APPLICATION OF CATIONIC

ANTIMICROBIAL PEPTIDES FOR

MANAGEMENT OF BACTERIAL PLANT
DISEASES
 INTRODUCTION
o Enormous losses world wide by plant diseases
o Caused by viruses, bacteria and fungi .
o Control relies mainly on chemical pesticides.
o Plant breeding strategies have been successfully used
to develop a large number of disease-resistant
varieties.
o Non-conventional strategies for the production of
disease-resistant crop plants have exploited gene
transfer technology for molecular resistance
breeding
NON-CONVENTIONAL STRATEGIES
o Expression of genes of plant defence response
pathway components.
o Expression of genes encoding plant, fungal or
bacterial hydrolytic enzymes.
o Expression of genes encoding elicitors of defence
response and small peptides
o Expression of antimicrobial peptides in plants
CATIONIC ANTIMICROBIAL PEPTIDES
Characteristics:
o Variety of sources (Prokaryotes to higher eukaryotes)
o more than 800 cationic, gene-encoded antimicrobial
peptides have been known
o Majority of peptides (96%) have a net positive charge
but some have a net negative charge.
o These endogenous peptide antibiotics constitute part
of the first line of host defence
o Cationic peptides can be induced and synthesized
much more rapidly than immunoglobulin upon
infection.
 STRUCTURAL FEATURES
o Diverse in size, sequence and structure.
o They range from 12 to 46 amino acids in length
o A net charge of at least +2 at neutral pH (Arginine or
Lysine residues in ammino acid sequence)
o Secondary structures contain hydrophobic and
hydrophilic domain.
o The basicity and amphipathicity are essential for their
antimicrobial activities.
o Hydrophilic (positively charged) surface facilitates the
interaction of the peptides with the negatively charged
bacterial surface
 Example:
Lipopolysaccharide on the outer membrane
of Gram-negative bacteria, teichoic acid on the
Gram- positive bacteria or negatively charged head
groups of the phospholipids in the lipid bilayer
NUCLEAR MAGNETIC RESONANCE
(NMR) TECHNIQUE
o Useful technique for studying structural details of
most of the known antimicrobial peptides.
o Analysis of the three-dimensional structure
o Helpful for understanding the function of
antimicrobial peptides.
 CLASSIFICATION OF
ANTIMICROBIAL PEPTIDES
Based on the NMR structures antimicrobial
peptides are broadly classified into five groups:
 Helical
 Cysteine-rich
 Sheet
 Antimicrobial peptides rich in amino acids
 Antimicrobial peptides with rare amino acids
 HELICAL ANTIMICROBIAL PEPTIDES

o Highly amphipathic helices with hydrophobic surfaces.

o Composed of 23 amino acid residues
o Well identified helical cationic peptide are,
 Cecropin-A (Moth)
 Magainins (Hyalophora cecropia)
 Xenopus laevis(Skin of the African clawed frog)
 CYSTEINE-RICH ANTIMICROBIAL
PEPTIDES
o Peptides that are rich in cysteine residues.
o Two or more disulfide bonds
o Well identified cystine rich peptide are,
 HNP-1, HNP-2 and HNP-3 (Human neutrophil
granules , were the first cysteine-rich peptides.
 Drosomycin (Drosophila)
 SHEET ANTIMICROBIAL PEPTIDES
o Composed of 20 amino acid residues.
o Contain one or two disulfide linkages
o Form a hairpin or loop structure as in,
 Horseshoe crab peptides
 Tachyplesin
 Polyphemusin
ANTIMICROBIAL PEPTIDES RICH IN
REGULAR AMINO ACIDS
oComposed of a high proportion of regular amino acids.
oStructure differs from the regular helical or sheet peptides
oExamples;
 Histatin
(Histidine rich peptides, Human saliva
and active against Candida albicans)
 Cathelicidins and Bactenecins-Bac- 5 & Bac-7
(Proline-rich peptides).
 Indolicidin & Tritripticin
(Tryptophan-rich peptides).
 PR-39
(Arginine -rich peptides).
 ANTIMICROBIAL PEPTIDES WITH
RARE MODIFIED AMINO ACIDS
o Composed of rare modified amino acids
o Peptides are those produced by lactic acid bacteria.
o Examples:
 Nisin (Lactococcus lactis)
 Leucocin A (Leuconostoc gelidum)
o Both (Nisin and Leucocin-A) composed of rare amino
acids
 MECHANISM OF PEPTIDE (ANTIBACTERIAL)
o Cationic peptides function by disrupting the cytoplasmic
membrane of bacteria
o Involve three steps:
 Binding to the cell surface
 Permeabilization of the outer membrane (in
Gram-negative bacteria) and the cytoplasmic
membrane
 Loss of cell viability as a result of cell lysis and
DNA damage.
o Cell lysis is initiated by the electrostatic interaction of
cationic peptides with the negatively charged cell surface
Contin….
For Gram-Negative bacteria:
The positively charged domain of the cationic
peptides binds to the divalent cation binding sites
of lipopolysaccharide. The displacement of the
native cations Ca2+ and Mg2+ disrupts the
structures of the outer membrane, due to the bulky
size of the cationic peptides. This disruption
subsequently results in the self-promoted uptake of
cationic peptides
Contin….
For Gram-Positive bacteria:
Cell wall contains covalently bound, negatively
charged teichuronic acid and carboxyl groups in the
peptidoglycan and these are the initial binding sites
for the cationic peptides. The interaction between
the peptides and the cytoplasmic membrane is
determined by factors such as the anionic lipid
composition of the bacterial membrane and the
presence of an electrochemical potential across the
membrane.
Contin….
o After positively charged cationic peptides bind to the
negatively charged lipid head groups under the influence
of a transmembrane potential (oriented internal
negative), the peptides insert into the membrane and
undergo conformational changes.
o Then aggregate to form multimers, which allow them to
form channels or pores with their hydrophobic faces
positioned towards the membrane and their hydrophilic
faces oriented towards the interior of these channels or
pores.
o Results in leakage of protons (causing dissipation of the
membrane potential) and of other small compounds
(causing cell death).
Contin….

o The same factors that are responsible for cell death

also seem to regulate the selectivity of cationic
peptides for bacterial membranes over eukaryotic
cell membranes.
o The composition of the eukaryotic membrane is
quite different from that of bacterial membranes
contain negatively charged lipids, such as
phosphatidylglycerol and cardiolipin whereas the
eukaryotic cell membrane is largely composed of
zwitterionic lipids, such as phosphatidylcholine and
sphingomyelin
 ANTIMICROBIAL PEPTIDES FOR BACTERIAL
RESISTANCE IN PLANTS
o Many different genetic strategies have been proposed.
o It includes;
 Inhibiting bacterial pathogenicity or virulence factors.
 Enhancing natural plant defences
 Artificially inducing programmed cell death at the site
of infection
 Producing antibacterial proteins of non-plant origin
o Genes encoding antibacterial proteins have been cloned and
expressed in plants
o Antimicrobial amphipathic peptides like cecropins and their
synthetic analogues(Shiva- 1 and SB-37) have been expressed in
transgenic potato and tobacco plants.
Contin….

o Transgenic tobacco plants expressing the Shiva-1 gene

showed delayed symptoms and reduced mortality
Ralstonia solanacearum and Pseudomonas syringae
o Expression of tachyplesin along with a signal sequence in
plants has been shown to confer resistance against
Erwinia soft rot in potato
o Attacins, isolated from the giant silk moth, introduced
into apple plants, have also shown a reduced
susceptibility to Erwinia amylovora.
Contin….
o Three different lysozyme genes (eggwhite, T4-
bacteriophage and human lysozyme) have been
expressed in plants.
 Extracellular extracts from transgenic tobacco
plants producing hen-egg-lysozyme inhibited
the growth of several species of bacteria.
 Partial resistance to Erwinia carotovora in
transgenic potato plants producing the T4-
bacteriophage lysozyme.
 A slight decrease in the symptoms caused by
P. syringae in tobacco plants producing a
human lysozyme.
Contin….
o Delayed the onset of symptoms caused by R.
solanacearum in tobacco having expression of a human
lactoferrin gene.
o Conferred resistance against Pseudomonas aeruginosa
when Esculentin from frog skin expressed in tobacco.
o Synthetic cecropin–melittin chimeric peptide and
modified temporin provided resistance against E.
carotovora in potato
o Indolicidin variants and polyphemusin variants (PV5
and PV8) showed enhanced resistance to Erwinia when
expressed in tobacco.
 DRAWBACKS OF PEPTIDE
EXPRESSION IN PLANTS
o Disease resistance depends on peptide expression levels
in plants, affected by homology-dependent gene
silencing.
o In vitro testing of the leaf extracts from the plants
expressing cationic peptides shows that the expressed
peptides are unstable or degraded by proteases
 FUTURE DIRECTIONS AND
CONCLUSIONS
• Broad-spectrum resistance using cationic peptides is the
co-expression of different molecules with
complementary modes of action that act at different
stages of disease development.
• Expression of defensive genes from a promoter that is
specifically activated in response to pathogen invasion is
highly desirable for engineering disease-resistant plants.