Post on 31-Mar-2023
RESEARCH ARTICLE
Defense Transcriptome Analysis of Sugarcaneand Colletotrichum falcatum Interaction UsingHost Suspension Cells and Pathogen Elicitor
P. R. Rahul • V. Ganesh Kumar • R. Viswanathan • A. Ramesh Sundar •
P. Malathi • C. Naveen Prasanth • P. T. Pratima
Received: 7 August 2014 / Accepted: 5 November 2014
� Society for Sugar Research & Promotion 2014
Abstract Red rot, a stalk disease in sugarcane caused by
Colletotrichum falcatum an ascomycete fungus is a serious
production constraint in many Asian countries. However,
very limited studies at molecular level exist of the mech-
anisms related to interaction between sugarcane and the
fungal pathogen C. falcatum (Cf). In the conventional
system of pathogen inoculation, disease development is
influenced by prevailing environmental conditions in the
field. Hence an attempt was made to standardize an in vitro
system of using sugarcane suspension cells and crude
elicitor of Cf for transcriptome analysis and identifying
defense related genes. Suspension cells of sugarcane cv Co
93009 was treated with Cf-elicitor at 60 glucose equiva-
lents and transcriptome profile was monitored 30 min and
3 h later by differential display RT-PCR. From the
experiment 241 transcripts were found to be differentially
expressed and finally 75 of them were cloned and
sequenced. Among the up-regulated transcripts, about
37 % were found to be defense related and which was
followed by transcription and post transcription (13 %),
general metabolism (11 %), transport (9 %), cell structure/
growth/division (9 %) and signal transduction (5 %). The
down regulated transcript group constituted *27 % of the
differentially expressed transcripts and the grouping pat-
tern was different. Overall, the results revealed up regula-
tion of many potential defense related transcripts like
putative chitinase, glycine rich protein, 14-3-3-like protein,
xylanase inhibitor protein, calmodulin related protein,
Myb-related transcription factor CBM2-like, basal layer
antifungal peptide etc. Further by adopting RACE-PCR
approach, complete gene sequences of 14-3-3-like protein
and xylanase inhibitor were identified and the genes were
characterized to domain level. Our results demonstrate that
the transcript profile in in vitro system of sugarcane sus-
pension cells and Cf-elicitor is close to the cane tissue
challenged with the pathogen and useful to identify defense
related traits in sugarcane against Cf.
Keywords Differential display RT-PCR �Colletotrichum falcatum � Elicitor �Molecular interactions � Transcriptome analysis
Introduction
Sugarcane is a major commercial crop cultivated in more
than 20 million hectares in tropical and subtropical regions
of the world, producing up to 1.3 billion metric tons of
crushable stalks and accounts for nearly 60 % of the total
sugar produced in the world (www.faostat.org). Stalks are
the economically important part in sugarcane and on
mature internodes sucrose can accumulate up to 15 %
concentration. Sugarcane cultivation contributes substan-
tially to the agricultural GDP in India, as every year *300
million tonnes of cane are produced in the country, which
in turn are used by more than 500 sugar mills and thou-
sands of jaggery/khandsari units in different states (Nair
2008). In addition, the by-products molasses and bagasse,
serve as the raw material for ethanol production and power,
respectively. The potential ethanol production has been
realised from sugarcane, with Brazil at the forefront in
utilizing it as a bio-fuel (http://sugarcane.org). This calls
P. R. Rahul � V. Ganesh Kumar � R. Viswanathan (&) �A. Ramesh Sundar � P. Malathi � C. Naveen Prasanth �P. T. Pratima
Division of Crop Protection, Sugarcane Breeding Institute,
Indian Council of Agricultural Research, 641007 Coimbatore,
India
e-mail: rasaviswanathan@yahoo.co.in
123
Sugar Tech
DOI 10.1007/s12355-014-0356-8
for different approaches to supplement the present efforts
of increasing the yield using good agricultural practices as
well as developing new means of managing different biotic
and abiotic stress conditions.
Among the various diseases, red rot caused by Collet-
otrichum falcatum Went (Teleomorph: Glomerella tucu-
manensis [Speg.] Arx and Muller) has been implicated as a
cause for a nation-wide loss of 5–10 % in cane yield,
making it the most serious threat among the biotic stresses
in India and other south Asian countries. However, much
higher yield losses of up to 100 % from India has been
reported when the disease occurred in epidemic form
during different decades and many popular varieties like
Co 419, Co 997, Co 1148, Co 6304, CoC 671, CoC 85061,
CoC 92061, and CoJ 64 etc. were removed from cultivation
(Viswanathan 2010; Viswanathan and Samiyappan 1999).
Since red rot has been a serious constraint to sugarcane
cultivation, incorporating red rot resistance in sugarcane
has become an integrated part of the varietal development
programme. However, due to genetic complexities of
sugarcane genome, inheritance of red rot resistance was not
established. Earlier through pathogenesis-related (PR)
proteins and 3-deoxyanthocyanidin phytoalexins, bio-
chemical basis of red rot resistance has been established
(Malathi et al. 2008; Viswanathan et al. 1996, 2003, 2005).
However, only limited information on the molecular
aspects i.e. on the expression of various resistance/defense
genes during the sugarcane—C. falcatum interaction is
available. The large genome size of sugarcane implies that
genome sequencing is currently not viable and this has led
many researchers to work on expressed sequence tags
(ESTs).
Currently, a large number of sugarcane EST sequences
are available in public databases; however these databases
are lacking information on C. falcatum induced transcripts.
In our previous studies, transcript level changes in sugar-
cane stalk tissues were assessed after C. falcatum inocu-
lation. In these assays matured canes were challenged with
the pathogen under field conditions (Prathima et al. 2013;
Viswanathan 2010). Usually such inoculation is done
during monsoon season for conducive environment for
disease development and better phenotypic expression of
genotypes to the pathogen. The system depends on patho-
gen inoculation during specific crop stage and favourable
environment. If the environment is not conducive, pheno-
typic expression becomes skewed hence an alternative
system to study host-pathogen interaction for sugarcane-
C. falcatum was necessitated. At the cellular level, the
response of the host plant against a pathogen infection
requires complex changes in gene expression patterns. The
timing and activation of these defense responses after the
infection is the most important factor for the success of
host resistance. This activation of defense in the plant
system has been replicated in the suspension culture by the
use of elicitor isolated from the pathogen (Menard et al.
2004). In the present study, attempts were made to study
the defense related transcriptomes in an in vitro system of
sugarcane suspension cells and C. falcatum elicitor through
differential display (DD) RT-PCR approach. The study
identified several defense related transcripts and two of
them were characterized to domain level.
Materials and Methods
Plant and fungal material used: the sugarcane cultivar Co
93009, highly resistant to red rot was used for the experi-
ments throughout the study. The plants were raised and
maintained under field conditions as per the recommended
agronomic practices at the Institute in Coimbatore (Sund-
ara 1998). C. falcatum pathotype Cf 671 (isolated from the
cv CoC 671) maintained at the red rot culture collection of
the institute was used for isolation of the crude elicitor for
treatment in cell suspension culture. The pathogen was
multiplied on oatmeal agar (oatmeal 40 g, agar 10 g, water
1 l) for 7 days and conidial suspension (106 conidia ml-1)
prepared in sterile water. For the isolation of elicitor, the
monoconidial culture of the fungus was inoculated on
oatmeal broth and incubated under room temperature
(28 ± 2 �C) for 12 days.
Isolation of Elicitor from C. falcatum
The elicitor was isolated from the mycelial cell wall of the
fungus, following the method of Anderson-Prouty and
Albersheim (1975). The mycelial mats were harvested
from oat meal broth culture by filtering through two layers
of muslin cloth. The harvested mycelial mat was rinsed
with sterile distilled water three times and homogenized to
powder using liquid nitrogen in a pestle and mortar. 5 ml
of water was added per gram wet weight of mycelia to the
powdered mycelial mat and was further ground for a few
minutes. The homogenate was filtered through two layers
of muslin cloth and the residue was homogenized thrice in
water, once in a mixture of chloroform and methanol (1:1)
and finally in acetone. This preparation, when air-dried,
represents the fraction referred to as the mycelial walls.
Elicitors were extracted from the mycelial walls by sus-
pending 1 g of walls in 100 ml of distilled water and
autoclaving for 20 min at 120 �C. The autoclaved sus-
pension was filtered and the filtrate was clarified by cen-
trifugation and then concentrated to 1/10th v/v in a freeze
drier (Labconco, MO, USA). This preparation is termed as
the crude elicitor.
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Establishment of Cell Suspension Culture
Murashige and Skoog (1962) nutrient agar (MS) medium
was used for initial induction of callus from the inner leaf
whorls of sugarcane variety Co 93009. 100 ml coconut water
(liquid endosperm) per one litre of MS medium was added to
the media prior to pH adjustment. The medium was sup-
plemented with 4 mg l-1 concentration of 2, 4-D for initial
callus induction and for subcultures, reduced concentrations
of 2 mg l-1 and 1 mg l-1 were used. pH of the medium was
adjusted to 5.8 ± 0.2 prior to sterilization. The medium was
solidified using agar at 0.8 % (w/v) and sterilized at 121 �C,
15 lbs for 20 min in an autoclave. The liquid MS medium for
suspension culture was prepared similarly without addition
of agar and 2,4-D being replaced with IAA at 1 mg l-1. Inner
leaf whorls of the cv Co 93009 were excised under aseptic
conditions, cut into several small pieces and cultured on MS
agar medium supplemented with 4 mg l-1 concentration of
2,4-D. The rapidly growing embryogenic suspension-cul-
tured cells were established by transferring friable calli
(Fig. 1) to 100 ml conical flasks containing 30 ml MS liquid
medium and agitated on an incubated rotary shaker at 40 rpm
at 25 �C under continuous darkness. The friable embryo-
genic calli were sub-cultured by transferring the callus into a
fresh medium at every 7 days interval and used for the
studies on priming suspension cells with fungal elicitor.
Cf-elicitor Response Study
The elicitor activity of Cf-elicitor was measured in terms of
accumulation of phenolic content in the sugarcane suspen-
sion cells and in leaf bioassays (cv Co 93009) upon treat-
ment using different concentrations of elicitor (30–200 and
20–100 lg of glucose equivalents, respectively). The crude
elicitor was filter sterilized prior to addition to the suspen-
sion culture and later grown in MS liquid medium. After
adding different concentrations of elicitor to respective
100 ml conical flasks containing 30 ml suspension cells
grown in MS liquid medium, the flasks were incubated in a
rotary shaker at 150 rpm at 25 �C under continuous dark-
ness. The suspension cells were harvested 12 h after elicitor
treatment and phenolic content of the cells was estimated
(Zieslin and Ben-Zaken 1993). One gram of harvested cells
were homogenized in 10 ml of 80 % methanol and agitated
for 15 min at 70 �C. 1 ml of the methanolic extract was
added to 5 ml of distilled water and 250 ml of Folin–Cio-
calteau reagent (1 N) and the solution was kept at 25 �C for
3 min. 1 ml of Na2CO3 (saturated solution) and 1 ml of
distilled water were added and the reaction mixture was
incubated for 1 h at 25 �C. The absorption of the developed
blue colour was measured at 650 nm in a UV spectropho-
tometer (T80? , PG Instruments, UK).
Total RNA Extraction
Total RNA was extracted from control, 30 min and 3 h
elicitor treated samples of cv Co 93009 using TRI reagent
(Sigma, USA). 500 mg of sample tissue was ground to a
fine powder using liquid nitrogen, transferred to 30 ml
DEPC treated sterile centrifuge tubes and added 7 ml of
TRI Reagent. It was mixed vigorously by rapid shaking and
it was kept at 4 �C until all the samples were homogenized.
Fig. 1 Schematic representation of development of sugarcane sus-
pension culture a embryogenic callus, the induced calli developed as
embryogenic calli and transferred on to a fresh medium for further
growth and proliferation; b suspension cultured cells, suspension cells
raised from the friable calli in liquid MS medium
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Differential Display
The DD protocols were followed as per manufacturer’s
instructions with certain modifications (RNA image� kit,
GenHunter, USA). The method involved the generation of
three subsets of mRNAs by reverse transcription with
anchor oligo-dT primers followed by PCR with combina-
tions of respective anchor oligo-dT and eight random
primers in presence of a radiolabelled dNTP and subse-
quently displayed the cDNA produced on a polyacrylamide
gel matrix and visualization by autoradiography (Liang and
Pardee 1992; Liang et al. 1995, 2007).
Reverse Transcription of mRNA
Three reverse transcription reactions for each RNA sample
were setup on ice in separate 0.2 ll thin walled PCR tubes.
The anchor oligo-dT was designed with the following
sequence 50-AAGCTTTTTTTTTTTX-30 where a Hind III
restriction site was provided at the 50 end to facilitate the
cloning and X denotes either A/G/C in their respective
anchor primers. Reverse transcription was done in total
volume of 20 ll containing 200 ng of freshly diluted RNA,
125 mM Tris–HCl, pH 8.3, 188 mM KCl, 7.5 mM MgCl2,
25 mM DTT, 250 mM dNTPs and 2 mM of HT11X
(where X may be G, A or C). RNA was initially heated at
65 �C for 5 min to unfold any secondary RNA structures
and then the temperature was brought down to 37 �C,
followed by addition of 1 ll MMLV reverse transcriptase
(100 U/ll) and incubated at 37 �C for 60 min. After the
first strand cDNA synthesis, the reaction mix was incu-
bated at 75 �C for 5 min to destroy the reverse transcrip-
tase activity and finally held at 4 �C.
Polymerase Chain Reaction
The PCR was carried out for three subsets of cDNA sam-
ples using the combination of the respective anchor oligo
dT primer along with an arbitrary primer (from among
eight) (Table 1). In total, for each sample 24 PCR reactions
were performed. These arbitrary primers were designed to
have Hind III restriction sites at the 50 ends and the rest of
sequences were designed for maximising the coverage of
genes that might be expressed at the given point of time.
From the RT reaction, 2 ll of cDNA was added to 1.6 ll
dNTP (25 lM), 2 ll 109 Taq polymerase buffer, 2 ll
anchor oligo dT primer (2 lM), 2 ll H-AP primer (H-AP1-
8) (2 lM), 0.2 ll [33P] dATP (0.2 mCi) (BARC, Mum-
bai), 0.2 ll Taq polymerase enzyme (5 U/ll) (Qiagen,
Germany) in a final volume of 20 ll DEPC treated water.
The PCR cycling condition involved an initial denaturation
for 2 min at 94 �C followed by 40 cycles of denaturation
for 30 s at 94 �C, annealing at 40 �C for 2 min and
extension at 72 �C for 1 min. The final extension was
carried out for 10 min at 72 �C.
PAGE and Re-amplification of Differential Bands
The amplified products from the DD-RT-PCR reactions
were resolved on a 6 % urea polyacrylamide gel (Sam-
brook et al. 1989) cast on a sequencing gel unit (Sequi-Gen
GT Sequencing Cell, Bio-Rad, USA). The bands were
compared between the control and elicitor treated samples
in the autoradiogram. The differentially displayed bands
were cut out from the gel using the autoradiogram as a
template. The x-ray sheet was aligned with the gel using
the incisions made when exposing the film and was
clamped together using paper clips/cellophane tape. Pin
pricks were made on the edges of bands through the x-ray
film on to the gel sheet. The differential bands that were
marked as pin pricks were carefully excised, soaked in
100 ll of sterile water for 10 min, boiled for 15 min and
cooled. Supernatant containing the DNA fragment was
collected after centrifugation and was stored at -20 �C
until re-amplification.
Re-amplification was carried out in a 40 ll reaction
volume containing 0.5 ll of template, 4 ll 109 PCR
buffer, 3.2 ll dNTP (250 lM), 4 ll of respective anchor
and arbitrary primers (2 lM), 0.4 ll of Taq polymerase
(5 U/ll) and the reaction volume was made up to 40 ll
with water. The PCR cycling conditions were the same as
those during differential display. The products were then
electrophoresed on a 1.8 % agarose gel along with a
100 bp DNA ladder. The gel was visualised under UV
trans illuminator and approximate size of the fragments
was determined. The bands were then carefully excised and
used for further processing by elution, cloning and
sequencing.
Rapid Amplification of cDNA Ends
Rapid amplification of 50 cDNA ends (50-RACE) was
carried out to isolate full length gene sequences from the
partial sequences of transcripts specifically induced during
cell suspension-C. falcatum elicitor interaction. The RNA
ligase mediated-RACE (RLM-RACE) technique was per-
formed using GeneRacerTM kit (Invitrogen, USA). The
method involved designing of two gene specific primers
(i.e. GSP1, GSP2) to carry out 50 RACE. FastPCR primer
designing software was used for primer designing with
parameters like 50–70 % GC content, high annealing
temperature ([72 �C), 23–28 nucleotides length, GC con-
tent at 30 ends and no self-complementary sequences within
the primer or to the primers supplied in the kit. The
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protocol for RACE was followed according to the recom-
mendations of GeneRacer kit (Invitrogen, USA). Total
RNA from 12 h treated sample was used for the RACE-
PCR. The procedure had the following steps calf intestinal
phosphatase (CIP) treatment of RNA samples, RNA pre-
cipitation by phenol: chloroform method, in which the final
pellet was suspended in 8 ll of DEPC water and 1 ll was
run on 1.8 % agarose gel to check the quality, removal m-
RNA cap structure from full length mRNA in the sample
by treatment with tobacco acid phosphatase enzyme (TAP)
and RNA precipitation and isolation by phenol chloroform
method, ligation of RNA oligos and reverse transcription of
mRNA by SuperscriptTM III. The following reaction was
set up for this step in a PCR tube: 1 ll of gene specific
primer 1 (GSP1) primer (10 lM), 1 ll of dNTP mix, 1 ll
DEPC water. The tube was incubated at 65 �C for 10 min
followed by snap cooling on ice for 5 min. This was fol-
lowed by addition of following components to the reaction
mixture; 4 ll 59 first strand synthesis buffer, 1 ll of 0.1 M
DTT, 1 ll of RNaseout (40 U/ll) and 1 ll of Super-
ScriptTM III enzyme (200 U). This reaction mix was
incubated at 55 �C for 1 h followed by 15 min incubation
at 70 �C and finally on ice for 10 min. This was followed
by addition of 1 ll of RNase H (2 U) and incubation at
37 �C for 20 min and finally the cDNA was stored at
-20 �C till being used for PCR reactions.
The PCR reaction mixture contained 35.5 ll sterile
milliQ water, 3 ll of GeneRacer 50 primer, 1 ll of gene
specific primer 1 (GSP1), 0.5 ll of cDNA template, 5 ll
of 109 XT-PCR buffer, 4 ll dNTP (2.5 mM each) and
1 ll (3 U/ll) of XT-5 Taq DNA polymerase enzyme
(Merck, India). The reaction condition followed was
94 �C for 2 min for 1 cycle followed by 94 �C 30 s,
72 �C for 2 min for 5 cycles followed by 94 �C 30 s,
70 �C for 2 min for 5 cycles followed by 94 �C 30 s,
65 �C for 30 s, 72 �C for 2 min for 20 cycles followed by
final extension at 72 �C for 15 min. Nested PCR was
carried out in the same manner replacing GSP1 with
GSP2 in the above reaction using different dilutions of
first round PCR products as template. The gene products
were run on 1.5 % agarose gel and the visualized pro-
ducts were cloned and sequenced.
Results
Establishment of Cell Suspension Culture
The callus was initiated from the inner leaf whorls of the
sugarcane cv Co 93009. After two weeks of callus initia-
tion, the calli were separated from leaf whorl and sub-
cultured on a fresh MS medium. The friable calli were
formed and were transferred to fresh medium for further
growth and proliferation. A suspension cells were raised
from the friable calli in liquid MS medium (Fig. 1).
Cf-elicitor Purification and Dose Response
Glucose equivalents and protein content in the C. falcatum
crude elicitor purified from the fungal mycelial wall were
estimated to be 3.7 and 1.2 lg/ll, respectively. The activity
of the crude elicitor was measured in terms of accumula-
tion of phenolics in the sugarcane suspension cells (cv Co
93009). It was found that 60 lg of glucose equivalents
induced maximum activity in terms of phenol accumula-
tion and it was reduced at elicitor concentrations of more
than 80 lg of glucose equivalents. Similarly elicitor
activity of Cf-elicitor was confirmed on sugarcane leaves
(cv Co 93009) and the study also indicated that 60 lg of
glucose equivalent to maximum activity at level of elicitor
(Fig. 2). Based on these assays, 60 lg of glucose equiva-
lents per 30 ml suspension culture was used in the further
studies for the induction of defense response.
Downstream Processing of Differentially Expressed
Transcripts
About 241 transcripts were found to be differentially
expressed in DD images and from the DD-RT-PCR gel,
about 75 fragments were re-isolated followed by re-
amplification from the gel matrix using the same primer
pairs as in the DD-RT-PCR. These differential transcripts
were reamplified, cloned into pTZ57R/T plasmid vector
and sequenced (Genbank Acc.no. HO 209116-209182)
(Fig. 3, 4). Transcripts of less than 100 bp were not
processed.
Table 1 Sequences of arbitrary
primers used in differential
display
List of arbitrary primers Forward Reverse
H-AP 1 & 2 50-AAGCTTCATTGCC-30 50-AAGCTTCGACTGT-30
H-AP 3 & 4 50-AAGCTTTGGTCAG-30 50-AAGCTTCTCAACG-30
H-AP 5 & 6 50-AAGCTTAGTAGGC-30 50-AAGCTTGCACCAT-30
H-AP 7 & 8 50-AAGCTTAACGAGG-30 50-AAGCTTTTACCGC-30
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Sequence Analysis
Blast in NCBI/SUCEST/TIGR databases revealed many
potential defense-related fragments like putative chitinase,
glycine-rich protein, 14-3-3-like protein, xylanase inhibitor
protein, calmodulin-related protein, Myb-related tran-
scription factor LBM2-like, non-symbiotic haemoglobin
and T-snare like protein to be differentially regulated upon
elicitor treatment (Table 2). Of the 75 differential tran-
scripts isolated, 73 and 27 % were up-regulated and down-
regulated transcripts, respectively. ESTs with matches in
the databases were categorized into six groups, primarily
based on putative functions. Among the known proteins,
the defense protein included the highest percentage (37 %)
of up-regulated sequences, followed by transcription and
post-transcription (13 %), general metabolism (11 %),
transport (9 %), cell structure/growth/division (9 %) and
signal transduction (5 %). Among the down-regulated
transcripts, cell structure/growth/division and transcription/
post transcription group included the highest percentage
(20 % each), followed by transport (15 %), defense
(10 %), general metabolism and signal transduction (5 %
each) (Fig. 5).
Full Length Sequence Isolation of DD-RT-PCR
Transcripts by RACE-PCR
In this technique compatible gene specific primers were
designed at the 30 end using the identified partial sequence
information (Table 3). The full length sequences of 14-3-3
like protein and xylanase inhibitor were successfully
amplified, cloned and sequenced. The *1.1 kb fragment
of 14-3-3 like protein transcript was successfully cloned,
sequenced and has been characterized using bioinformatics
Fig. 2 Dosage optimization
study of Cf-elicitor in terms of
accumulation of phenolics. The
elicitor activity of Cf-elicitor
estimated in terms of
accumulation of phenolic
content (cv Co 93009) using
a suspension cells (12 h) and
b sugarcane leaves (24 h)
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tools (Genbank Acc.no. HO222097). The CDD search in
the NCBI database revealed the presence of conserved 14-
3-3 superfamily domain. The open reading frame in the
cloned full length sequence was identified using the ORF
Finder tool from the NCBI website. The information
revealed the presence of 50-UTR (1–86 bp), ORF (87–
857 bp) and 30-UTR (858–1094 bp) in the full length
sequence of 1,094 bp. The gene product had 256 amino
acids in length and the calculated molecular weight was
found to be 28.8 kDa. A 1.1 kb fragment encoding xy-
lanase inhibitor cDNA was amplified, sequenced (Genbank
Acc. no. HO222096) and had 301 amino acids in the
translated protein sequence. Conserved domain analysis by
CDD search algorithm revealed the presence of active site
flap and inhibitor binding site as part of xylanase inhibitor I
(XIP-I) like protein.
Discussion
The new approach to study interaction between sugarcane
and C. falcatum has allowed identification of many
Fig. 4 Representative gel
indicating reamplified
fragments from differential
display-RT-PCR gels (Lane 1–
10 and12–20 are reamplified
fragments, lane 11:100 bp DNA
ladder)
Fig. 3 Autoradiography image
of differential display pattern of
sugarcane cv Co 93009 (elicitor
treated cell suspension)
amplified using 30. Anchor
primers H-T11 (A/C/G) and
eight Arbitrary primers 13-mer
random primers (HAP-1-8)
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Table 2 Differential transcripts identified from sugarcane suspension cells and Colletotrichum falcatum elicitor interaction by differential
display
No. Clone Sequence homology Organism Accession No. Identity
(%)
Function
1 U1 Putative class III chitinase S. officinarum TA36375 90 PR-protein
2 U2 Class III chitinase S. officinarum TC139607 86 PR-protein
3 U3 Non-symbiotic haemoglobin S. officinarum CO373684 96 Nitric oxide signalling
4 U4 Putative xylanase inhibitor S. officinarum CA294642 98 Inhibition of microbial xylanases
5 U5 Xylanase inhibitor S. officinarum CA134685 96 Inhibition of microbial xylanases
6 U6 Putative class III chitinase Zea mays CA264825 90 PR-protein
7 U7 Putative CTV.22 S. officinarum TA28316 63 Transcription cofactor
8 U8 Eukaryotic initiation factor 4A-8 S. officinarum CA280838 60 Translational initiation factor
9 U9 Myb-related transcription factor LBM2-like S. officinarum TC152417 92 Transcription factor for
defense related genes
10 U10 UDP-glucose pyrophosphorylase S. officinarum TA26045 95 Biosynthesis of polysaccharides
11 U11 Carbonic anhydrase S. officinarum TA31757 70 Involved in photosynthesis
12 U12 NADH ubiquinone oxidoreductase subunit S. officinarum TC116208 95 Electron transport chain
13 U13 Phosphoenolpyruvate carboxylase S. officinarum CA174542 67 CO2 fixation/n metabolism
14 U14 14-3-3-like protein S. officinarum TC143247 98 Signalling
15 U15 14-3-3-like protein S. officinarum TC138037 95 Signalling
16 U16 PHD finger protein-like S. officinarum CA079908 97 Epigenetics and chromatin-
mediated transcriptional regulation
17 U17 Leucine rich repeat family protein S. officinarum CA262024 90 Signalling
18 U18 Phosphatidylinositol-4-phosphate 5-kinase S. officinarum CA274716 60 Signalling and in membrane traffic
19 U19 Calmodulin-related protein S. officinarum CA186211 92 Ca2? signal transduction
20 U20 Proline-rich protein S. officinarum TC138513 59 Structural function
21 U21 MSI type nucleosome/chromatin
assembly factor C
S. officinarum TA31492 97 Nuclear assembly
22 U22 Glycine-rich protein S. officinarum CA279589 60 Structural function
23 U23 Os11g0112300 protein S. officinarum TC134956 98 Unknown
24 U24 OSJNBa0008A08.11 protein Sorghum bicolor TA30115 85 Unknown
25 U25 BAC22218.1 hypothetical protein Oryza sativa NP657646 69 Unknown
26 U26 UniRef100_Q7XDP3.Rep: Expressed protein S. officinarum TC138202 93 Unknown
27 U27 Hypothetical protein Sorghum bicolor XM002436389 93 Unknown
28 U28 Hypothetical protein Sorghum bicolor XM002466510 93 Unknown
29 U29 Putative hydroxyproline-rich glycoprotein S. officinarum TA26639 98 Structural protein
30 U30 Putative actin-depolymerizing factor S. officinarum TA28888 97 Actin dynamics
31 D1 Senescence-associated protein-like S. officinarum CA267854 100 Senescence
32 D2 T-snare S. officinarum TC132900 92 Vesicle-mediated transport
33 D3 Actin-7 S. officinarum CA083832 59 Structural function
34 D4 Chromosome undetermined scaffold_183 S. officinarum TC134078 97 Unknown
35 D5 SWIB/MDM2 domain containing
protein mRNA
Zea mays EU958069 68 Unknown
36 D6 Hypothetical protein P0705A05.114 S. officinarum TA33537 98 Unknown
37 D7 Expressed protein Oryza sativa TC458006 84 Unknown
38 D8 VIP1 protein S. officinarum TC133534 97 Brassicosteroid
signalling pathway
39 D9 Putative serine carboxypeptidase II S. officinarum CF570413 91 Proteolysis
40 D10 Putative EBNA1-binding protein homolog S. officinarum CA281884 98 Pre-rRNA processing
41 D11 Putative polyprotein S. officinarum CA206145 83 Unknown
42 D12 Putative histidine amino acid transporter Sorghum bicolor TA24413 90 Amino acid transport
43 D13 Gamma-tubulin interacting protein-like Oryza sativa TA58812 69 Microtubule assembly
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differentially regulated genes. A significant proportion of
these genes resembled genes described as being involved
directly in plant defense. Other high proportion of genes
showed a similarity to genes encoding LRR receptor-like
proteins, signal transduction protein kinases and tran-
scription factors from different species described to be
related with the signaling mechanism leading to plant
defense against pathogen infections (Table 2). Differences
in gene expression between sugarcane tissues upon infec-
tion with C. falcatum and differences in gene expression
between sugarcane suspension cells upon infection with C.
falcatum elicitor were found to be similar. Previous DD
studies with cane tissue and C. falcatum also revealed
major composition of defense, stress and signal transcripts
among differentially expressed transcripts (Prathima et al.
2013).
Differences in gene expression pattern between tissues
and suspension cells of the same plant can be considered as
a result of both innate differences in plant tissue architec-
ture and composition and the pathogen attack strategy. The
pathogen’s primary strategy can be easily based on down
regulation of those plant genes whose products are partic-
ularly dangerous (acting as a first line of defense) to the
growth and development of conidia, appressorium and
other pathogen structural components to establish a suc-
cessful infection (Casado-Dıaz et al. 2006). In other words,
pathogens must first be able to disable plant defenses to
enable them to grow. Because of differences in structural
components, chemical and biological composition of plant
tissues, this pathogen strategy can proceed quicker and be
successful in some plant tissues rather than in others.
Furthermore, these results suggest that a set of these gene
expression profiles can be valuable as a biotechnological
tool because they open the possibility of exploiting them as
‘molecular signatures’ to identify host resistance to C.
falcatum.
Earlier Ramesh Sundar et al. (2002) reported isolation of
a high molecular weight elicitor from the mycelial walls of
C. falcatum. It was characterized as a glycoprotein and the
activity of elicitor resides in the carbohydrate moiety. The
partially purified elicitor induced phenolics accumulation
and the activities of phenylalanine ammonia-lyase (PAL)
and peroxidase (POX) in both sugarcane leaves and sus-
pension- cultured cells. The suspension cells responded to
the elicitor similar to sugarcane leaves in inducing defence
responses. When Cf-elicitor was compared with C. lind-
emuthianum—a non-pathogen elicitor, differential induc-
tion of POX isoforms in suspension-cultured cells of
sugarcane cv. CoC 671 was found (Ramesh Sundar and
Vidhyasekaran 2003a). For studying the defense gene
activation in sugarcane, the intact plant-pathogen system
may not be ideal because the time course of pathogen
infection along with the concomitant accumulation of host
defense could not be monitored precisely. Cell cultures and
elicitors instead of whole plants and live pathogens
respectively are found to be suitable models to study the
defense gene activation in bean, tobacco, rice and in many
other plants because of their high degree of reproducibility
and rapid experimental cycles (Bradley et al. 1992, Brisson
et al. 1994, Chappell et al. 1997, Cordelier et al. 2003,
Janisch and Schempp 2004, Kieffer et al. 2000, Ndimba
et al, 2003, Yamaguchi et al. 2000). Further, the homog-
enous cell suspension is uniformly exposed to the elicitor
preparation and hence the response of cells is relatively
uniform.
Studies of Ramesh Sundar and Vidhyasekaran (2003a,
b) confirmed that the elicitor molecules from C. falcatum
are responsible for specific recognition of the pathogen by
the host and resistance is determined by the rapidity of the
downward signalling of defense pathway. Probably varia-
tion in initiation of signalling process between the resistant
and susceptible genotype determines the pathogen coloni-
zation and disease development in host resistance. The
present study is in continuation of this study to further
explore the molecular interaction of the pathogen elicitor
with sugarcane. The genetic differences in terms of resis-
tance among different varieties could be due to variations
in the synthesis, transport and breakdown of PR-proteins,
other metabolites, reception or signal transduction of hor-
mones (Viswanathan et al. 1996, 2005). At this juncture
concerted efforts are required to isolate and characterize
such specific genes for disease resistance/pathogen recog-
nition in cultivated sugarcane varieties/germplasm. This is
first of its kind in a host pathogen relationship involving
sugarcane and a fungal pathogen. However this approach
was followed in many other plant systems like rice,
tobacco, tomato, French bean, Arabidopsis, soybean etc.
(Wasternack, and Kombrink 2010).
Table 2 continued
No. Clone Sequence homology Organism Accession No. Identity
(%)
Function
44 D14 Auxin efflux carrier protein-like Sorghum bicolor TA28620 88 Auxin specific membrane transport
45 D15 Putative secretory carrier membrane protein Sorghum bicolor TA28044 95 Membrane trafficking
46 D16 Putative ubiquinone oxidoreductase S. officinarum CA271223 97 Electron transport chain
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The use of suspension cells for the study instead of field
samples has eliminated the possibility of other environ-
mental factors to influence the results. The controlled con-
dition has provided a suitable platform for understanding the
recognition of the elicitor moiety by the host system. How-
ever, only few differentially expressed transcripts were
identified when 50 primers targeting defense related and
signaling transcripts through genome scanning (Viswana-
than, Unpublished). In a similar work carried out using same
set of primers in the cane tissues showed differential
expression for six transcripts. These included chitinase,
metallothionein, resistance protein R30, receptor protein
kinase, reversibly glycosylated protein and signal sequence
hydrophobic region (Viswanathan et al. 2009). This neces-
sitated the use of a technique that could inclusively identify
all the possible transcripts that are differential in a given
treatment/tissue. The DD-RT-PCR is an ideal technique
satisfying this criterion. So far there are no reports on works
carried out in sugarcane in this regard and suspension culture
was not used to study transcriptomics in sugarcane. This
makes the data from DD-RT-PCR all the more important, as
it is able to reveal large number of transcripts as differential
Fig. 5 Relative abundance of
differential display transcripts
categorized based on putative
functions (cell suspension)
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in the autoradiograph in contrast to the selected genes used in
the semi quantitative RT-PCR study (Data not shown).
The study of resistance response of any host plant
against a pathogen infection requires understanding the
complex changes in gene expression patterns. This is
mediated by the recognition of specific moiety (elicitor/
avirulence factors) derived from pathogen. Cell surfaces
are believed to play a key role in early interactions between
plants and microorganisms. This was the reason for
choosing the cell suspension as the material that can be
treated with elicitor from C. falcatum. Ayers et al. (1976)
applied this hypothesis who reported that fungal cell walls
contain active fragments that elicit defense responses in
plants. Many elicitors have been isolated since then from
fungi. Fungal elicitors may originate from the cell wall or
may be secreted into the extra cellular space of the
mycelium. Cell wall elicitors are best demonstrated by the
b-1,3-glucans from Phytophthora sojae that are released
from the mycelial cell wall (Hahlbrock et al. 1995) and a
34 kDa protein elicitor from Phytophthora nicotianae
(Delmas et al. 1997). Secreted molecules have also been
purified from few fungi (Joosten et al. 1994; Kamoun et al.
1998). Among them, the peptide elicitors of Cladosporium
fulvum and Rynchosporium secalis and elicitins of Phy-
tophthora spp. are particularly interesting, because they
have been shown to be the products of avirulence genes
(Joosten et al. 1994; Kamoun et al. 1998). Knowledge of
these peptides and the corresponding genes, notably the
avr4 and avr9 genes of C. fulvum, and the INF1 of P.
infestans have greatly contributed to the understanding of
race-cultivar specificity at the molecular level. Several
protein elicitors were isolated from Magnaporthe grisea,
Alternaria spp., Rhizoctonia solani, Aspergillus spp,
Botrytis spp, Penicillium spp. and Trichoderma spp (Qiu,
2004, 2005).
The timing and activation of the defense response after
pathogen recognition is the most important factor for the
success of host resistance. After the pathogen recognition
has been addressed by the host system, downstream pro-
cess of signalling and cascade of events is unleashed. This
may take effect in different forms. For example, glyco-
protein elicitor CSBI from hyphal cell walls of M. grisea
induced defense related enzymes in rice (Li et al. 2004).
These glycoproteins activated plant defense system and
promoted plant disease resistance, representing a potential
tool to engineer disease resistance against broad spectrum
of pathogens. In order to target the genes involved in these
downstream events of host system, primers were designed
from such defense pathways. Interestingly they were the
same transcripts that were identified as differentially
expressed in both the studies (Fig. 5) This means that a
common mode of action is predominantly active in both the
systems i.e. cane tissue vs pathogen and cell suspension vs
elicitor. The results clearly indicated that defense related
transcripts were the major group that was up-regulated
considerably in response to elicitor treatment in suspension
culture in contrast to cane tissue. This may be due to better
accessibility of the cells in suspension to elicitor, leading to
a rapid response with lesser time needed for signal
transduction.
Earlier (Borras-Hidalgo et al. 2005) applied amplified
fragment length polymorphism (AFLP) analyses of cDNA
to identify differentially expressed genes in disease resis-
tant but not in susceptible somaclones of sugarcane in
response to inoculation with either Sporosorium scitami-
neum or Bipolaris sacchari causing smut and eye spot,
respectively. They identified 62 differentially regulated
genes, of which 52 were induced and 10 were down reg-
ulated and most of the induced transcripts were related to
defense or signaling. Similarly the same approach has been
used to identify genes from sugarcane somaclones
expressed during the interaction with another fungal path-
ogen Puccinia melanocepahala causing brown rust (Car-
mona et al. 2004). This study also revealed expression of
transcripts mostly of defense, disease resistance, cell rescue
and signal transduction. Oberschmidta et al. (2003) used
DD technique to study a F2 population of sugar beet that
segregated for Heterodera schachtii resistance. A novel
cDNA Bp23 was isolated by comparing resistant and sus-
ceptible sugar beets in this study.
Table 3 - List of gene specific primers used for 50-RACE-PCR
Sl. no Gene name Primer name Sequence (50–30) Size
(bp)
GC (%) Tm (�C)
1 Xylanase Inhibitor GSP1 GCCTCACTTTATTCTTGCACACGCAC 26 50 78
2 GSP2 CATTGAATGGCGTAGCTGCTGTAGTTG 27 48 80
3 14-3-3 like protein GSP1 AGAGGCTCAGCTCGTTAAACCGC 23 57 72
4 GSP2 GCTAGAGCGACCACAGACATGC 22 59 70
5 GSP3 AGTTTCAGGATGCCATCACAGATGTTGC 28 46 82
6 GeneRacer 50 primer – CGACTGGAGCACGAGGACACTGA 23 61 74
7 GeneRacer 50 nested primer – GGACACTGACATGGACTGAAGGAGTA 26 50 78
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Among the 21 screened transcripts by reverse northern
blot seven transcripts viz., 14-3-3-like protein, xylanase
inhibitor protein 1 precursor, chitinase, leucine rich repeat
family protein, basal layer antifungal peptide, actin depo-
lymerizing factor, putative hydroxyproline-rich glycopro-
tein were identified as differential transcripts. Subsequently
full length sequences of 14-3-3 like protein and xylanase
inhibitor were sequenced and characterized using various
bioinformatics tools. Chitinase gene was characterized as
class IV of family 19 glycosyl hydrolases based on 3D
structure prediction through transcriptomic and bioinfor-
matics tools (Rahul et al. 2013). Further characterization of
these genes functions in relation to disease resistance
would serve as potential candidate genes to understand red
rot resistance in sugarcane. The study on sugarcane—C.
falcatum interaction using DD-RT-PCR represents the first
such attempt to understand the activation of sugarcane
genes by using crude elicitor in suspension cell culture.
The identification of transcripts expressed during elicitor
treatment of suspension cell using DD-RT-PCR has given a
better understanding of the interaction at molecular level.
The study has been able to throw light on the different
categories of transcripts that were activated during the
sugarcane—C. falcatum interaction. The full length char-
acterization of DD derived transcripts will help in their
application in sugarcane transgenic and further character-
ization in terms of their exact role during pathogenesis. To
conclude, the DD-RT-PCR technique has been successfully
used to identify 75 potential candidate genes induced in
sugarcane suspension culture upon elicitor treatment. The
differential expression was confirmed by the reverse
northern study and the full length sequence of the potential
differential transcripts were identified by RACE technique.
Acknowledgments The authors are grateful for Dr. N.V. Nair for
the encouragement and providing facilities. The work was supported
by Department of Biotechnology, New Delhi (BT/PR4970/NDB/51/
051/2004).
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