Biotech Today (January—June 2016) 6(1): 60-64
DOI : 10.5958/2322-0996.2016.00009.0
RESEARCH ARTICLE
Reciprocal relationship between expression of MAP Kinase 4
and MAP Kinase 6 during pathogenesis of Alternaria Blight
in Arabidopsis thaliana
Rakesh Mondal, Dinesh Pandey, Gohar Taj and Anil Kumar
Published : 02 January, 2016
Abstract Arabidopsis thaliana was shown to be an al-
ternate host plant for the disease, Alternaria blight of
Brassica and used as a model plant to delineate the rela-
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tionship between MAP Kinase 4 (MAPK4) and MAP
Kinase 6 (MAPK6) during pathogenesis of Alternaria
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blight. Reverse Transcriptase Polymerase Chain Reac-
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tion (RT-PCR) based approach was used to determine
the change in transcript profiling of MAPK4 and MAP-
K6 in leaves of Arabidopsis thaliana ecotype Columbia
during different stages of disease progression of Alter-
naria blight. It was observed that the expression of MAP
kinase 4 decreases whereas that of MAP kinase 6 in-
creases as disease progresses through different stages of
infection. Thus an inverse relationship between expres-
sion of MAPK4 and MAPK6 was observed during path-
ogenesis of Alternaria blight in Arabidopsis. Since
MAPK4 positively regulates Jasmonic acid (JA) de-
pendent defense, against the necrotrophic pathogens,
down regulation of MAPK4 suggests that Alternaria
brassicae pathogen perhaps overcomes JA mediated
defense to favour it’s necrotrophic colonization. Upreg-
ulation of MAPK6 which is regulator of basic plant
defense indicates host plant responds to pathogen attack
by strengthening of basal plant defense which is, how-
ever, insufficient to curtail necrotrophic colonization of
pathogen.
Highlights
 Arabidopsis thaliana ecotype Columbia acts as host
plant for the disease, Alternaria blight of Brassica
crops.
Rakesh Mondal, Dinesh Pandey*, Gohar Taj and Anil
Kumar
Department of Molecular Biology & Genetic Engineer-
ing, College of Basic Sciences & Humanities, Govind
Ballabh Pant University of Agriculture & Technology,
Uttarakhand, India
Corresponding Authors E-mail: dineshpandeymbge@
gmail.com
 MAP Kinase 4 is downregulated whereas MAP
Kinase 6 is upregulated as disease progresses in
Arabidopsis thaliana, representing reciprocal rela-
tionship between them.
Key words Alternaria blight, Arabidopsis thaliana ,
MAP Kinase 4, MAP kinase 6
Introduction
The disease Alternaria blight, incited by the pathogen
Alternaria brassicae, seriously hampers productivity of
rapeseed and mustard, which are the economically im-
portant oilseed crops of India (Ram et al.. 1998). Plant
breeding methods have so far been unsuccessful to de-
velop disease resistant varieties due to unavailability of
resistant germplasm within Brassica genus. It is being
realized that Biotechnological methods could help in
development of disease resistant varieties if the molecu-
lar mechanism of pathogenesis is delineated.
Mitogen activated protein kinases (MAPK) are im-
portant mediators in signal transmission, connecting the
perception of external stimuli to cellular responses.
MAPK cascades not only mediate responses to various
biotic and abiotic stress signals, but also regulate vari-
ous developmental processes of plants. (Karin and Hirt
2001). MAP kinase cascade is composed of three fami-
lies of kinases, MAP kinase kinase kinase (MAPKKK
or MAP3K), MAP kinase kinase (MAPKK or MAP2K)
and MAP kinase (MAPK), which are sequentialy phos-
phorylated during transmission of signal from receptor
to downstream targets (Ichimura et al., 2002). On the
basis of genomic information, 20 MAPKs, 10 MAP2Ks
and 80 MAP3Ks have been identified in Arabidopsis,
which engage in various modules to regulate different
responses in plants (Colcombet and Hirt, 2008). Out of
three families of MAP kinases, some members of
MAPK family ,which act at downstream level, have
been reported to positively or negatively regulate de-
fense response against some pathogens in Arabidopsis
61
 Fig.1. (a) Arabidopsis thaliana ecotype Columbia showing the symptoms of Alternaria blight disease
(b) Alternaria brassicae spores isolated from infected leaves of Arabidopsis plants
thaliana. For example, MAPK4 has been shown to neg-
atively regulate systemic acquired resistance but posi-
tively regulate Jasmonic acid (JA) mediated induced
systemic defence response (ISR) (Petersen et al., 2000).
Recently, in silico studies have shown MAP kinase 4 as
target molecule of defense pathway against Alternaria
blight of Brassica ( Marmath et al., 2013). On the other
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hand, MAPK6 is known to play some role in resistance
against some pathogens in Arabidopsis, but it does not
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affect the ability of plant to develop SAR or ISR
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(Menke et al., 2004). Keeping the evolutionary con-
served function of MAPK cascade, it was speculated
that some of these MAPK’s could also determine de-
fence response against Alternaria blight. It was realized
that Arabidopsis thaliana which exhibits all of the major
defence responses found in other flowering plants, and
whose genome has been completely sequenced (The
Arabidopsis genome initiative, 2000) can be utilized as
model plant to derive knowledge on regulation of de-
fense related MAP kinases. In the context of above, the
present study was initiated to test if Arabidopsis thali-
ana acts as alternate host for Alternaria blight disease
and to investigate the role of MAPK4 and MAPK6 dur-
ing different stages of disease progression.
Materials And Methods
Plant Material and Growth conditions
Fig.2. Infected leaf sample at different stages of diseases (a) Healthy stage (b) Initial stage (c) Middle stage
and (d) Late stage of disease progression
The seeds of Arabidopsis thaliana ecotype Columbia
were sown in the autoclaved soil in pots and plants were
grown in green house at 220C with 16h light/8h dark
cycle and 80-85% relative humidity.
Isolation of Alternaria brassicae spores
The leaves of Brassica juncea cv. Varuna , infected with
Alternaria blight disease were collected from Crop Re-
search Centre, G.B. Pant University of Agriculture and
Technology, Pantnagar. The spots were cut, surface steri-
lized with freshly prepared aqueous mercuric chloride
solution (1:1000) for one minute and washed with steri-
lized water for 3-4 times. Surface sterilized leaf spot
pieces were then aseptically transferred into the petri
dishes containing Potato Dextrose Agar (PDA) medium
and incubated at 24 ± 20C for seven days. After incuba-
tion, mycelium from margins of apparently distinct col-
onies of A. brassicae on the medium were inoculated in
to PDA and incubated at 24 ± 20C with 12 h light/ 12 h
dark cycle for 15 days to develop pure cultures.
Inoculation of Arabidopsis thaliana leaves with Alter-
naria brassicae
The sporulating culture of Alternaria brassicae was
scrapped in to 1 ml distilled water to adjust the concen-
tration of spores to 104 ml-1. 10µl of spore suspension
was sprayed on the leaves of Arabidopsis plants with
the help of atomizer. The control plants were sprayed
with distilled water. The inoculated plants were incubat-
62
 Fig .3 (a) Determination of change in transcript profiling of MAPK4 gene by semi-quantitative RT-PCR
from infected leaves of Arabidopsis thaliana eco. Col. having different stages of infection ( Lanes M : 100 bp
ladder; lanes 1-4 = MAPK4 and Actin gene expression from Healthy; Early; Middle and Late stage of dis-
ease progression )
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Fig.3. (b) Densitometry analysis of the expression
profile of MAPK4 from Arabidopsis thaliana leaves
harvested from different stages of disease progres-
sion
ed at 280C with 90-100 percent relative humidity for 3
days in humid chamber and then transferred in to
growth room.
Screening of Arabidopsis plants for infection and
collection of leaf samples
Infected Arabidopsis plants were checked for the pres-
ence of Alternaria blight like disease symptoms and
presence of Alternaria brassicae pathogen was checked
by observing the spores for the same. For analysis of
MAP Kinase 4 and 6, the infected leaf samples were
collected at the interval of 3 days after appearance of
first necrotic lesion (4 DAI) and categorized in to leaves
from Healthy, initial, middle, and late stage of disease
progression depending upon number and area of chlo-
rotic/necrotic lesions as described in Table.1 and
Fig.2..Leaf material was harvested, frozen and stored at
-800C until further experiments.
RNA Isolation
The sample for RNA isolation consist of five leaves of
each stage obtained from three plants. Total RNA was
isolated using QIAGEN Plant RNA isolation kit, as per
manufacturer’s instructions. The extracted RNA was
purified by RNase free DnaseI treatment as per manu-
facturer’s instructions (Genei, Banglore) and spectro-
Fig .4(a) Determination of change in transcript profiling of
MAPK4 gene by semi-quantitative RT-PCR from infected leaves
of Arabidopsis thaliana eco. Col. having different stages of infec-
tion ( Lanes M : 100 bp ladder; lanes 1-4 = MAPK6 and Actin
gene expression from Healthy; Early; Middle and Late stage of
disease progression )
photometrically quantified.
Designing of primers
Gene specific primers were designed using primer Blast
(the online primer designing tool) after retrieving cds
sequences from NCBI for MAP Kinase 4
(>NM_116367.2) and MAP Kinase 6
(>NM_129941.3) . The sequences of primers are as
follows : MPK4F 5΄-AAACCA CCG CAG AGA GAG
AA-3΄ ; MPK4R 5΄-GGG CTC TCG TGT CAT TGT
TT-3΄; MPK6F 5΄-CAT CGT TTG TTC GGC TAT GA
-3΄; MPK6R 5΄-AGT TTG CGT TCA GGA GGA GA-
3΄ . The sequences forward and reverse primers for Ac-
tin gene, which was used as positive control, include
ACT.F 5΄-CTT AGG TAT TCG AGA CCG TAT GAG
C-3΄; ACT.R 5΄-GTT TTT ATC CGA GTT TGA AGA
GGC T-3΄ as reported earlier by Nozomi (2005).
Semi-quantitative RT-PCR analysis
In order to analyze the expression pattern of MAPK4
and MAPK6 at different stages of disease progression,
one step semiquantitative RT-PCR was performed by
using 0.2µg RNA as template along with gene specific
primers as per the manufacturer’s instructions
(QIAGEN, USA). The coamplification of actin and in-
dividual MAPK gene was performed under the follow-
63

Fig.4(b) Densitometry analysis of the expression profile
of MAPK6 from Arabidopsis thaliana leaves harvested
from different stages of disease progression
ing PCR conditions : Reverse transcription at 500C for
30 min followed by 35 cycles of amplification (initial
denaturation at 94°C for 1 min, annealing at 54°C for 1
min and extension at 72°C for 1 min) with final exten-
sion at 72°C for 10 min. The RT PCR products were
analyzed on a 1.5% agarose gel and integrated density
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values (IDV) of amplicons were measured using AL-
PHA IMAGER tool. RT PCR product with expected
size was sent for sequencing to Chromous Biotech for
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verification of their identities.
Results And Discussion
Development of disease symptoms on Arabidopsis
thaliana
Upon inoculation of Arabidopsis plants with spore sus-
pension of Alternaria brassicae, small necrotic spot was
seen after three days of inoculation which gradually
increased to give rise chlorotic and necrotic lesions at
the later stages resembling symptoms of Alternaria
blight in Brassica. (Figure 1a) Lower leaves showed
the symptoms first which later progressed to upper
leaves of the plant. The presence of spores of Alter-
naria brassicae spores was detected when the suspen-
sion from infected leaf sample was examined under
microscope. (Figure 1b). Thus results confirmed that the
establishment of Alternaria blight in Arabidopsis was
due to infection by Alternaria brassicae and that Ara-
bidopsis acts as alternate host for the pathogen of Alter-
naria blight.
Collection of leaf samples for analysis of MAPK4
and MAPK6
During pathogenesis process of Alternaria blight, first a
small necrotic spot develops which subsequently in-
creases in size. In the next stage, the necrotic lesion is
surrounded by the chlorotic lesion. In the later stages,
Table 1. Collection of leaves at different stages of disease progression
Days after Leaves Description
inoculation
0 Healthy No necrotic and chlorotic lesions
4 Initial stage One small necrotic spot (1 mm)
7 Middle stage One necrotic spot (3mm) along with little chlorosis
10 Late stage One necrotic spot (>5mm) along with large amount of chlorosis
both chlorotic and necrotic lesions increase in size to
produce full disease symptoms of Alternaria blight. In
order to study the relationship between production of
disease symptoms with the regulation of MAP kinase 4
and MAP kinase 6, the leaves representing different
stages of infection in terms of symptoms were collected
(Table 1 & Figure 2).
Expression profiling of MAPK 4 at different stages
of pathogenesis of Alternaria blight in Arabidopsis
thaliana
Reverse Transcriptase Polymerase Chain Reaction (RT-
PCR) analysis for MAP Kinase 4 in Arabidopsis leaves
at different stages of infection has indicated that the
expression of MAPK4 (399bp) continuously decreases
as disease progresses from initial to late stage of infec-
tion of Alternaria blight. The constitutive expression of
Actin (470 bp) was observed with all the samples indi-
cating there is no difference in the quantity of RNA
used from different samples. (Figure 3 a & b). The RT
PCR amplicon was sequenced and subjected to homolo-
gy search for other sequences present at the NCBI data-
base (http://www.ncbi.nlm.nih.gov) using BLASTn
tool. The amplified products for MAPK4 showed 97%
homology with existing MAPK4 sequence of Arabidop-
sis thaliana confirming the identity of amplicon.
The transcript level of MAP K4 was found to be higher
in healthy leaf than all infected samples. This suggests,
the pathogen continuously cross-talks with plant cell to
weaken the plant defense and plant, in response to infec-
tion, tries to defend the pathogen through downregula-
tion of MAP Kinase 4. It is worth to recall thatt MAP
K4 genes are activated in response to attack of fungal
pathogens (Nuhse et al., 2000; Desikan, 2001) and are
differentially regulated during defense response of
plants to pathogen. For example, Peterson et al., 2000
have demonstrated that the inactivation of Arabidopsis
MAP kinase 4 leads to constitutive systemic acquired
resistance (SAR) including elevated salicylic acid (SA)
levels, increased resistance to virulent pathogens, and
constitutive pathogenesis-related gene expression. How-
ever, the induction of jasmonic acid responsive genes
was blocked by inactivation of MAPK4, suggesting that
it is required for jasmonic acid dependent induced sys-
temic defense response (ISR). In the light of above, it
could be interpreted that SA dependent resistance to
Alternaria brassicae plathogen increases but JA de-
pendent resistance decreases as Alternaria blight disease
progresses from initial to late stage of infection in Ara-
bidopsis thaliana. Since Alternaria brassicae is a ne-
crotrophic fungi and JA dependent pathway is operative
against necrotrophs, it suggests that downregulation of
MAPK4 during host pathogen interaction may facilitate
64
 more and efficient colonization of the pathogen of Al-
ternaria blight.
Expression profiling of MAPK6 at different stages of
pathogenesis of Alternaria blight in Arabidopsis tha-
liana
Reverse Transcriptase Polymerase Chain Reaction (RT-
PCR) analysis for MAP Kinase 6 in Arabidopsis leaves
at different stages of pathogenesis has indicated that the
expression of MAPK6 (390bp) continuously increases
as disease progresses from initial to late stage of infec-
tion of Alternaria blight. (Figure 4 a & b) The constitu-
tive expression of Actin (470 bp) was observed with all
the samples indicating there is no difference in the
quantity of RNA used from different samples. The RT
PCR amplicon was sequenced and subjected to homolo-
gy search for other sequences present at the NCBI data-
base (http://www.ncbi.nlm.nih.gov) using BLASTn
tool. The amplified products for MAPK4 showed 97%
homology with existing MAPK4 sequence of Arabidop-
sis thaliana confirming the identity of amplicon.
It is worthwhile to mention here that resistance to an
avirulent strain of Peronospora parasitica and avirulent
and virulent strains of Pseudomonas syringae were
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compromised in MAPK6 silenced Arabidopsis plants
(Menke et al., 2004). Although MAPK6 enhances the
basal resistance to some pathogens, it does not affect the
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ability of plant to develop SAR or ISR. This suggests
that upregulation of MAPK6 during disease progression
of Alternaria blight is an indication that basal resistance
of plant increases during pathogen encounter, but it is
not sufficient to curtail the pathogen infection.
In conclusion, preliminary investigations, in the present
study, show reciprocal relationship between expression
of MAP Kinase 4 and MAP kinase 6 during pathogene-
sis of Alternaria blight in Arabidopsis leaves at different
stages of infection. The observation in the results of
present work suggests that there is simultaneous down
regulation of MAP Kinase 4 and up regulation of MAP
kinase 6 during interaction of Arabidopsis plant with the
pathogen Alternaria brassicae. As a result, there could
be induction of JA mediated defense pathway and
strengthening of basal defense against the pathogen.
However, the defense pathways triggered by these mod-
ulations might not be sufficient to curtail pathogen at-
tack, but perhaps, pathogen uses some other mecha-
nisms to regulate other MAP Kinases so as to overcome
plant resistance and to colonize the plant tissue.
Acknowledgement
Authors are greatful to DBT for providing the financial
support in the form of R & D support under the project
Programme mode support in Agricultural Biotechnolo-
gy.
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