Unit3

Signal recognition particle (SRP): a ribonucleoprotein (protein-RNA)

complex to which signal peptides of integral membrane proteins bind and are
targeted to the endoplasmic reticulum in eukaryotes or the plasma membrane in
prokaryotes

Effector protein: toxins introduced directly into host cells by specialized

secretion systems; also called Exoenzymes.

 effector protein alone was sufficient to cause cytotoxicity as long as the

secretion apparatus was functional.
 Yersinia pathogenesis: direct translocation of the Yop effector protein
YopE from the bacterial cell into the eukaryotic cell.


Translocon: it is a complex of proteins associated with translocation of

protein/polypeptides thereby forming conducting channel through the cell
membrane.

It has transmembrane subunit, which is hydrophobic in nature.

It is a part of the general secretory pathway;
Ex SecYEG

Multidrug efflux system: integral membrane protein transporters that use energy
to actively pump or excrete antibiotics and other small molecules out of the
cell.

They have been reported to transport a variety of structurally unrelated

substrates from a cell.
Ex. Mycobacterium tuberculosis: initially developed to protect against
environmental toxins but evolution repurposed it for intracellular growth.

Leader sequence: a sequence of 15 to 26 amino acids at the N termini of

proteins that target them for secretion via the general secretory system
What are chaperones? Chaperones in secretion:
A protein that aids in the folding of proteins after translation or of RNA
molecules after transcription; a protein that binds and stabilizes another pro-
tein or brings another protein to a particular site in the cell; a protein that
potentiates pairing between small RNAs and mRNAs

Feature:

 These are auxillary cytoplasmic proteins

 They bind to nascent peptides exiting ribosomal translational apparatus
and
a. Protect them from premature aggregation
b. Protect them from degradation
c. Direct them towards correct secretion pathway

Role of chaperones in secretion:

Many bacterial proteins must be first exported through the cytoplasmic

membrane to take up residence in the bacterial envelope. Mostly such protein
precursors remain unfolded and not secreted until they are associated with
molecular chaperones.

• The secreted proteins are synthesized as precursors with a 15- to 26-

amino-acid signal sequence (also called a leader sequence) at the N
terminus, which contains 10 to 12 hydrophobic residues preceded by 1
or 2 charged residues and followed by a protease cleavage site.
• The signal sequence is cleaved by a leader peptidase during the process
of transport from the cytoplasm across the membrane.

• The hydrophobic residues of the leader sequence trap the cleaved

peptide in the membrane.

• Mutations in the protease cleavage site do not prevent transport across

the membrane but result in the protein being tethered to the membrane
instead of being released into the medium or periplasm.

• The signal sequence is required for the proteins to be recognized and

transported by the Sec system

• The hallmark of the Sec system is that it exports unfolded proteins

posttranslationally.

• Two cytoplasmic proteins are responsible for binding and directing the
protein to be secreted.

 SecB is a protein chaperone (less than 20 KDa) that binds to proteins

containing signal peptides as they exit the ribosome during translation.
The SecB tetramer contains a chamber that is thought to bind segments
of the proteins and keep them unfolded until they can be delivered to
SecA.

The function of SecB chaperone is to form highly specific

transient interaction with the substrate to promote T3SS and
translocation.

 The SecA dimer is a molecular motor whose ATPase activity provides

part of the energy for the translocation process (the rest of the energy
seems to be provided by the proton motive force across the membrane).

 SecA binds to the heterotrimeric translocase complex composed of

SecYEG (sec Translocon), which forms a protein-conducting channel
through the cellular membrane.

 SecB delivers the unfolded protein to the ADP-bound SecA-SecYEG

complex.

 Additional proteins (SecD, SecF, and YajC) help to stabilize the entire
complex.

 The amino terminus of the protein containing the signal peptide is then
threaded through a pore in SecA and remains bound to SecA at its
interface with SecYEG.

 The signal peptide remains tethered in the complex and is cleaved by the
activity of a leader peptidase, after which the peptide is released and
 refolds.

 In gram-positive bacteria, the mature protein folds as it emerges

through SecYEG and is released to the surfaces of the bacteria or
directly into the medium.
 In gram-negative bacteria, the transported protein folds in the
periplasmic space, where it may remain or be transported across
the outer membrane by one of the transport mechanisms

As integral membrane proteins exit from the ribosome, they are sorted so that
their signal peptides bind to the signal recognition particle (SRP) complex,
which consists of 4.5S RNA and a protein called Ffh. The SRP in this complex
then docks to the membrane-bound SRP receptor protein, called FtsY, which
hydrolyzes GTP and delivers the signal peptide to the SecYEG complex.

In pathogenic gram-negative bacteria, the host invasion is done by

translocation of effector proteins directly into the target cells. They are needle
like structure is used as the sensory probe to detect the presence of eukaryotic
organisms and help the bacteria to infect their host.

a complex process that requires the participation of a number of auxiliary

proteins.

The exported proteins have specific bacterial cytoplasmic chaperones that bind
them and guide them to the injectosome for secretion and translocation.
Epiphytic fitness and mechanism in plant pathogenesis

Fitness is the ability of a pathogen to survive and re- produce. The fitness of a
pathogen or parasite can be quantified by measuring its reproductive rate, rate
of multiplication, efficiency of infection, and amount of disease caused
(aggressiveness). Fitness seems to be the driving force in the stability and
evolution of a pathosystem in agriculture. In a freely mating system, excess
virulence genes in a pathogen population constitute a genetic load or drag so
that future selection favors genotypes free of excess genes. Even the presence
of excess genes for virulence imposes a fitness penalty to the pathogen.
Therefore, a mutation from avirulence to virulence occurs only if it is needed to
overcome an R gene for resistance, i.e., only if it is absolutely necessary for the
pathogen to survive. So, for a specific interaction between a pathogen with an
avirulence gene and a host with a matching R gene for resistance, a mutation to
virulence will occur because it increases the fitness of the pathogen to survive
while the R gene is present. If, however, the mutation from avirulence to
virulence gene carries a fitness penalty, the pathogen will suffer from reduced
fitness on the host in the absence of the R gene.

Epiphytic bacteria are capable of living (i.e., multiplying) on plant surfaces [1] and
their colonization is controlled by biological factors such as the host plant growth and
the life cycle of epiphytes [2,3]. Epiphytes occupy a narrow ecological niche because
of their existence at the interface of vegetation and atmosphere, a variation in climatic
conditions including moisture, humidity, temperature, wind speed, radiation and
rainfall may influence epiphyte diversity.

Epiphytic bacterial aggregates on plant surfaces may vary in size and composition
depending on nutrient availability at a given site. Epiphytic bacteria physically
interact with plants through surface colonization, in which they adhere to external
plant surfaces as individual cells, in clusters or biofilm.

Gene for gene hypothesis

Mechanism of hypersensitive response to plant pathogens: role of avr and r
gene
TYPES OF PLANT RESISTANCE TO PATHOGENS

Plants are resistant to certain pathogens because they belong to taxonomic groups that
are outside the host range of these pathogens (nonhost resistance), because they
possess genes for resistance (R genes) directed against the avirulence genes of the
pathogen (true, race- specific, cultivar-specific, or gene-for-gene resistance), or
because, for various reasons, the plants escape or tolerate infection by these pathogens
(apparent resistance).

The variation in susceptibility to a pathogen among plant varieties is due to different

kinds and, perhaps, different numbers of genes for resistance that may be present in
each variety.
Types of resistance:
1. True Resistance - Disease resistance that is controlled genetically by the
presence of one, a few, or many genes for resistance in the plant is known as
true resistance. The host and the pathogen are more or less incompati- ble with
one another, either because of lack of chemical recognition between the host
and the pathogen or because the host plant can defend itself against the
pathogen.

There are two kinds of true resistance:

a. Partial, Quantitative, Polygenic, or Horizontal Resistance

All plants have a certain, but not always the same, level of possibly unspecific
resistance that is effective against each of their pathogens. Such resistance is
sometimes called partial, race nonspecific, general, quantitative, polygenic,
adult-plant, field, or durable resistance, but in the past it was referred to most
commonly as horizontal resistance.

Partial resistance is probably controlled by several genes, thereby the name

polygenic or multigene resistance.

b. R Gene Resistance, Race-Specific, Monogenic, or Vertical Resistance

Depending on the race of the pathogen used to infect a variety, the variety may
appear strongly resistant to one pathogen race and susceptible to another race
(race specific) under a variety of environmental conditions. Such resistance
differentiates clearly between races of a pathogen, as it is effective against
specific races of the pathogen and ineffective against others . Race-specific
resistance is always controlled by one or a few genes (thereby the names
monogenic or oligogenic resistance). These genes, referred to as R genes,
control a major step in the recognition of the pathogen by the host plant and
therefore play a major role in the expression of resistance. Race-specific
resistance inhibits the development of epidemics by limiting the initial inocu-
lum or by limiting reproduction after infection.

Complete resistance may be provided by a single resistance gene. Often, it is

desirable to combine, or pyramid, more than one resistance gene (R1R2,
R1R3, R1R2R3) in the same plant, which then is resistant to all the pathogen
races to which each of the genes pro- vides resistance.

GENETICS OF VIRULENCE IN PATHOGENS AND OF

RESISTANCE IN HOST PLANTS
Infectious plant diseases are the result of the interaction of at least two
organisms, the host plant and the pathogen, which is governed by their genetic
material, the DNA,
When the same race of a pathogen is inoculated on two appropriately chosen
varieties of a host plant, one variety is susceptible while the other is resistant to
the same pathogen (table)

This clearly indicates that, in the first case, one race possesses a genetic
characteristic that enables it to attack the plant, while the other race does not,
and, in the second case, that the one variety possesses a genetic characteristic
that enables it to defend itself against the pathogen so that it remains resistant,
while the other variety does not. When several varieties are inoculated
separately with one of several races of the pathogen, it is again noted that one
pathogen race can infect a certain group of varieties, another race can infect
another group of varieties, including some that can and some that cannot be
infected by the previous race, and so on

 Studies of the inheritance of resistance versus susceptibility in

plants prove that single genes control resistance and their absence
allows susceptibility.
 Studies of the inheritance of avirulence versus virulence in
pathogens prove that single genes control avirulence and their
absence allows virulence.
 Studies of their interactions prove that R genes in the plant are
specific for avr genes in the pathogen.

The Gene-for-Gene Concept (H.H.Flor)

The coexistence of host plants and their pathogens side by side in nature
indicates that the two have been evolving together. Changes in the virulence of
the pathogens appear to be continually balanced by changes in the resistance of
the host, and vice versa. A dynamic equilibrium of resistance and virulence is
maintained, and both host and pathogen survive over considerable periods of
time. The stepwise evolution of virulence and resistance can be explained by
the gene- for-gene concept, according to which for each gene that confers
virulence to the pathogen there is a corresponding gene in the host that confers
resistance to the host, and vice versa.
The gene-for-gene concept was first proved in the case of flax and flax rust.

 In the host the genes for

 resistance are dominant (R),
 whereas genes for susceptibility, i.e., lack of resistance, are
recessive (r).
 In the pathogen, however, genes for
 avirulence, i.e., inability to infect, are usually dominant (A)
 whereas genes for virulence are recessive (a)

When two plant varieties, one carrying the gene for resistance R to a certain
pathogen and the other lacking the gene R for resistance, i.e., carrying the gene
for susceptibility (r) to the same pathogen, are inoculated with two races of the
pathogen, one of which carries the gene for avirulence (A) against the
resistance gene R and the other of which carries the gene for virulence (a)
against the resistance gene R, the gene combinations and reaction types shown
in Table 4-3 and Fig. 4-11 are possible.

 Of the four possible gene combinations, only the AR interaction is

incompatible (resistant), i.e., the host has a certain gene for
resistance (R) that recognizes the corresponding specific gene for
avirulence (A) of the pathogen.
 In the Ar combination, infection results because the host lacks
genes for resistance (r) and so the pathogen can attack it with its
 other genes for virulence (after all, it is a pathogen on this host).
 In aR, infection results because although the host has a gene for
resistance, the pathogen lacks the gene for avirulence that is
recognized specifically by this particular gene for resistance and
therefore no defense mechanisms (resistance) are activated.
 In the ar interaction, infection results because the plant has no
resistance (r) and the pathogen, being a pathogen and therefore
virulent (a), attacks it.

The pathogen gene is then thought of as the “avirulence” gene (avrA) of the
pathogen that corresponds to the plant resistance gene R.

Recognition of avrA gene product (the elicitor molecule) by the receptor coded
by the R gene triggers the hypersensitive response reaction in the plant that
keeps the plant resistant.

A new gene for virulence that attacks the existing gene for resistance appears
by mutation of an existing avirulence gene, which then avoids gene-for-gene
recognition, and the resistance of the host breaks down. Plant breeders then
introduce another gene for resistance (R) in the plant, which recognizes the
protein of the new gene for virulence of the pathogen and extends the
resistance of the host beyond the range of the new gene for virulence in the
pathogen.

Plant breeders apply the gene-for-gene concept every time they incor-
porate a new resistance gene into a desirable variety that becomes
susceptible to a new strain of the pathogen. With the diseases of some
crops, new resistance genes must be found and introduced into old
varieties at relatively frequent intervals, whereas in others a single gene
confers resistance to the varieties for many years.
the hypersensitive response is a localized self-induced cell death at the site of
infection of a host plant by a race or strain of a pathogen that cannot develop
extensively in this particular resistant plant cultivar. Thus, the plant species as a
whole may be a host to the pathogen species, but individual cultivars (varieties)
of the plant may be hosts (susceptible) or nonhosts (resistant) to a particular
race or strain of the pathogen.

Mechanism of hypersensitive response to plant pathogens:

role of avr and r gene
The hypersensitive response is a localized self-induced cell death at the site of
infection of a host plant by a race or strain of a pathogen that cannot develop
extensively in this particular resistant plant cultivar. Thus, the plant species as a
whole may be a host to the pathogen species, but individual cultivars (varieties)
of the plant may be hosts (susceptible) or non-hosts (resistant) to a particular
race or strain of the pathogen.

 Resistance through the hypersensitive response has been shown to be the

result of gene-for-gene systems in which an avirulence (avr) gene in the
pathogen corresponds to a resistance (R) gene in the host plant.
 Whatever the type of pathogen, it is believed that resistance through the
hypersensitive response is the result of recognition by the plant of
specific signal molecules, the elicitors, produced by the avirulence
genes of the pathogen and recognized by R gene-coded specific receptor
molecules in the plant.
 Such recognition causes the activation of a cascade of host genes, which
result in a burst of oxidative reactions, disruption of cell membranes,
and release of phenolic and other toxic compounds, which then lead to
the hyper- sensitive response, programmed cell death, inhibition of
pathogen growth, and thereby resistance (Fig 4.13)
 It also leads to the activation of numerous other defense- related genes
that result in other types of resistance, including horizontal resistance
and systemic acquired resistance.

Pathogen-Derived Elicitors of Defense Responses in Plants

Avirulence (avr) Genes: One of the Elicitors of Plant Defense Responses

Avirulence (avr) genes, first identified by H. H. Flor in the 1950s, were only
rather recently isolated from bacteria (1984) and fungi (1991), but since then
numerous bacterial and fungal avr genes have been identified.

Features:

 The avr genes make a pathogen avirulent, that is unable to induce

disease on a specific variety of the host plant because their protein
product warns the plant of the presence and impending attack by the
pathogen and the host plant then mobilizes its defenses and blocks infec-
tion by the pathogen.
 In this way, avr genes, by warning the host and thereby inhibiting
infection by the pathogen, determine the host range of the pathogen at
the species and at the race-variety level.
 As the gene-for-gene concept implies, in the majority of cases a
matching dominant resistance gene (R) in the resistant host corresponds
to each avirulence gene in the pathogen.
 Some avr genes, when transferred artificially to other pathovars, are
active in the new pathovars, making the recipient pathogen unable to
infect their previously susceptible hosts and, instead, causing the
hypersensitive response in these plants. In some host–pathogen systems,
avr genes determine not only which cultivars of a species the pathogen
 can attack, but also which plant species it can attack. For example, an
avr gene (avrBsT) in the tomato-infecting group of strains of the
bacterium Xanthomonas campestris pv. vesicatoria, the pathogen of
bacterial spot in tomato and pepper, enables the bacterium to induce the
hypersensitive response on all cultivars of pepper. Loss of avrBsT from
such tomato-infecting strains allows these strains to cause disease on
normally resist- ant pepper cultivars.
Characteristics of avr Gene-Coded Proteins

 The biochemical basis for explaining the gene-for-gene concept

is the elicitor– receptor model according to which an avirulence
(Avr) gene of a pathogen encodes an elicitor (Avr) protein that is
recognized by a receptor protein encoded by the matching
resistance (R) gene of the host plant.
 The simplest way of recognition would be if the pathogen-
produced elicitor interacted with the protein encoded by the
matching resistance gene of the host.
 Recognition of the elicitor protein by the host plant leads to
activation of a cascade of defense responses, which often include
cell death around the infection site. The death of cells around the
point of infection is known as the hypersensitive response and is
characteristic of gene-for-gene-based resistance.
 To date, the AvrD protein, produced by the AvrD gene of the
bacterial spot of tomato pathogen P. syringae pv. tomato, is the
only Avr protein for which a biochemical function has been
clearly defined. This function is the ability of the AvrD protein to
direct the synthesis of low molecular weight syringolide elicitors,
which elicit the hypersensitive response on soybean. A
syringolide-binding protein has been identified in resistant
soybean plants, possibly representing the protein of the matching
R gene of the host plant.
 Avr proteins seem to be generally hydrophilic and, therefore,
water soluble, lacking stretches of hydrophobic amino acids that
would enable them to be anchored in cell membranes. Avr
proteins also lack stretches of amino acids known as “signal
sequences” that would allow the proteins to be secreted into the
external medium by the general secretory pathway.

Resistance (R) Genes of Plants

 Even when a plant becomes attacked and diseased by a pathogen,

however, a number of defense response (resistance) genes are activated.
 As a result, in most cases, the plant manages to limit the spread of the
pathogen into a smaller or larger spot, lesion, canker, and so on through
defense compounds and structures that block the further expansion of
 the pathogen.

In many cases, the hypersensitive response is followed by the development of

various levels of systemic acquired resistance (SAR), which is expressed in the
vicinity of attack as well as in distant parts of the plant.
Multi- drug efflux pumps: pumps that excrete antibiotics of more than one
type. Recent structural work has been aimed at addressing how such pumps are
nonspecific for more than one antibiotic class, whereas they do not export
small molecules essential to the bacterium.

 mutations in gram-negative porins or some types of antibiotic efflux,

confer resistance to more than one type of antibiotic.
 Efflux pumps are membrane proteins that use energy to pump small
molecules out of the bacterial cytoplasm. If this small molecule is an
antibiotic, resistance results, because the antibiotic is prevented from
reaching a high enough concentration in the cytoplasm to be effective.
Bacteria contain multiple efflux pumps, many of which can contribute to
reducing cytoplasmic concentrations of antibiotics. For example, the
genomes of E. coli and P. aeruginosa each encode over 30 different
efflux pumps. These pumps normally play roles in maintaining
homeostasis by pumping metabolites and toxic substances out of the
bacterial cell. Some efflux pumps are highly specific for a metal or
compound (e.g., the TetA tetracycline efflux pump), whereas oth- ers
pump out many compounds (e.g., tetracyclines and macrolides),
resulting in resistance to multiple an- tibiotics. Some efflux pumps move
horizontally be- tween different bacterial pathogens on mobile ele-
ments or are part of pathogenicity islands.
 Efflux pumps fall into two structural classes:
 The majority are antiporters that use the uptake of protons (H ) as the
source of energy to pump the antibiotics and other small molecules from
the cytoplasm.
 Some efflux pumps are ABC transporters, which are multisubunit
complexes that use ATP hydrolysis to power the pump.
 The structures of efflux pumps of gram- positive bacteria are usually
relatively simple, because these pumps only need to remove the
antibiotic to the outside of the cell.
 In contrast, efflux pumps of gram- negative bacteria often consist of
proteins in the inner membrane, periplasm, and outer membrane that
channel the antibiotic outside of the cell.

The first efflux mechanism to be characterized extensively mediates

resistance to tetracyclines. The resistance protein is an antiporter pump
(called TetA, TetB, etc., in different bacteria) located in the cyto-
plasmic membrane. These pumps catalyze energy- dependent transport
of tetracycline out of the bacterium. Since tetracycline is removed as
rapidly as it is taken up, the intracellular concentration of tetracy- cline
remains too low to inhibit protein synthesis. Efflux pumps for every
class of antibiotic have now been discovered, and they cause serious
clinical problems by imparting resistance to -lactams, macrolides,
fluoroquinolones, and streptogramin, as well as tetracycline antibiotics,
especially in Staphylococcus species. They consequently represent an
extremely serious form of antibiotic resistance that has been hard to
overcome.
The best example of this alarming trend is the escalating spread of
MRSA strains, which can be acquired in the community as well as in
hospitals. MRSA strains, which were initially identified for their
resistance to the -lactam methicillin, are now often resistant to multiple
antibiotics besides -lactams, including macrolides, tetracycline,
aminoglycosides, and antiseptics.
