Plant Defenses

 Pre-formed (Constitutive) Defenses

 Inducible Defenses
 Constitutive defenses can be either
structural, chemical or both ……

Physical Barriers

Cuticle
- cuticular wax
- cutin polymer

Cell wall
- polysaccharide matrix
- phenylpropanoid esters/amides
- cell wall proteins
Lacey Samuels - UBC
Lacey Samuels - UBC
 cuticle

Lacey Samuels - UBC

Waxes - long chain alkanes and alcohols

Cutin / Suberin - 3-d polyester polymers

CO
O O
C
O
Cell wall polysaccharides

microfibrillar cellulose

neutral hemicelluloses -
xyloglucans
mixed beta 1-3, 1-4 glucans

acidic polysaccharides -
partially methyl-esterified homogalacturonans
and rhamnogalacturonans
Cell wall proteins

Hydroxyproline-rich glycoproteins (HRGP);

extensins – major structural proteins of the plant cell
wall

Oxidizing enzymes – peroxidases and laccases can oxidize

and cross-link phenolic molecules
- oxalate oxidase and amine oxidases
can generate reactive oxygen species (ROS) such as H2O2
 Cell wall proteins

hydroxyproline-rich glycoproteins (HRGP)

high content of hydroxyproline, lysine and tyrosine

-OH -NH3+

sidechains
OH

short oligosaccharide sidechains attached to -OH groups

Oxidizing enzymes peroxidases
laccases

 2x

OH

OH H- OH

OH
Pre-formed Chemical Defenses
“Phytoanticipins”

- antimicrobial peptides
- phenolic esters
- lignin
- flavonoids ‘Secondary’
- isoprenoid lipids metabolites
- glucosinolates
- cyanogenic glycosides
Inducible Chemical Defenses
“Phytoalexins”

- antimicrobial peptides
- phenolic esters
- lignin
- flavonoids ‘Secondary’
- isoprenoid lipids metabolites
- glucosinolates
- cyanogenic glycosides
Phytoalexins were originally defined formally as

low molecular weight

anti-microbial (at physiological concentrations and
against the relevant organism)
not pre-existing

plant secondary metabolites whose synthesis/

accumulation is induced by pathogen challenge

Problems:
• ‘not pre-existing’ is a technology-dependent parameter
• often not very effective as stand-alone anti-microbials
• often induced by other biotic and abiotic stresses
Antimicrobial peptides

Short cationic peptides that are able to form

pores in microbial membranes. Highly variable
structures and activities.

Some are constitutively expressed (especially in seeds)

while others are induced by stress/pathogens

• Thaumatin-like proteins
• Lipid transfer proteins
• Anti-freeze proteins
• Defensins
Secondary metabolites

Hydroxycinnamic acid
Chlorogenic acid
Oxidizing enzymes: polyphenol oxidase

2H- HS - Enz
H2N - Enz
OH O

OH O
ortho-quinone
S - Enz

OH
covalent
conjugation OH

product - denatured
Preformed anti-microbial defense chemicals can
sometimes be a two-edged sword……

Example :
The fungus Septoria lycopersici causes ‘tomato leaf
spot disease’ on susceptible host genotypes.

Tomato leaves contain alpha-tomatine, a steroidal

glycoalkaloid with potent anti-fungal activity.

S. lycopersici produces tomatinase, an enzyme that

cleaves a sugar unit off the alpha-tomatine, converting
it to the less toxic beta2-tomatine.
Secondary metabolites
 S. lycopersici strains lacking tomatinase appear to be
unaffected in their ability to colonize tomato leaves.

However, the initial stages of infection are associated

with a stronger resistance response from the host.

It could be shown that beta2-tomatine actively

suppresses the plant’s defence response mechanisms,
thus contributing to the ultimate success of the fungus.

Bouarab et al Nature 418: 889 (2002)

Inducible Defense Mechanisms
 Ion fluxes
 Production of reactive oxygen species (ROS)
 Activation of signal transduction kinases
and phosphatases
 Cytoskeletal rearrangement
 Callose deposition
 Cell wall strengthening
 Transcription of defense genes
 Accumulation of defense metabolites
and new signals (SA and SAR)
 Programmed cell death / HR
Pathogen (or elicitor) challenge usually induces a
rapid (within sec/min) flux of ions across the
plasmalemma

proton uptake (medium alkalinization)

anion loss (Cl-)
Ca++ uptake

This is presumed to reflect receptor-mediated

activation of membrane ion channels, perhaps by
phosphorylation or de-phosphorylation
Another very early event following challenge is the
apoplastic and cytosolic accumulation of reactive
oxygen species (ROS) -
hydrogen peroxide (H2O2)
superoxide anion (O2)
hydroxyl radical (OH )
These may be generated by multiple mechanisms in the
apoplast:
NADP(H) oxidase
amine oxidase
oxalate oxidase
peroxidase

This phenomenon is often referred to as the

“oxidative burst”
 Different ROS can be rapidly interconverted by
enzymatic and non-enzymatic processes

superoxide
dismutase (SOD)
O 2 H 2O2

catalase Fe++/Cu+

O2
OH 

Since ROS are able to oxidize and disturb the function

of other biomolecules (protein, DNA, membrane lipids),
plant cells maintain active ROS suppression mechanisms
These include:

enzymes such as SOD, catalase and ascorbate peroxidase

chemical reductants such as ascorbate and glutathione

free radical scavenging metabolites such as tocopherol,

flavonoids and thiols.

The ultimate source of reductant is either photosynthesis

or respiration or both
(NAD(P)H; reduced ferredoxin; thioredoxin)

but, note that these same processes act as ROS generators

ROS production in a plant cell (in vitro culture) at
the site of attempted penetration by an avirulent
pathogen.
Histochemical staining for superoxide anion / hydrogen peroxide.
Histochemical
detection of ROS
accumulation in the
apoplast and
intercellular space
surrounding an
invading bacterial cell

Plant Cell 8:1774

1996
It is thought the the longer-lived ROS (e.g. hydrogen
peroxide) might also serve as a diffusible second
messenger within the cell, or even between
neighbouring cells

The pathway activated by ROS acting as messengers is

not fully defined yet, but MAPK modules seem likely to
be involved.
 Pathogen challenge
Signal

Sensor ROS

?
?
? ? G-, , 
MAPKKKK MAPKKKK?
?

MAPKKK ANP1

MAPKK AtMKK4?

MAPK AtMPK3/6

GST6, HSP18.2, HMGR, PAL (+)

Target genes
GH3, ER7 (-)
Modified from Tena et al., Curr Opin Plant Bio, 2001
 Pathogen challenge
Signal

suramin

Sensor ROS

?
?
G-alpha
MAPKKKK MAPKKKK? G-beta
?

MAPKKK ANP1

PD98059 (a MAPKK inhibitor)

MAPKK MAPKK?

MAPK AtMPK3/6
AtMPK3/6

GST6, HSP18.2, HMGR, PAL (+)

Target genes
GH3, ER7 (-)
Modified from Tena et al., Curr Opin Plant Bio, 2001
 The proposed dual role of ROS implies that the cell must
maintain a careful balance between ROS suppression and
ROS generation

What happens to disease resistance if we suppress

the ability of the cell to control intracellular hydrogen
peroxide levels?

Test: Create transgenic tobacco plants carrying an

antisense version of cytosolic ascorbate peroxidase (cAPX)
 Antisense suppression of gene expression

target antisense copy

transcripts

no translation
translation
Lesion formation
develops at lower
levels of bacterial Increased cell death
inoculum (measured as ion leakage)
at lower inoculum levels
 Fungal spore App

One of the early

host responses
is re-organization
of the challenged
cell’s cytoskeleton,
which becomes
focused on the
point of attack
Callose is an amorphous beta-1,3 glucan polymer
that is rapidly formed in plant cells challenged by
pathogens

Aniline blue
staining and
UV fluorescence

Plant Cell 8 (1996)

Callose synthase is activated by increased calcium ion levels
Fungal spore App
Papilla formation
beneath the site
of attempted
penetration by a
fungal pathogen

Plant Cell 8:1774

1996
 lignin polymer

Bidlack et al Proc.Okla.Acad.Sci. 72:51-56 (1992)

Lignin deposition
around perimeter
of challenged cells

infected cell

Plant Cell 8:1774

1996
Longer-term changes
associated with resistance
responses (6-48 hr)
are typically the
result of changes in
gene expression
Phenylpropanoid metabolism

flavonoid synthesis
phenylalanine ammonia-lyase (PAL)
chalcone synthase (CHS)
isoflavonoid synthesis
(CHS)
isoflavone synthase
stilbene synthesis
PAL
stilbene synthase
hydroxycinnamic acid ester synthesis
PAL
4-coumarate:CoA ligase (4CL)
lignin synthesis
PAL
cinnamyl alcohol dehydrogenase (CAD)
Secondary metabolites

flavonoid
Secondary metabolites

isoflavonoid
Secondary metabolites

stilbene
TMV infection
of susceptible
(left) and resistant
(right) genotypes
of tobacco
TMV-challenged
tobacco leaves
(resistant
genotype)
viewed under
UV light
Biosynthesis
of scopolin
through the
phenylpropanoid
pathway

coumarins

aglycone glucoside
Lower scopolin accumulation correlates with
larger lesions and heavier TMV infection in
AS plants
Isoprenoid lipid metabolism

Sequiterpene phytoalexin synthesis

Triterpenoid synthesis
HydroxymethylglutarylCoA reductase (HMGR)
Prenyl transferases
Specific cyclases
Secondary metabolites

triterpene
Secondary metabolites

sesquiterpene
Global profiling of gene
expression is now
possible.
However, it is still
not possible to link all
the observed changes
to known biological
processes

Pto-regulated
transcriptome
in tomato
PR proteins
Pathogenesis-related Proteins

Novel low molecular weight proteins whose accumulation

is induced in plant cells by pathogen challenge.

Both acidic (PRa) and basic (PRb) classes found.

PR1a accumulation (usually measured as gene transcription)

is a classical marker for a resistance response.

Biochemical functions have been proposed for some PR

proteins (e.g. glucanase, chitinase, tyrosine decarboxylase)
but many remain unidentified
When a strong/fast resistance reaction is induced,
this is usually associated with a host cell death pattern
whose consequence is the isolation of the invading
pathogen from healthy host tissue.
The resulting lesion can be as small as a single cell, but
usually encompasses more (10-1000 cells), depending
on the specific pathosystem.

The current view is that this pattern of cell death is a

genetically controlled event, since mutants have been
found that appear to control the speed, character and
extent of the cell death phenomenon.

It is therefore referred to as programmed cell death

(PCD), or apoptosis.
 Fungal spore

Appressorium

Hypersensitive response in a potato leaf epidermal cell

attacked by an avirulent fungal pathogen