Enhanced Resistance of Oryza sativa to
Magnaporthe grisea via Overexpression of
OsWRKY45 Allele Regulated by Benzothiadiazole-
Induced Salicylic Acid Signaling Pathway
Lisette Catangui, Justine Paul Llamson, Allen Melvin Pontejos and Glen Paulo Trinidad
Bicol University – College of Engineering, lisette.catangui, justinepaul.llamson, allenmelvin.pontejos,
glenpaulo.trinidad@bicol-u.edu.ph
Abstract – Magnaporthe grisae as one of the most serious
and widespread infectious diseases of cultivated rice
decreases the rice production. The researchers analyzed
the pathway for the naturally occurring defense Salicylic
Acid (SA) in order to lessen the effect of the fungus to the
plant. Benzothiadiazole, a plant activator, enhances the
defense response by acting on the SA signaling pathway.
The transcription factor improves the disease resistance
in the rice plant by genetic manipulation. Overexpression
of WRKY45 also enhances the resistance to the fungus.
Our study revealed that the modification of the pathway
of SA via overexpressing OsWRKY45 by increasing BTH
dosage can increase the resistance to the said fungus.
Index Terms – Benzothiadiazole, Magnaporthe grisae,
Overexpression, Salicylic acid
INTRODUCTION
Rice (Oryza sativa) is the most widely consumed staple food
for a large part of the world’s population and well-known
species for plant research. To overcome the increasing
demand of rice production, high-yielding rice lines that are
tolerant to biotic and abiotic stresses are being developed
over the years. Rice blast disease, caused by the fungus
Magnaporthe grisae, is one of the most serious and
widespread infectious diseases of cultivated rice, and
continuously threaten rice production worldwide.
Recent advances in research on plant–pathogen
interactions have provided insights into the mechanisms of
plants’ defenses against potential pathogens. One approach is
to modify the defense Salicylic Acid (SA) signaling pathway
by transgenic manipulation of the genes encoding defense-
related signaling molecules such as protein kinases and
transcription factors (TFs).
Specifically, this paper aims to discuss the mechanism
how benzothiadiazole, a chemical defense inducers (plant
activator) activates and induces disease resistance
mechanism of rice plant (Oryza sativa) to blast disease caused
by fungus Magnaporthe grisae via overexpression of
OsWRKY45 allele, a key transcription factor in the SA
signaling pathway.
METHODS
Researches gathered related studies in order to modify
the pathway for the defense SA and to increase the resistance
to the fungus M. Griae.
Plants in general, are able to muster kind of reserve
defense system against different kinds of attacks, ie. Fungus.
Plants responds to diseases by activating various immune
responses.
Seed treatment is one of the method in counter the
disease. It refers to the application of fungicide, insecticide,
or a combination of both, to seeds so as to disinfect and
disinfect them from seed-borne or soil-borne pathogenic
organisms and storage insects. The different types of seed
treatment are seed disinfection, seed disinfestation, and seed
protection. Foliar feeding is a technique of feeding plants by
applying liquid fertilizer directly to their leaves
Gene Overexpression may be in the cell where the gene
is originally located or in other expression systems. The
fundamental principle is to add regulatory elements to the
upstream of the target gene through artificial construction, so
that genes can be transcribed and translated efficiently under
controlled conditions. The experiment processes for gene
overexpression are as follows: (1) obtain target gene, (2)
construction of Expression Vector (3) cell transfection, (4)
cell collection (5) characterization of mRNA expression
level, and (6) characterization of protein expression level.
RESULTS AND DISCUSSION
The researchers have reviewed some of the recent advances
in the studies on the SA pathway in rice, with particular focus
on potential practical applications of the signalling
components. The SA pathway in rice branches into OsNPR1
and OsWRKY45-dependent pathways. Experimental results
showed that overexpression of OsWRKY45 confers high
resistance to blast and blight diseases in rice. The
OsWRKY45 pathway is the potential target for genetic
manipulation of rice in order to develop a broad-spectrum
disease resistance. In this section, the fundamental
knowledge necessary in understanding the topic and recent
advances in this study are discussed.
I. Overview of rice plant systematic resistance to pathogens such that the growth is affected negatively [3]. Moreover, the
results highlight the potential of BTH as an effective activator
The cell wall is the primary barrier protecting the rice
to make the SA pathway most dominant during the attack of
plants from pathogen invasion. Fungus, specifically
pathogens.
Magnaporthe grisea, penetrates the cell wall after they induce
rice synthesis of Indole-3-acetic acid (IAA), a major form of IV. The transcription factor OsWRKY45 role in plant
auxin in most plants that promote disease symptoms. disease resistance
Pathogens also secrete their own IAA, which induces the
Transcription factors generally control the multiple
host’s gene expression. The host then encodes proteins that
genes that drive the defence response and modulation of key
loosen the cell wall [1]. After the first local infection, a series
TFs will lead to wide range of expressional changes.
of signalling pathways responsible for the defences are
Therefore, the key TFs are suitable targets for genetic
enabled with the aid of plant activators. The resistance
manipulation to improve the disease resistance of rice plants.
mechanisms of the plant innate immune system can be
Takatsuji et al. characterized the BTH responsive genes and
implemented through the following major pathways:
identified the key TFs involved in the BTH-induced disease
Salicylic acid (SA) pathway; Jasmonic (JA) pathway and;
resistance in the rice plant. Gene encodings, 4 WRKY-types
Ethlyene (ET) pathway. In this plant, SA pathway is
were identified. Blast resistance tests showed that the key TFs
associated with resistance to biotrophic pathogens while JA
that expressed the OsWRKY45 cDNA exhibited greatly
and ET pathways are generally for combating necrotrophic
enhanced resistance to fungus. Also, blast tests on
pathogens. However, the relationship between these
OsWRKY45 knockdown reduced the blast resistance
pathways are mutually antagonistic with corresponding
induced by parabenzole and tiadinil.
trade-offs of respective resistances. For instance, activating
the pathway to inhibit the growth of a biotrophic pathogen V. Antagonistic crosstalk of abscisic acid (ABA) pathway in
negates the resistance of the plant to other necrotrophic Magnaporthe grisea interaction
pathogen. As a result, activation of defences against a
The major problem in the proposed defence response
particular pathogen increases the susceptibility against other
acting on the SA pathway is that it does not respond
types of pathogens [2].
effectively after the Magnaporthe grisea infection. Jiang et al
II. Salicylic acid (SA) signaling pathway in rice examined the possibility that the chemical ABA is derived
from the fungus. Hormone analysis showed that substantial
In the systemic acquired resistance of rice, the proposed
amount of ABA in the culture filtrates. This indicates that the
defence-signaling pathway mediated by SA pathway plays an
ABA was secreted in the culture medium. The ABA has been
effective role in the natural defence system of the plant
shown to antagonize the SA signaling pathway. As a result,
against the attack of pathogens. Recent studies revealed that
the disease susceptibility of the plant is enhanced. Increased
the SA pathway interacts with the transcription factors (TFs)
in ABA concentration, suppresses the SA-induced
that activate the target defence genes [3]. As shown in Figure
transcriptional up-regulation OsNPR1 and OsWRKY45
1, the SA pathway is branched into the transcription factors
dependent sub-pathways.
OsNPR1 and OsWRKY45 dependent sub-pathways. Because
of the mutual antagonism, while OsNPR1 increases the VI. Overexpression of OsWRKY45 allele to enhance
defence and resistance of the plant, the JA and ET pathways resistance to blast disease
are suppressed. Also the some cellular activities such as
Overexpression of WRKY45 in rice enhances rice
photosynthesis are down-regulated, because the resource
allocations are playing a role in the defence mechanism. resistance to M. grisea by strengthening the SA pathway. To
Moreover, the WRKY45 generally promotes the major further validate the antagonistic interaction of ABA with the
rice SA pathway and to determine the point at which ABA
disease resistance of the plant [4].
acted in the SA signaling pathway, Jiang et al. tested the
III. Benzothiadiazole (BTH) as a plant activator inducing effects of ABA application on the blast resistance of the
the SA pathway transgenic lines overexpressing WRKY45. The plant
seedlings were pre-treated with ABA for 24 hours by foliar
The plant activator BTH is effective on protecting many
spraying and then inoculated with compatible blast fungus
plant species from pathogen colonization. This chemical does
(race 007.0). ABA application resulted in only a minor
not have direct effect on the pathogen but it activates and
reduction in the blast resistance in the two transgenic rice
enhances the defence response of the plant by acting on the
lines overexpressing WRKY45 but did not restore blast
SA signalling pathway. In Figure 1, BTH signals and induces
susceptibility comparable with that of control wild-type
the SA pathway to mediate after the pathogen local infection.
plants. These results indicate that the negative impact of ABA
High dosage of BTH constitutively activates the defence
on the SA-mediated defence signaling leading to the down-
genes including the up-regulations of other defences called
regulation of WRKY45 was partially suppressed by
direct defence. In contrast, low dosage of BTH plants defence
WRKY45 overexpression.
mechanism is activated effectively after the pathogen
infection; and the plant become primed for defence responses
 FIGURE I
CURRENT STATUS OF KNOWLEDGE ABOUT THE SA SIGNALING PATHWAY IN RICE.SA
To evaluate the degree of blast resistance of OsWRKY45
overexpressing rice plants, Takatsuji et al. compared them
with several blast-resistant rice varieties. A blast-resistance
test conducted with a compatible race indicated that rice
plants became more blast resistant. Moreover, OsWRKY45
overexpressing rice plants were also resistant to blast caused
by fungus Magnaporthe grisea. BTH also induces resistance
to bacterial leaf blight disease in rice plants. The researchers
also tested the leaf blight resistance of OsWRKY45
overexpressing rice plants and found that these plants were
highly resistant to the disease. These results suggest that
WRKY45 confers broad-spectrum resistance to rice, and they
are consistent with the notion that OsWRKY45 is a key
regulator of BTH-induced defense responses.
CONCLUSION
From the analysis of the written articles and journals, we can
conclude that the modification of one of the defense against
potential pathogens, Salicylic Acid (SA) via overexpressing
OsWRKY45 by increasing BTH dosage can increase the
resistance to the fungus Magnaporthe grisae. BTH as an
effective activator, protects many plant species by acting on
the SA signalling pathway. Transcription factor
OsWRKY45, which are suitable for genetic manipulations,
greatly enhanced the resistance to fungus. To counteract the
secretion of fungus with Abscisic Acid once the plant was
infected, the dosage of BTH should be increased whilst
overexpression of OsWRKY45 allele enhances the resistance
to blast disease.
RECOMMENDATION
The favourable traits of OsWRKY45 overexpression render
a promising target for gene manipulation to develop a broad-
spectrum disease resistance. However, some studies showed
that overexpression of OsWRKY45 driven by the promoter
ubiquitin results to growth retardation. This cannot be
neglected. Therefore, a realistic optimization approach is
essential to lower transgene expression and establishes a
more stable primed state. That is, to increase the rice plant’s
resistance to pathogens without compromising the resource
allocation for growth. Although OsWRKY45 is a powerful
tool against rice blast pathogens, a well-control use of
OsWRKY45 is still a challenge. Further researches are
encouraged that is not only applicable to rice but also to other
important crops.

ACKNOWLEDGMENT
This work will not be possible without the help and support
of the others. We would like to extend our gratitude to Engr.
Raymond Barajas for introducing Biotechnology to us and
allowing us to explore this field to create and modify some of
the essential metabolic pathways within our interests.
REFERENCES

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[3] Jiang et al. 2010. “Review: Salicylic Acid Signaling Pathway in Rice
and the potential Applications of its Regulators”. JARQ 44, pp217-223
http://www.jircas.affrc.go.jp
[4] Tao, Zeng et al. 2009. “A Pair of Allelic WRKY Genes Play Opposite
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