15 MI CROBI OLOGY AUSTRALI A MARCH 2012

Under the Microscope

Unlike animals, plants cannot flee, fight or hide from
predators. Plants lack mobile defender cells or an
adaptive immune system and have instead evolved
defences based on pre-formed barriers and inducible
cellular responses regulated by local and systemic
signals. The interaction between pathogen effectors and
these defences sets up an intriguing molecular arms race
between plants and pathogenic fungi, bacteria, viruses,
viroids and nematodes.
Plant surfaces present formidable physical and chemical barriers
as the first line of defences against pathogens. Waxy cuticles,
cellulosic or lignified cell walls and antimicrobial phytoanticipins
and defensins exclude most microbes. In a classic experiment, JC
Walker and colleagues showed that brown onions resist smudge
disease, caused by the fungus Colletotrichum circinans, because
of the antifungal phenolic protocatechuic acid in their dry outer
leaves
1
. White onions, lacking these compounds, are susceptible.
Following damage or infection, plant metabolites present in
inactive forms may be converted to more inhibitory substances,
including glucosinolates, cyanides and toxic quinones
2
.
Avenacins, triterpenoid saponins found in the outer cortex of
oat roots, confer resistance to take-all caused by non-specific
races of Gauemannomyces graminis
3
. The virulent oat-specific
pathogen G. graminis var. avenae produces the detoxifying
enzyme avenacinase, and races of the pathogen from other
cereals acquired virulence on oats when transformed with the
G. graminis var. avenae avenacinase gene
4
, while oat mutants
defective in avenacin biosynthesis became susceptible
5
.
Plant surfaces are also equipped with arrays of cell surface pattern
recognition receptors (PRRs) that detect pathogen-associated
molecular patterns (PAMPs). Recognition activates mitogen-
activated protein kinase (MAPK) cascades that both positively
and negatively regulate PAMP-triggered immunity (PTI) within
the cell
6-8
. PTI includes responses that disrupt parasitism, activate
defensive cell death programs, defence-related gene expression
and antibiotic accumulation, and reinforce plant cell walls
7,9
.
How plants defend themselves
David Guest
Professor of Plant Pathology
University of Sydney, NSW
[Link]@[Link]
Successful plant pathogens attempt to establish parasitic
nutrition by stealth biotrophy, or lethal force necrotrophy,
using effector molecules that suppress and overcome PTI. For
example, Oomycete pathogens such as Phytophthora release
RXLR effectors, similar to those used by the malarial pathogen
Plasmodium, to disrupt MAPK signalling and thus suppress
defences
10
. To counter this, plants have evolved another level
of defence by deploying variable proteins encoded by specific
resistance genes that recognise these effectors. Most are
membrane-associated NB-LRR proteins with a Leucine Rich
Repeat pattern receptor domain, and a regulatory Nucelotide
Binding domain that elicits effector-triggered immunity (ETI)
6
.
ETI is characterised by an amplified, more rapid and intense
PTI response that includes the hypersensitive response (HR), a
defensively-deployed form of programmed cell death
6,11,12
. The
HR is a central component of effective defence against biotrophs,
but its role in defence against necrotrophs is ambiguous as there is
striking evidence that these pathogens exploit the oxidative burst
and HR to promote their own necrotrophic requirements
13,14
.
A recent report suggests that autophagy restricts necrotroph-
induced cell death and lesion development by removing ROS-
induced pro-death signals
15
.
The sequence of cellular responses is similar in both PTI and
ETI. Within seconds of recognition the plant plasma membrane
depolarises and specific ion channels open, causing the rapid
uptake of Ca
2+
and H
+
into, and efflux of K
+
and Cl
-
from,
the cytosol
7
. Ca
2+
is a key second messenger in plant cells
that regulates calcium-dependent protein kinases and the
regulatory protein calmodulin, and activates membrane-bound
NADPH oxidase to release the superoxide anion (O
2
-
), triggering
an oxidative burst
16,17
. While basal resistance elicits a weak
transient oxidative burst, PTI and ETI elicit a second, sustained
and amplified burst that intensifies downstream resistance
responses
12
.
Protective antioxidant mechanisms in plant cells dismutate
superoxide to hydrogen peroxide (H
2
O
2
)
16
. H
2
O
2
is directly
antimicrobial, orchestrates the HR, and initiates cell wall
reinforcement at the point of pathogen penetration by cross-
linking cell wall structural proteins and the deposition of the
-1,3 glucan, callose
18
. The oxidative burst

damages cellular
macromolecules, releasing oxidation products that can activate
both local and systemic acquired resistance (SAR)
12,16
.
The oxidative burst also releases nitric oxide (NO), which
interacts with the other ROS to either amplify or suppress the
response
16
. The different components of the oxidative burst
cause profound changes in metabolism through disturbances
to cytosolic pH and redox homeostasis
19,20
. The ROS-scavenging
enzymes superoxide dismutase and catalase, and the ascorbate-
glutathione-NADPH cycle normally protect cells against damage
MI CROBI OLOGY AUSTRALI A MARCH 2012 16
Under the Microscope
caused by ROS, but changes in their levels or activities modulate
ROS-dependent signalling
12,19
. Down-regulation of antioxidant
enzymes, such as the salicylic acid (SA)-induced suppression of
catalase, or disturbance of the cellular redox balance following
the ROS-induced oxidation of glutathione, activates SA and
jasmonic acid (JA)/ethylene signalling pathways, the HR, and
defence gene expression
12,19
.
In another classic set of experiments, Mller and Borger
demonstrated that potato tuber slices undergoing a HR to an
incompatible race of Phytophthora infestans synthesise low
molecular weight antibiotics, collectively named phytoalexins
21
.
Phytoalexins are widespread throughout the plant kingdom and
are chemically diverse, including phenylpropanoid, terpenoid
and aliphatic molecules as well as inorganic sulphur
22
. Individual
phytoalexins are taxonomically restricted, but one plant species
may accumulate several phytoalexins.
While the biosynthesis and accumulation of phytoalexins
following pathogen challenge correlates with resistance, direct
evidence for a causal role is elusive. The cotton terpenoid
phytoalexin dehydrogossypol accumulates to toxic levels in xylem
tissues of a resistant cultivar in advance of hyphae of Fusarium
oxysporum [Link]. vasinfectum, the cause of vascular wilt
23
. In a
susceptible cultivar the phytoalexin only accumulates behind
the invading hyphae and thus fails to restrict infection. Recently,
intense accumulation of the oat phytoalexin avenanthramide A in
mesophyll cells responding hypersensitively to an incompatible
race of the rust pathogen, Puccinia coronata, was observed
at the time of attempted haustorial development, while it was
absent in a compatible interaction
24
. Given the critical role of
haustoria in effector release and parasitism, this observation
directly confirms many previous reports that phytoalexins
accumulate to toxic concentrations at the right time and place
to arrest pathogen development. While the phytoalexin-deficient
A. thaliana pad-3 mutant acquires susceptibility to Alternaria
brassicicola, its response to a range of other bacterial, oomycete
or fungal pathogens remains unchanged
25
. Thus, while
phytoalexins are decisive in some plant-pathogen interactions, in
many interactions they play a less decisive antiseptic mopping
up role, and in others they appear to be absent
22
.
Plants that deploy the hypersensitive response and survive the
initial pathogen attack develop systemic acquired resistance
(SAR) and their defences become primed against further attack.
In SAR, priming involves the systemic accumulation of salicylic
acid (SA) and the presence of the regulatory protein Non-
expressor of Pathogenesis-Related protein 1 (NPR1). Chemically-
induced SAR involves the accumulation of inactive MAPKs
26
that
become activated upon pathogen challenge,. Independent and
antagonistic JA/ethylene-mediated systemic responses follow
priming by rhizosphere microbes (ISR) and insects
27
.
A key feature of SAR is the systemic accumulation of multiple
families of pathogenesis-related (PR) proteins
28
. Many have
antimicrobial activity, including -1, 3-glucanase (PR-2) and
chitinase (PR-3, PR-8, PR-11) that catalyse the degradation of
microbial cell wall polysaccharides. PR-5 are thaumatin-like
proteins, PR-6 is a protease inhibitor, PR-9 has peroxidase
activity and is associated with the cell wall lignification response
and PR-13 are thionins. In addition, plants produce ribosome-
inhibiting proteins (RIPs) and polygalacturonase-inhibiting
proteins (PGIPs) that disrupt pathogenesis
29
.
Priming can be induced by some natural and synthetic compounds
and wounding. Functional analogues of salicylic acid such as
benzothiadiazole or 2,6 dichloroisonicotinic acid, and the non-
protein amino acid -amino butyric acid (BABA), directly elicit
defence responses and prime SA-dependent resistance against
subsequent challenges
30,31
. Even more interesting are the recent
reports that the phosphite anion, a competitive antagonist
of phosphate metabolism widely used to manage diseases
caused by Phytophthora spp. and other Oomycetes, primes
the SA signalling pathway (Massoud and Saindrenan, personnal
communication) by suppressing MAP kinase 4, a negative
regulator of SA-dependent defences in A. thaliana. With these
findings defence priming has emerged as a promising means for
sustainable disease management in the field.
Plants and pathogenic microbes are engaged in a complex
evolutionary arms race. The similarity of innate immunity across
eukaryote Kingdoms, involving receptor complexes and signalling
networks using Ca
2+
, ROS and MAPK cascades, suggests an early
evolutionary origin. To survive and flourish in potentially septic
and stressful environments plants have acquired sophisticated and
complex defence mechanisms regulated with an exquisite level
of control. Modern plant breeding has sometimes sacrificed plant
defences for yield, with disastrous and unforseen consequences.
Understanding how plants orchestrate their defences is vital to
sustaining food, fibre and biofuel production and to managing
our environment.
Acknowledgement
I thank Dr Patrick Saindrenan, Universit Paris-Sud-CNRS, France,
for his constructive comments on the manuscript.
References
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17 MI CROBI OLOGY AUSTRALI A MARCH 2012
Under the Microscope
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Biography
David Guest is the Professor of Plant Pathology and Associate
Dean (Development) in The Faculty of Agriculture, Food and
Natural Resources at The University of Sydney. His current
research interests include understanding plant disease resistance
mechanisms, and using this knowledge to manage Phytophthora
diseases in tropical horticulture and in Australian ecosystems. He
teaches undergraduate courses at all levels and has supervised
over 20 PhD and Research Masters students. His extensive
fieldwork activities involve partnerships with research institutes
and farming communities around the Asia-Pacific region. He is
President of the Asian Association of Societies of Plant Pathology
and a Past-President of the Australasian Plant Pathology Society.
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