Why study the interaction between plants and Microbes?

1.Estimated annual crop losses worldwide by disease only

approximately $220 billion.

Application of
Fungicides ,pesticides, costly and
non ecofriendly

New strategies

Plants own defense mechanism

2. Mutually beneficial relationship

Affect the agriculture productivity and health of the plants

(Yang et al.,2011)
 Introduction
 History

 Signal molecules

 Transduction of signals molecules

 Host response to signal

 Genetic approaches

 Conclusion

 References
Significances
This provide practical solutions for the control of plant-disease

Goal
Understand the basic molecular mechanisms of plant-microbe
interaction and plant pathogen resistance
History of signal transduction
development.
1878- USA-Burril-fire blight disease of apple & pear-Erwinia amylovora

1898 –Beijerinck-tobacco mosaic –contagium vivum fluidum

1971-Diner-potato spindle tuber-RNA

1933-Chesster-Hyper sensitivity reaction in plants

Later researchers proved that plants evolved response against microbes.

(Montesions, 2000)
 Two types of defense

1. Preformed (passive) defense

2. Induced (active) defense

 Passive defense

Recognize and resist invading pathogen by rapid defense response ,by

programmed cell death result in electrolyte leakage and oxidative
burst.
(Baker.1997,Yang,2011)

 Active defense
Signal transduction
(Baker 1997,Montesion 2000,Ritsema 2010,yang
2011)
 Elicitors-that evoke plant defence response

 Inducers-that initiates compactablle host parasite

interaction (-CHO,Fatty acids,phenolics)

 Supressor-that delay defense response(-

CHO,Glycopeptides)
(Yang et al., 2011,Castor et
al.,2009,Huang 2001)
 ELICITORS
Elicitors are proteins made by the pathogen
avirulence genes, or the products of those
proteins
Elicitors of Viruses
Coat proteins, replicases, transport proteins

Elicitors of Bacteria
40 cloned, 18-100 kDa in size

Elicitors of Fungi
Several now cloned- diverse and many unknown function

Elicitors of Nematodes
Unknown number and function
 Model for the action of Xa21
(rice blight resistance gene)

Leucine-rich receptor
Transmembrane domain

Kinase

Membrane
Elicitor Signal transduction
([Ca2+ ], gene expression)

Cell Wall Plant Cell

 Calciuum
 Jasmonates

 Inositol phosphate

 GTP binding proteins

 Cyclic AMP
 stimuli - include environmental signals produced by drought,
salinity, cold, mechanical perturbation, hormonal signals,
symbiotic and pathogenic microorganisms trigger increase con
of ca .
These results suggest that osmotic stress induced expression of
gene requires the [Ca2+ ] cyt elevation that is known to occur in
response to these stimuli (pas et al., 2005).

The specificity of the calcium responses depends on the Ca2+

signature of a given signal, characterized by its amplitude, duration,
frequency and location, and was shown to encode a message that,
after decoding by downstream effectors, contributes to the specific
physiological response. This explains the presence of the large
number of Ca2+ sensors in plant cells to decode different incoming
stimuli.
.

 Many Ca2+ signaling studies in plant cells are performed using

the
aequorin technology based on bioluminescence,fluorescence
resonance energy transfer (FRET)-based Ca2+- indicator
cameleon (vadassery et al., 2009, Boudsoeq et al., 2010) and
Patch clamp( Qi et al., 2009).

Role of secondary signal molecules in signal transduction pathways as affected by

abiotic stresses
Transgenic plants as a research tool for non-genetic studies
e.g. aequorin transformed plants to study calcium’s role as secondary
messenger

The aequorin gene from a luminescent jellyfish produces a protein aequorin.

When combined with a small chromophore, coelentrazine, the complex gives
off blue light at a rate dependent on [Ca2+ ].
When transformed in to tobacco, this
gene can be used to study the role of
[Ca2+ ] in signal transduction
Aequorin
Transient increase in

Luminescence
luminescence of
tobacco plant
challenged with
fungal elicitor.
Ca2+ involved in
pathogen recognition Time
Tobacco

Knight et al. 1991

 phytohormone jasmonic acid (JA) and its methyl ester
 regulate developmental processes, including embryogenesis,
pollen and seed development, and root growth (Farmer et al.,
2003, Liechti et al. 2006).
 As also mediate resistance to insects, microbial pathogens, and
abiotic stress responses to wounding and ozone (Norman-
Setterblad et al. 2000; Overmyer et al. 2000).
 terminal product of the octadecanoid pathway, it is not the only
one with biological activity.
 Recent studies suggest that a cyclopentenone precursor of JA, 12-
oxo-phytodienoic acid (OPDA), can also induce defense gene
expression (Farmer et al. 2003).
 Arabidopsis mutants impaired in the synthesis (fad3/7/8) or
perception (coi1) of JA exhibit enhanced susceptibility to a variety
of pathogens, including the fungi Alternaria brassicicola, Botrytis
cinerea, and Pythium sp., and the bacterium E. carotovora
(Thomma et al., 2001, Norman-Setterblad et al., 2000).
 These pathogens have a common virulence strategy; they kill plant
cells to obtain nutrients
The activated G protein subunits detach from the receptor and initiate signaling from
many downstream effector proteins. These include phosphodiesterases and adenylyl
cyclases, phospholipases, and ion channels that permit the release of second
messenger molecules such as cyclic-AMP(cAMP), cyclic-GMP (cGMP), inositol
triphosphate (IP3), diacylglycerol (DAG), and calcium (Ca2+ )ions (Estrella et al.,1994,
Clark et al., 2001, Xing et al., 2000).
cAMP is generated as a second (intracellular) messenger in eucaryotes. A ligand binds to a receptor causing
a conformational change that allows the G protein to associate with it. This interaction leads to a change in the
conformation of the G as subunit causing GTP to replace GDP which leads to the dissociation of the GTP-Gαs
from the G β/γ subunits. As shown in this figure, the Gαand Gγ subunits are linked to the membrane by lipid
groups. The "s" on the Gα subunit indicates it stimulates cAMP synthesis by adenylyl cyclase. In keeping with
this, the GTP-Gαs subunit binds to adenylyl cyclase (AC). This activates AC leading to the synthesis of cAMP
from ATP
 The completion of Arabidopsis thaliana (AT)
genome sequence has helped
in the
identification and isolation of gene families
encoding MAPKs and their
immediate
upstream regulators,
MAPK kinase (MAPKK) and MAPKK .
 Nine MAPK genes have been identified from rice.
Each MAPK encodes a
distinct protein
kinase that plays a role in mediating
drought tolerance .
.
 CH
CH CH
3
3 3

OH
 Small,lipophyllic phytohormone COOH
 Plant growth, and development
 Cellular response to stress-pathogen attack,
O CH

cold, salt, wounding ,UV radiation

3

 In has been investigated that influence of abscisic acid (ABA) on

Physcomitrella patens at the level of the proteome using two-dimensional gel
electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry
(LC-MS/MS).
 Sixty-five protein spots showed changes in response to ABA treatment. Among
them, thirteen protein spots were down-regulated; fifty-two protein spots were up-
regulated including four protein spots which were newly induced. These proteins
were involved in various functions, including material and energy metabolism,
defense, protein destination and storage, transcription, signal transduction, cell
growth/division,transport, and cytoskeleton.
 Specifically, most of the up-regulated proteins functioned as molecular
chaperones, transcriptional regulators, and defense proteins. Detailed analysis of
these up-regulated proteins showed that ABA could trigger stress and defense
responses and protect plants from oxidative damage
Diagrammaticwith black boxes in the organization of nod gene cluster of Rhizobia
and the arrows with different nod gene indicate the direction of transcription.
representation of phenolic compounds signal exchange between the legumes and
rhizobia during nodulation. Plant signal activates the nod D product that controls
the transcription of other nod genes. The nod boxes are highlighted
A simplified model for signal transduction in plant defense responses. Host recognition of
pathogen elicitors initiates early signaling events such as protein
phosphorylation/dephosphorylation, ion fluxes and oxidative burst. Subsequent
transcriptional and/or posttranslational activation of transcription factors leads to
induction of plant defense genes such as GST and PAL, and biosynthesis of
endogenous secondary signals such as SA. In addition, the activated NADPH oxidase
complex generates reactive oxygen species (ROS) such as 02"- and H202 that alter the
redox status of plant cells and affect defense signaling. SA, ROS, as well as defense
genes, all contribute to the development of HR and SAR during plant-pathogen
HOW HOST RESPONSE TO SIGNAL?
Signal-transduction pathways.
a)A signal on a cell’s surface is converted into specific cellular response
using a series of steps, e.g., greening = decreased rate of stem elongation,
leaves expand, roots elongate, shoot produces chlorophyll; requires reception
of signal, transduction of signal, response to signal.
b) Transduction: secondary messengers transfer and amplify signal, e.g.,
phytochrome conformation changes and it makes/activates many second
messengers, e.g., G-proteins which activate many enzymes, e.g., kinases
or activates calmodulins that bind to calcium ions which activate kinases.
c) Response: cellular activities are changed by enzymes that are activated
due to either transcriptional regulation that can activate positive transcription
factors or deactivate negative transcription factors or posttranslational
modification of enzymes by kinases in a phosphorlytion cascade.
d). Reception: conformational change in protein shapes, e.g., phytochrome
pigment within cytoplasm absorbs light (even very low levels of light).
(Nair,1993)
 Infectious-Nonself Model
 Charles Janeway (1989)
 Danger Model
 Polly Metzinger (1994)
 Guard Model
 Jeffrey Dangl & Jonathan Jones
(2001)
 Detection of conserved molecular
patterns (pathogen-associated
molecular patterns, PAMPs) by pattern
recognition receptors (PRR).
 Upon recognition of PAMPs by …
 Membrane-associated PRR: signaling
pathways that induce antimicrobial
effectors and inflammation is activated.
 Soluble PRRs: pathogens bound and flagged
for destruction by phagocytosis or the
complement system.
 The immune system is activated in response to
substances that cause damage, rather than those
that are simply foreign. These alarm/danger
signals are molecules or molecular structures,
released or produced by cells undergoing stress
or abnormal death. These signals are perceived
by resting APCs.
 The alarm signals can be either endogenous or
exogenous, intracellular or extracellular/secreted.
They may function as primal initiators or simply
give positive-feedback to enhance or modify an
ongoing response.
 Resting DCs are activated by cells killed by acute necrotic death but
no by cells dying of physiological apoptotic death.
 Crystalline, but not soluble, uric acid can activate DCs.
R proteins associate physically and specifically with cellular targets
(or “guardee”) of bacterial type III effectors (Avr). The interaction
between guardee and Avr is recognized by the R protein, which is
thus activated to initiate disease resistance. Guardees are likely to
be plant defense components or host proteins whose function is
modified to nourish the extracellular bacterial pathogen. In the
absence of a specific R protein, the host target is not guarded from
the virulence function of Avr, and disease ensues.
AvrRpt2 is a Cysteine Protease.
 Gene-for-Gene theory of Complete
Resistance by FLOR

Pathogen Plant has resistance

has gene
virulence (a)
and
avirulence RR rr
A a
(A) genes

If the pathogen has an Avirulence gene and the host a

Resistance gene, then there is no infection
 Prior to results obtained in animals, in 1993, Greg Martin
cloned the Pto gene in tomato confers resistance to
races of Pseudomonas syringae pv. tomato that carry
the avirulence gene avrPto.
 In 1994, the Ausubel and Staskawicz groups clones the
resistance gene RPS2 from Arabidopsis. In the same
year, Barbara Baker’s group cloned the N gene from
tobacco.
 The N and RPS2 proteins, like Toll and TLRs has the LRR
and TIR domains. They define a large group of
cytoplasmic R proteins called the NBS-LRRs. They
contain a central nucleotide binding site and a C-
terminal LRRs called the NBS-LRR.
 A third class of R genes are membrane-anchored
glycoprotein with extracytoplasmic LRRs.
Gene for gene specificity.
 Classical method
Biochemical, genetic, molecular biological and (Wang
et al., 2010).

 plant pathology approaches,

 systems biology approaches such as genomics and

proteomics (Kav et al., 2007).

 Mutational analysis (Yang et al., 2011).

 Provide generic disease assessment protocols for breeders & variety
testing (gen + path specific)
 System protocols for pathologists Systems level information that is
likely to link closely with other working groups and be important includes:
nutrient status, climate, morphology/canopy, seed rate, weeds, rotational
position, LAI, soil type, disease complexes.
 Systems are defined by hierarchies of information. Therefore coordinate
basic plant pathological details from :
 Variety resistance ring tests
 Pathogen virulence surveys
 Seed borne disease experiments

 Sampling, analysis and assessment protocols for diseases in

heterogeneous crops is complicated and will be addressed by the group.
 Breeding pathogen resistant crop plants
 Discovery of environmentally safer agrochemicals
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