylovora and Erwinia chrysanthemi.

12. Type 2
secretion
system
 The T2SS secretion system known as the sec dependent system translocate folded
proteins across the inner membrane either by sec pathway or Tat pathway to the
periplasm and then to extracellular environment.
 The plant pathogenic bacteria uses such a system to export hydrolytic enzymes
involved in degrading different plant substances including xylanases, amylases and
proteases.
E.g. Erwinia carotovora pv atroseptica, Pseudomonas fluorescens, Xanthomonas
compestris pv compestris and X. oryzae pv oryzae (Peaboby et al., 2003).

13. Type 3
secretion
system
 Plant bacterial pathogens have evolved a strategy of delivering an array of effectors
and toxins proteins directly into the cytoplasm of host cell known as the type III
secretion system.
 These virulence determinants have the capacity to modulate the physiological
functions.
 The type III secretion system apparatus is composed of more than of 20 proteins
consisting of basal body spanning both inner and outer membrane of bacterial cells,
and extra needle with the tip complex extending into the host cell.
 T3SS is encoded by hypersensitive response and pathogenicity (hrp) gene involved in
the transfer of Avr proteins in the host cell (Galan and Collmer. 1999).
E.g. Xanthomonas spp., Erwinia spp., Pseudomonas syringae and Ralstonia
solanacearum.

15. Type 4
secretion
system
 The type IV secretion system is present in both the Gram-negative and positive plant
pathogenic bacteria (Wallden et al., 2010).
 This translocation system that deploy the sec gene to transport the pathogenicity
factors from the inner bacterial cell into the extracellular environment or directly into
the host cell and also involved in the translocation of either single stranded DNA, the
multi subunit toxins or the monomeric proteins including the permeases into the plant
cell (is related to a conjugation).
E. g. Agrobacterium tumefaciens that target the oncogenic DNA-protein complex in the
plant cell.

16. Type 5
secretion
system
 This type V secretion system is present in Gram-negative bacteria (Tseng et al.,
2009). It is one of the simplest secretion pathway.
 The T5SS translocation system is dedicated to transfer a single specific
polypeptide known as the passenger domain in two step process:
1. Sec translocator across the inner membrane.
2. The transportation of the passenger through the outer membrane by forming a
protective module called a ß barrel.
The virulence factors associated with T5SS passengers includes biofilm formation,
adhesions, toxins, enzymes productions and cytotoxic activity (Huang and Allen.
1997).
E. g. Xylella fastidiosa, E. chrysanthemi.

17. Type 6
secretion
system
 The structure of the T6SS secretion system presents a significant similarity
with the bacteriophage tails which injects their effector protein either directly
into the host cell or in the extracellular membrane.
 This system also includes in formation of biofilm, the quorum sensing and
antibacterial toxins (Benali et al., 2014).
E. g. A. tumificiens, Pectobacterium atrosepticum and Xanthomonas oryzae.

18. Production of plant cell
wall degrading enzymes
 Plant cell walls consist of three major polysaccharides such as cellulose,
hemicellulose and pectin, in woody and some other plants, lignin.
 The number of genes coding cell wall degrading enzymes varies include pectinases,
proteases, cellulases and xylanases. Proteases are secreted by the T1SS, whereas the
rest of the above said enzymes by the T2SS (Preston et al., 2005).
 Pectinases to be most important in pathogenesis, because they are responsible for
tissue maceration by degenerating the pectic substances in the middle lamella and
eventually, for cell death.
 Four major types of pectin degrading enzymes are secreted viz. pectate lyase, pectin
lyase, pectin methyl esterase and polygalacturonase.
 Among these pectinase enzymes, pectate lyases (Pels) are largely involved in the
virulence of soft rot Pectobacterium species

19.  The expression of pectinase genes is triggered by the fragmented products of
pectin and pectate, particularly 2-keto-3-deoxygluconate, 5-keto-4-deoxyuronate
and 2,5-diketo-3-deoxygluconate (Jha, et al., 2005).
 Cell wall degrading enzymes are believed to play a role in pathogenesis by
facilitating penetration and tissue colonization, but they are also virulence
determinants responsible for development of symptom once growth of the bacteria
has been started.
 A few Xanthomonads, e.g., X. campestris pv. campestris, the causal agent of black
rot of crucifers, have genes for two pectin esterases and polygalacturonases, four
pectate lyases, five xylanases and nine cellulases.
 Other deprived pectinolytic bacteria include A. tumefaciens, which has only four
genes encoding pectinases of any form and Xylella, which has only one gene
coding for a polygalacturonase.

20. Productions of bacterial
toxins
 Toxins play a vital role in pathogenesis and parasitism of plants by several plant
pathogenic bacteria.
 Plant pathogenic bacteria are known to produce a wide range of both specific and
nonspecific host phytotoxins. Some are polypeptides, glycoproteins others are
secondary metabolites.
 These toxins acts by using diverse mechanisms from modulating and suppressing
plant defence response to alternation and inhibition of normal host cellular metabolic
process.
 P. syringae pv. syringae, the cause of many diseases and kinds of symptoms in
herbaceous and woody plants, generates necrosis-inducing phytotoxins,
lipodepsipeptides, which are generally categorized into two groups, such as mycins
and peptins (Melotto et al., 2006).
 Chlorosis inducing phytotoxins include coronatine formed by P. syringae pv.
atropurpurea, glycinea.

21.  Coronatine biosynthesis plays an important role in virulence of toxin-
producing P. syringae strains.
 Coronatine is also believed to induce hypertrophy of storage tissue, thickening
of plant cell walls, accumulation of protease inhibitors, compression of
thylakoids, inhibition of root elongation and stimulation of ethylene
production (Alarcon-Chaidez et al., 1999).

22. Production of extracellular
polysaccharides
 Extracellular polysaccharides (EPS) may be connected to the bacterial cell as a
capsule, be produced as fluidal slime, or be present in both forms.
 EPS play a significant role in pathogenesis of many bacteria by both direct
interference with host cells and by providing resistance to oxidative stress.
 EPS1 is the chief virulence factor of the bacterial wilt disease caused by R.
solanacearum in solanaceous crops (Milling, et al., 2011).
 EPS1 is a polymer made of a trimeric repeat unit consisting of N-acetyl
galactosamine, deoxyl-galacturonic acid and trideoxy-d-glucose, where it is
produced by the bacterium in huge quantity and constitutes more than 90% of
the total polysaccharides.
 Xanthan, the major exopolysaccharide secreted by Xanthomonas spp., plays a
key role in X. campestris pv. campestris pathogenesis.

23. Production of phytohormones
 Biosynthesis of the phytohormones, auxins (e.g. indole-3-acetic acid-
IAA) and cytokinins are major virulence factors for the gall-forming plant
pathogenic bacteria, Pantoea agglomerans pv. Gypsophilae.
 Ethylene, the gaseous phytohormone formed by several microbes
including plant pathogenic bacteria, can also be considered a virulence
factor for some of them. P. savastanoi pv. Phaseolicola (Weingart et al.,
2001).

24. Bacterial virulence by avr
genes
 avr genes in bacteria are expected to encode or to direct the synthesis of
molecules that are recognized by the host plants and bring out the rapid
induction of defence responses on resistant host plants.
 In many plant-bacteria combinations, it has been demonstrated that the proteins
(Avr proteins) encoded by avr genes, encourage growth of pathogens and
development of diseases in susceptible hosts (Collmer, 1998).
 Avr proteins can interfere with the resistance mediated by the avr genes. Since
the Avr proteins are encoded by the avr genes, it is obvious that avr genes can
alter the signaling of host defence systems in resistant host plants.
 However, different avr genes, even of the same bacterium, contribute varying
degrees of susceptibility/aggressiveness to bacteria that harbour these genes.
 It reveals that the particular Avr protein functions inside the host plant cell and
enhances bacterial virulence (Luderer and Joosten, 2001).

25. Quorum sensing and biofilm production:
 It is a bacterial communication mechanism that regulates the density of microbial
population using the gene expression in response to the environment (Kanda et al.,
2011).
 This molecules are also known as autoinducers. Quorum-sensing signal N-acyl
homoserine lactones are known to regulate numerous virulence factors including
enzymes production and exopolysaccharides in many plant pathogenic bacteria.
E. g. in E. amylovora a series of regulators namely MqsR, QseBC and exporter
TqsA.
 Biofilm is a complex multilayer cellular structure attached to a tissues and
embedded with an exopolysaccharide. Several plant pathogenic bacteria have been
considered as biofilm producer as virulence factors including X. compestris and P.
syringae (Keith et al., 2003)

26. Bacterial virulence factors and the respective phytopathogens and
diseases
Phytopathogen Disease Virulence Factors
Erwinia amylovora Fire Blight Extracellular
polysaccharides (EPS) –
amylovoran and levan
6-Thioguanine (6-TG)
Desferrioxamine (DFO)
Pseudomonas syringae pvs. Bacterial-LeafSpot,
Bacterial Speck, Bacterial
Blight
Coronatine (COR)
Phaseolotoxin Tabtoxin
Syringopeptin
Syringomycin
Xanthomonas spp. Cassava Bacterial Blight,
Black Rot Disease,
Bacterial Leaf streak
Xanthan, Extracellular
polysaccharides,
Lipopolysaccharide,
Xanthoferrin,
Cyclic β-(1,2)-glucans

27. Disease symptoms caused by some bacterial pathogens of plants and representative
virulence mechanisms used by these pathogens

28. HOST –BACTERIAL PATHOGEN INTERACTION:
A pathogen interact with the host and causes infection, leading to the development
of disease in the host. How microbes sustain themselves within host organisms on
a molecular, organismal or population level.
 Plants are a rich source of nutrients and water for microbes, and they are
infected by many bacterial pathogens like Proteobacteria (such as
Agrobacterium, Erwinia, Pseudomonas, Ralstonia and Xanthomonas).
 These pathogens are spread by wind, rain, insects or cultivation practices. They
enter plant tissues either by wounds or through natural openings such as
lenticels, hydathodes or stomata, and they occupy the inter cellular spaces
(apoplast) of various plant tissues or the xylem.
 Plant-pathogenic members of the Proteobacteria cause diverse disease
symptoms, including specks, spots, blights, wilts, galls and cankers, and they
can cause host-cell death in roots, leaves, flowers, fruits, stems and tubers

29.  Plant-pathogenic bacteria use virulence strategies that are either specialized to
plant tissues or are broadly conserved among pathogens.
 Bacterial virulence is manifested as increases in the rate of growth or final
population size, as well as by enhanced disease symptoms, which promote the
spread of the pathogen through the plant.
 The bacterial cells survive and colonize the plant host which firmly adhere
(extracellular strands (fimbriae) and extracellular polysaccharides ensures the
adherence) to the xylem vessel walls (Genin and Boucher, 2004).
 The fibrous network holding the bacterial cells together could also function as
protection against the host defence.
 Virulence factors such as extracellular polysaccharides, lipopolysaccharides,
adhesins, substrates of virulence-associated secretion systems, including
T1SS and T2SS.

30. Bacterial elicitation of host basal defences
 Plants, have evolved PRRs, which function to recognize certain PAMPs. Recognition
of a PAMP activates several early, ‘frontline’ plant defences against bacterial
pathogens.
 Plant responses to pathogen attack can be differentiated into ‘basal’ and ‘resistant (R)-
gene-mediated’ defences. Basal defences occur early in the plant–pathogen interaction.
 Assays for basal defences in suspension cell or protoplast systems include detection of
increased extracellular pH (caused by a rapid efflux of K+ ), increases in Ca2+,
ethylene, reactive oxygen and nitrogen species, activation of mitogen-activated protein
kinases (MAPKs) and increased expression of certain genes (for example, FRK1 and
WRKY29).
 R-gene-mediated defences are typically detectable later in the plant–pathogen
interaction (2–3 hours) after the delivery of type III effectors into the host cytoplasm
(Mudgett, 2005).
 The most characteristic feature of R-gene-mediated defences, which is generally not
associated with basal defence, is the development of localized programmed cell death
(the hypersensitive response).

31.  Recognition capacity for certain PAMPs such as flagellin. The best characterized
phytobacterial PAMP is flagellin, a structural component of the bacterial
flagellum.
 A 22-amino-acid epitope of flagellin, flg22, was found to be recognized by the
FLS2 leucine-rich repeat (LRR) receptor kinase.
 Plants that lack FLS2 are more susceptible to Pseudomonas infection. FLS2
recognizes flg22. the FLS2 protein was shown by crosslinking and
immunoprecipitation to directly bind to the flg22 peptide (Felix, et al., 1999).
 Host glycosidases such as β-galactosidase, together with host proteases, release
immunogenic peptides from flagellin of plant pathogenic. Other bacteria also
trigger host responses, including LPS, xanthan gum, peptidoglycan, cell wall-
degrading enzymes, elongation factor Tu and quorum sensing molecules.
 FLS2 localized in roots, stems and flowers. Consistent with a role in early
pathogen detection, FLS2 was also present in leaf epidermal cells and stomatal
guard cells — typical entry points for bacterial pathogens.

32. Xanthomonas spp. stimulate PTI and ETI. Host
immunity is triggered by flagellin, potentially
through several PAMP receptors133–136. FLS2
encodes the flagellin receptor, which
recognizes the immunogenic component of
flagellin133,137.
Model depicting the activation of PRR-mediated basal defences and their
suppression by type III effectors

33.  Effector proteins confer specificity at the levels of pathogen race and host
cultivar, and these proteins might also function as determinants of host species
and tissue specificity.
 Effectors achieve functional redundancy apparently by targeting common host
components through different molecular strategies (Fu et al., 2007).
 Effectors are secreted in tiny amounts, so enzymatic turnover of substrates
amplifies this signal by allowing one effector molecule to process multiple
substrate molecules (Shao et al., 2003).
 Examples of known effector enzymatic activities include protease activity, E3
ligase activity, ADP-ribosyl transferase activity, and phosphothreonine lyase
activity.

34.  The T3SS is considered the primary secretion system responsible for virulence.
T3SS modulate host physiology to obtain nutrients, facilitate infection and/or evade
host immune responses (Shao et al., 2003).
 Example in Xanthomonas: T3SEs are integral to Xanthomonas pathogenicity,
effectors have evolved to target different components of the pathogen or damage
associated molecular pattern.
 A catalytically active protein kinase, promotes disease development by manipulating
MAPK signaling through phosphorylation. Effectors eliciting ETI have
conventionally been identified as avirulence genes.
 In Xanthomonas XopDXe, XopDXcc8004, XopJXe and XopAH (also known as
AvrXccC) interfere with hormone signaling pathways involved in plant defences or
disease susceptibility.
 Type III effectors have also been shown to modulate JA signaling to inhibit plant
defence.

35.  TAL (Transcription activator-like) effectors are one of many types of proteins
delivered into plant cells through the specialized type III secretion system of
gram negative pathogenic bacteria during infection.
 This proteins are secreted by some beta- and gamma-proteobacteria. In
Xanthomonas spp. have evolved a distinct family of T3SEs known as
transcription activation-like effectors (TALEs), which increase plasticity in
adaption of the bacteria to host plants.
 A prominent example of TALEs and their cognate susceptibility are TALEs of X.
oryzae pv. oryzae and SWEET genes of rice, which are responsible for a
pronounced phenotype in bacterial blight of rice.
 Eight major TALEs are known in X. oryzae pv. oryzae that target one of three
SWEET alleles of the clade III SWEET members; host targets that are
convergently activated by multiple TALEs are referred to as susceptibility hubs
(Makino, et al., 2006).
 In the absence of SWEET gene expression, bacteria fail to effectively colonize
rice leaves. TALE-mediated ETI involving nucleotide binding, leucine-rich repeat
(NLR) resistance genes has been identified in tomato and rice.

36.  Pathogenic bacteria utilize a number of mechanisms to cause diseases in plant
hosts. The plant pathogenic bacteria expresses virulence factors in each specific
stage of pathogenesis. The virulence of plant pathogen is a multifactorial
phenomenon which involves host-pathogen interaction.
 To better understand the complex interactions between plants and bacterial
pathogens. There is a broad spectrum of outcomes that have been defined for
plant–pathogen interactions, ranging from non-host and R-protein-mediated
resistance responses, to weakly or fully susceptible disease responses. The T3SS
are major factors influencing pathogenicity and virulence.
CONCLUSION

37. THANK YOU