cDNAcDNA- ---AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT...
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Chapter IIChapter IIChapter IIChapter II
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MOLECULAR MARKERS RELATED TO MOLECULAR MARKERS RELATED TO MOLECULAR MARKERS RELATED TO MOLECULAR MARKERS RELATED TO
BLISTER BLIGHT TOLERANCE IN BLISTER BLIGHT TOLERANCE IN BLISTER BLIGHT TOLERANCE IN BLISTER BLIGHT TOLERANCE IN TEATEATEATEA
2.1. INTRODUCTION
Molecular data from the investigation of compatible/ incompatible
plant-pathogen interactions in tea genotypes is scarce and no reports related
to transcriptome studies for such interactions are available as yet. There has
been no large-scale molecular analysis for blister blight disease so far and
the pathogen genomic information is also limited. Understanding the
molecular basis of tolerance and susceptibility would greatly assist in the
development of new control strategies and the identification of pathogen and
host factors required for disease progression. One useful approach for the
molecular analysis of plant-pathogen interactions is the determination of
changes in the host transcriptome during infection. In the present study,
cDNA-AFLP based transcriptional profiling technology has been employed
for screening and identification of transcripts expressed upon blister blight
infection in tea, in order to identify gene-derived markers potentially
associated with resistance or tolerance to the disease. In the era of PCR
based marker techniques, cDNA-AFLP is a useful method, not only for
transcript profiling but also for the development and identification of
molecular markers associated with important traits in plants. More recently,
cDNA instead of genomic DNA has been utilized for the development of
AFLP markers to amplify fragments from the selected (coding) regions of the
genome (Gupta and Rustgi, 2004). Using cDNA as template, gene rich parts
of the genome can be targeted for marker development or for subsequent
direct marker generation. Availability of markers for resistance genes will help
in identifying plants carrying these genes at an early stage and without
subjecting them to pathogen attack, thus facilitating marker assisted selection
(MAS). Although this approach has not been commonly utilized for
identification of molecular markers, it has great potential towards MAS in
breeding programmes. The results generated in the study will contribute to
further research towards utilization of cDNA-AFLP technology for large scale
transcriptome studies as well as development of molecular markers for
resistance/ tolerance to blister blight and other diseases of the tea plant.
2.2. MATERIALS AND METHODS
2.2.1. Plant materials
For the present study, two groups of plant materials or cultivars were
taken into consideration based on their resistance/tolerance and
susceptibility characteristics to blister blight disease. These cultivars were
released by Tea Research Association (TRA) for Darjeeling gardens and are
being maintained at the Clonal Proving Station (CPS) of Ging Tea Estate,
Darjeeling. The cultivars have been named after the gardens for which they
were released. The different cultivars used in the study and their salient
features are as follows:
Phoobsering-1258 (P-1258): A standard China hybrid clone having compact
and spreading frame. Leaves are semi-erect, light yellowish green of medium
size, shoots of medium weight, with average flavour and briskness. It is
mildly susceptible to red spider and other mites but has good resistance to
blister blight and drought.
Phoobsering-312 (P-312): A China hybrid clone with medium size leaves,
semi-erect, dark green and matte foliage having pronounced serrations. It
flowers profusely and has a compact frame, wide spread and medium weight
shoot. It is fairly tolerant to blister blight.
Tukhdah-78 (T-78): A very vigorous China hybrid cultivar with erect leaves
of dark green colour and a fairly good spreader with a lax frame. It has a very
good flavour. It has good resistance to drought but is susceptible to blister
blight and red spider mite.
Banoph-157 (B-157): A medium size, dark green, glossy leafed China hybrid
clone with a medium sized frame having many trailing lower branches. It has
a very good flavour. It is fairly resistant to red spider mite and drought but
highly susceptible to blister blight.
Runglee Rungliot-17/144 (RR-17/144): A China hybrid clone with semi-
erect, dark green and medium size leaves, shoots having high pubescence
with a highly spreading compact frame. It is susceptible to blister blight and
red spider mite but has good drought resistance.
The cultivars were divided into two groups, the first group consisting of
blister blight resistant/tolerant cultivars viz. P-1258 and P-312, and the
second group consisting of the susceptible cultivars T-78, B-157 and RR-
17/144.
2.2.2. Pathogen infection: Blister blight experiment
Pathogen infection was carried out at the CPS, Ging Tea Estate, at
plot no. CPS-A3. The experiment for artificial inoculation of the selected plant
materials with the pathogen E. vexans was set up in the 1st week of August,
2008, during which the atmospheric conditions in Darjeeling were very much
favourable for blister blight incidence. The atmospheric conditions of
temperature, relative humidity (RH), rainfall, and sunshine hours were found
to be quite conducive for pathogen inoculation (Table 2.1). Three year old
plantlets of each cultivar which were grown and maintained in polyethylene
sleeves of 10 cm diameter and 15 cm height, in glasshouse under natural
conditions of daylight (11 hours), temperature (26°C±2) and RH (65-85%),
were chosen for the experiment. To validate the results of the pathogen
inoculation experiment, the same was repeated for all the selected plant
materials in the following year (1st week of August, 2009) under similar
conditions.
For inoculation, basidiospores were collected from sporulating blister
lesions from naturally infected plants at the CPS. In view of the fact that the
strains of E. vexans infecting the different Darjeeling tea cultivars have not
been identified and characterized yet, the spores collected only from
respective cultivars in the field were used for artificial infection of the
plantlets. For example, for inoculating the T-78 plantlets basidiospores
collected only from T-78 plants in the field were utilized. The morphology of
the fungal spores collected for inoculation was studied under a microscope.
Spore suspensions were prepared in sterile distilled water containing 106
spores ml-1 (Baby et al., 2004), separately for each cultivar from
basidiospores collected 6 hrs after sporulation in the field. The leaves were
surface sterilized by wiping with 90% ethanol and the spore suspension was
dropped on to the 1st, 2nd and 3rd leaves of the plant materials with the help of
a micropipette. Inoculation was performed early in the morning (during 6:00
AM to 7:00 AM) when the stomata are most likely to remain open (Squire,
1978). Control plants were inoculated with sterile water. The plantlets were
maintained at 100% RH by covering with polyethylene-bags for 72 hours in
shade (avoiding direct sunlight) (Figure 2.1) after which they were transferred
to glasshouse. The inoculation experiment was carried out in 5 replicates for
each of the cultivars under study. The incidence and symptoms of infection
were monitored daily and observations were made for development of
blisters. Lesions resembling different stages of symptoms that occurred in the
field-grown plants were observed on the infected leaves within 4 weeks of
inoculation. Pathogenicity of the fungal isolates used for the infection
experiment was assessed by following the Koch’s postulates and examining
the morphology of the basidiospores. The fungus was recovered from the
diseased tissues excised from all the inoculated materials, satisfying Koch's
postulates. For the microscopic study, sections from at least two different
areas of each leaf were examined for the presence of E. vexans spores and
hyphae. Collection of leaf materials was done at the oil spot stage of
infection, after 10 days of inoculation. The collected materials were
immediately immersed in liquid nitrogen. These were stored at -80 °C until
extraction of total RNA was done. Uninfected samples from control plantlets
were also collected for further use.
Disease severity percentage in the different infected cultivars was
assessed on the 10th day after inoculation. For determining disease severity,
healthy leaf area and the area covered by diseased lesions were calculated
according to the method by Debnath and Paul (2005). The leaf area was
measured by using a transparent square paper. Sizes of the lesions were
assessed by taking the diameter of the blisters. The total number of lesions
formed in each cultivar was also recorded. Disease severity was calculated
according to the following formula (Debnath and Paul, 1994):
Disease severity (%) = (Area of diseased lesion/ Total leaf area) X 100
Atmospheric Parameters (Mean) August, 2008 August, 2009
Maximum Temperature 29 ± 2°C 29.5 ± 2°C
Relative Humidity 85% 89%
Rainfall 345 mm 347 mm
Sunshine Hours 3 hours 3 hours
Table 2.1. Atmospheric parameters recorded at CPS, Ging T.E., Darjeeling
during the period when inoculation experiments were conducted.
Figure 2.1. Experimental set-up for infection of tea plantlets with E. vexans.
After inoculation, the plantlets were maintained at 100% RH by covering with
polybags for 72 hours in shade.
2.2.3. Isolation of RNA from leaf tissues
Total RNA was extracted from 1gm of leaf tissue (first two leaves and
the bud were chosen for extraction) following Guanidine-HCL extraction
method (Logemann et al., 1987) with modifications. Plant materials were
finely ground in liquid nitrogen and resuspended in 8M RNA extraction buffer
containing β-mercaptoethanol (1.4% (v/v) of total extraction volume).
Following three phenol-chloroform-isoamylalcohol (25:24:1) extractions, RNA
was precipitated with 1 volume of 2M LiCl2 solution and washed with 70%
ethanol. The RNA pellet was air dried and resuspended in RNA storage
buffer. All the centrifugation steps were carried out at 14500 rpm at 4 °C. The
glass and plastic wares used in RNA extraction were pre-treated with, and all
the solutions and buffer used were prepared in DEPC (Diethylpyrocarbonate)
treated sterile distilled water.
Total RNA samples were analyzed for quality and quantity by running
a small aliquot on a 1% (w/v) agarose gel stained with ethidium bromide and
visualized under UV light. Concentration of the RNA samples was
determined by reading absorbance at 260nm in a spectrophotometer
followed by isolation of polyA+ mRNA using PolyA-Tract mRNA Isolation Kit
(Promega, Madison, USA) following manufacturer’s instructions.
2.2.4. cDNA synthesis
First and second strand cDNA synthesis was done from 0.2µg of
mRNA using SMART full length cDNA library construction kit (Clontech,
TAKARA Bio, Japan) according to the instructions provided by the supplier.
The resulting double stranded cDNA (ds cDNA) was purified by proteinase K
digestion. Samples were finally dissolved in 30µl of nuclease free water.
Concentrations were determined using a spectrophotometer and the samples
were stored in -20°C until used.
2.2.5. cDNA-AFLP analysis
cDNA-AFLP analysis was carried out as described by Bachem et al.
(1996) with some modifications.
a) Restriction enzyme digestion and adapter ligation
Restriction enzyme digestion and adapter ligation were carried out in
the same reaction for 600ng of ds cDNA. For restriction digestion, two
restriction enzymes, BstYI and MseI (New England Biolabs, Beverly, Mass)
were used and for ligation, adapters for the two restriction enzymes were
used. The reaction mix (10U BstYI, 10U MseI, 10X Ligase buffer, 5U/µl T4
DNA Ligase, 10X BSA, 0.5 M NaCl and sterile distilled water in a total
volume of 20 µl) was incubated at 37 °C overnight. The product was
incubated at 65 °C for 20 minutes (min) to inactivate the enzymes and
subsequently diluted in sterile distilled water in 1:1 ratio.
b) Pre-amplification
Pre-amplification was carried out using Bst (5'-GAC TGC GTA GTG
ATC-3') and Mse (5'-GAT GAG TCC TGA GTA A-3') primers complementary
to the adapter ligated sites. The Mse primer carried no selective nucleotides
but the Bst primer had a selective nucleotide ‘C’ at the 3’ end. 5µl of the
restriction digested/ligated product was used for the reaction (10X PCR
buffer, 10mM dNTPs, 50mM MgCl2, 5U/µl Taq DNA polymerase, 20 µM of
each primer and sterile water in a total volume of 20µl). Cycling conditions
consisted of 25 cycles at 94 °C for 30 seconds (s), 56 °C for 1 min and 72 °C
for 1 min. Successful completion of pre-amplification was determined by gel
electrophoresis of the amplified products on a 1% (w/v) agarose gel and
visualized under UV light. Samples were then diluted in 1:50 ratio in sterile
distilled water.
c) Selective amplification and polyacrylamide gel electrophoresis
Selective amplification was carried out using Bst and Mse primers with
2 or 3 additional selective nucleotides. The Mse primer was end labeled with
32P for visualization of bands through autoradiography. The selective
amplification reaction mixture contained 10X PCR buffer, 2.5mM of each
dNTP, 50mM MgCl2, 20µM of each labelled Mse primer and unlabelled Bst
primer, 5U/µl Taq DNA polymerase, sterile distilled water and 5µl of diluted
pre-amplification product. Amplification was carried out following the cycling
profile: 1 hold at 94 °C followed by 10 cycles of 30 s at 94 °C, 30 s at
65 °C and 1 min at 72 °C; followed by 23 touch-down cycles at 0.7 °C/cycle
(30 s at 94 °C, 30 s at 56 °C and 1 min at 72 °C). Upon completion of
amplification, reaction products were mixed with an equal volume of
formamide loading dye (95 % deionized formamide, 20mM EDTA and
0.8mg/ml bromophenol blue). The mixture was denatured for 5 min at 94 °C
and then immediately chilled on ice for at least 10 min. 2µl of each sample
was loaded on a 6% polyacrylamide gel and electrophoresed on a Sequi-Gen
GT Sequencing Cell (Bio-Rad Laboratories, Hercules). Electrophoresis was
carried out for 3 hours at 1200 W. After completion of electrophoresis, the gel
was transferred to a 3mm Whatman paper and dried in a Bio-Rad gel dryer at
80 °C for 1 hour. For visualization of bands, the dried gel was subjected to
autoradiography by exposing to an X-Ray film for 15 hours.
2.2.6. Isolation, sequencing and analysis of transcript derived
fragments (TDFs)
Polymorphic transcript derived fragments (TDFs) exhibiting a
differential banding pattern between the tolerant and susceptible groups of
plant materials were identified and eluted using the GenElute Gel Extraction
Kit (Sigma-Aldrich, Switzerland). The isolated fragments were reamplified
using the PCR profile and conditions as followed for selective amplification.
These were then cloned into pGEM-T easy vector (Promega, Madison, USA)
and transformed into DH10β chemical competent cells. The transformed
colonies were selected through blue-white screening on LB (Luria broth) -
agar plates containing Ampicillin (50mg/ml) selection. Plasmid isolation was
done from the transformed white colonies through alkaline lysis method
(Sambrook et al., 2000). Subsequently, sequencing was performed by using
the BigDye Terminator v3.1 Cycle Sequencing Kit in the 3130xl Genetic
Analyzer (Applied Biosystems, California, USA). The TDF sequences were
compared against all available sequences in the non-redundant (nr)
databases using the blastx and blastn algorithms of NCBI (National Center
for Biotechnology Information). These were then classified into different
functional categories based on their putative functions and the different
biochemical processes they are associated with. For validation of expression
of the TDFs through qRT-PCR analysis, primers were designed on the
sequences using the online tool Primer3 (http://frodo.wi.mit.edu/cgi-
bin/primer3/) (Appendix A).
2.2.7. Confirmation of differential gene expression by qRT-PCR
Quantitative Real Time-PCR (qRT-PCR) reactions were carried out on
total RNA derived from two independent biological experiments. Each sample
was a pool of identical quantities of RNA from the two experiments. All
samples were examined in three technical replicates. First-strand cDNA was
synthesized from DNase-treated total RNA using Transcriptor First Strand
cDNA Synthesis Kit (Roche, Mannheim, Germany). Specific primer pairs
were designed on seven TDFs (Appendix A), and tested and standardized by
semi-quantitative RT-PCR. Primers specific for the C. sinensis 18S and 26S
rRNA housekeeping genes were used as reference genes for the
normalization of qRT-PCR reactions. Experiments were carried out using the
LightCycler 480 SYBR Green I Master kit (Roche) on a LightCycler 480 II
System (Roche) according to the manufacturer’s instructions. The optimized
conditions and the PCR parameters followed for qRT-PCR reactions are
given in Chapter IV. Data were analyzed using the software GeNorm v3.5
(http://medgen.ugent.be/genorm/) and the normalization factor obtained for
each of the treated sample was used to normalize the expression of the
TDFs. The normalized expression values were represented by hierarchical
clustering using the software GenePattern (http://genepattern.broadinstitute.
org/gp/pages/index.jsf).
2.2.8. Statistical analysis
For disease severity, mean lesion diameter, average no. of lesions per
leaf and quantified gene expression normalized values by qRT-PCR, the
results are presented as mean values for three individual samples with
standard errors. Statistical analyses were performed using Student’s t-test
and Principal Component Analysis (PCA) functions of the XLStat-Pro 7.5
(Addinsoft, New York, USA) software. A Pearson correlation coefficient was
used to perform PCA analysis. The correlation matrix between the variables
and the eigenvalues of the factors are presented in Appendix E (a) and (b).
2.3. RESULTS
2.3.1. Morphological studies and collection of infected materials
Observations on development of blisters were made for upto 28 days
after inoculation. The infected plants produced the same symptoms as those
found on naturally infected plants on the field (Figure 2.2). Appearance of the
symptoms started with the occurrence of pin-hole size spots on the leaves 3
to 5 days after inoculation. On the susceptible cultivars (T-78, B-157 and RR-
17/144), the spots were found to gradually increase in size to become oil-
spot like lesions to finally formation of pale pinkish-white blisters on the lower
leaf surface within 7 to 15 days. The blisters started to sporulate after 15 to
18 days of infection. Sporulation was observed till 20 to 25 days after
inoculation, after which, the blisters gradually turned brown and finally
necrotic with the death of the tissues at the infected regions. In case of the
tolerant cultivars, the infected regions gradually became necrotic and the
surrounding tissues were distorted. In the cultivar P-312, very little infection
was seen with the formation of small blisters. In P-1258, which is known to be
more tolerant than P-312, infection was rather restricted and no blister
formation was observed but the spots gradually turned necrotic within 7-10
days.
All tea leaves are not equally prone to attack by E. vexans but the
sprouting buds and young leaves are most susceptible (Gadd and Loos,
1948). In our experiment also, infection was observed on the inoculated
younger leaves but not on the mature leaves. For recording the size and
numbers of lesions formed and estimation of disease severity, the third leaf
of the plant materials was selected as it has been found to carry relatively
more number of blister lesions and is the index for blister blight assessment
(Satyanarayana et al., 1978). Observations were made on blister
Figure 2.2. Leaves of the different tea cultivars, A) B-157; B) T-78; C) RR-
17/144; D) P-1258; and E) P-312, artificially inoculated with E. vexans, after
12-15 days of infection.
development on the different plant materials after 12 days post-inoculation
and disease severity was calculated (Table 2.2). Under favourable
environmental conditions, the lesions grew faster in the susceptible cultivars
while they were restricted in the tolerant ones. It is evident from Table 2.2
that, P-1258 is the most tolerant cultivar to blister blight infection while RR-
17/144 is the most susceptible one. Disease severity has been found to be
the highest in the cultivar RR-17/144, the mean percentage being 6.2%,
while in P-1258 it has been estimated to be the least of 0.78%. The mean
lesion diameter and distribution in P-1258 were recorded to be 2.5 mm, and 0
to 2 lesions per leaf respectively. On the other hand RR-17/144, which has
been found to be the most susceptible in the lot, recorded a mean lesion size
of 9.24 mm with more than 10 lesions per leaf.
Collection of the materials was done after 10 days of infection when
the blister spots enlarged to become oil-spot like lesions, 3-12 mm in
diameter, in the susceptible cultivars. In the tolerant cultivars, however, the
spots became necrotic with twisting of the tissues at the site of infection after
10-12 days of inoculation. The oil-spot stage of infection was chosen
because the compatible interaction is well established and the mycelia
produced at this stage are abundant enough to allow the detection of
pathogen transcripts, even though the plant cell is still active, since various
plant functions are needed to maintain pathogen survival (Polesani et al.,
2008).
2.3.2. cDNA-AFLP analysis
In the present study, cDNA-AFLP analysis was carried out for tea
cultivars artificially infected with E. vexans to identify genes differentially
expressed in the tolerant cultivars which are potentially involved in blister
blight tolerance, and elucidate the defence responses during an apparently
incompatible interaction between the tea plant and the pathogen. For
selective amplification, Mse and Bst primers carrying 2 or 3 additional
selective nucleotides were used. With these primers, we got a total of 36
possible primer combinations (Table 2.3) out of which, 15 gave a differential
Tea Cultivars Mean Disease Severity (%)
Mean Lesion Diameter (mm) ± S.D.
No. of lesions per leaf
P-1258 0.78 2.50 ± 0.2 0-2
P-312 0.98 3.36 ± 0.42 2-4
T-78 3.40 8.80 ± 0.55 6-10
B-157 4.65 8.78 ± 0.25 8-10
RR-17/144 6.20 9.24 ± 0.75 10-12
Table 2.2. Observations made on development of blisters on the different tea
cultivars artificially infected with E. vexans, after 12 days of inoculation.
BstYI +
CA
BstYI +
CG
BstYI +
CT
BstYI +
CAA
BstYI +
CAG
BstYI +
CAT
MseI +
A A X CA A X CG A X CT A X CAA A X CAG A X CAT
MseI +
T T X CA T X CG T X CT T X CAA T X CAG T X CAT
MseI +
AA AA X CA AA X CG AA X CT AA X CAA AA X CAG AA X CAT
MseI +
AT AT X CA AT X CG AT X CT AT X CAA AT X CAG AT X CAT
MseI +
TA TA X CA TA X CG TA X CT TA X CAA TA X CAG TA X CAT
MseI +
TT TT X CA TT X CG TT X CT TT X CAA TT X CAG TT X CAT
Table 2.3. Primer combinations used for cDNA-AFLP analysis. Of the 36
primer combinations tried, 15 (highlighted in yellow) showed a differential
polymorphic amplification pattern, and were selected for further analysis.
polymorphic amplification pattern. These 15 primer combinations (which have
been highlighted in Table 2.3) were selected for isolation of differentially
expressed TDFs. cDNA-AFLP analysis was carried out on ds cDNA samples
of infected materials at the oil spot stage of E. vexans infection, as well as
the control materials. Approximately, 20 to 50 TDFs were visualized as bands
in each sample, ranging from 50 to more than 1000 bp in size, depending
upon the primer combination. When primers with 2 selective nucleotides
were used for amplification, a large number of bands were seen on the gels
but, when primers with 3 selective nucleotides were used, the band number
was reduced to a great extent, thus, enabling easier differential analysis,
scoring, as well as detection and elution. The banding patterns of the cDNA-
AFLP gels were reproducible, which were confirmed with four independent
replicates. Figure 2.3 represent cDNA-AFLP gels from four different primer
combinations showing differential amplification patterns among the cultivars
used in the study.
From the cDNA-AFLP gels it was observed that, most of the TDFs
induced in inoculated tolerant genotypes were also induced in inoculated
susceptible genotypes, showing that modulations in gene expression overlap
to a great extent between host-pathogen interactions occurring in a tolerant
and a susceptible genotype. Interestingly, a higher number of TDFs were
found to be induced in an interaction with a susceptible genotype compared
to that with a tolerant genotype thereby pertaining to the fact that a significant
amount of response reactions occur in the susceptible genotypes, while
experiencing pathogen challenge. On an average, about 65% of the total
number of TDFs amplified was found to be constitutively expressed in both
sets of the cultivars post-inoculation. Seeing that the blister blight pathogen is
a biotroph, the transcriptional changes taking place in a susceptible genotype
could be the result of an integration of events that were required for
suppression of cell death while allowing growth of the fungus, along with the
expression of genes related to general defence responses. However, as the
experiment was conducted at a single time-point, we could not infer whether
the resistance responses occurred early or late in the genotypes. Since, we
Figure 2.3. cDNA-AFLP gels for the primer combinations: A) AA x CAA; B)
AA x CAG; C) AA x CAT; and D) AT x CAG, showing differential banding
pattern among the cultivars. Arrows are indicating bands recovered from the
gels.
A) MseI-AA X BstYI-CAA
B157i RR17/144i T78c T78i P312c P312i P1258c P1258i
B) MseI-AA X BstYI-CAG
B157i RR17/144i T78c T78i P312c P312i P1258c P1258i
C) MseI-AA X BstYI-CAT
B157i RR17/144i T78c T78i P312c P312i P1258c P1258i
D) MseI-AT X BstYI-CAG
B157i RR17/144i T78c T78i P312c P312i P1258c P1258i
were more concerned with the resistance responses occurring in the tolerant
genotypes, we concentrated our studies on the transcriptomic changes taking
place in the plant materials tolerant to E. vexans during the host-fungus
interaction.
2.3.3. TDF isolation, sequencing and characterization
Through cDNA-AFLP, we could screen for candidate transcripts which
are differentially expressed between tolerant and susceptible genotypes
under blister blight disease stress. We identified and isolated several TDFs
that were differentially expressed in the tolerant genotypes as compared to
the susceptible ones. A total of 287 numbers of bands presented a
differential banding pattern between the tolerant and susceptible groups of
cultivars. Out of these, 176 no. of bands were present in either P-1258 or P-
312, or both, post-infection, but were absent from or markedly under-
expressed in the susceptible cultivars. As these bands or TDFs were
supposedly expressed or upregulated as a result of blister blight infection in
the tolerant cultivars but were unexpressed in the susceptible ones, we
hypothesized that these might have a possible role in conferring resistance
responses to the disease. So, these TDFs were selected for further analysis,
of which we could recover a total of 162 bands from the dried gels. The
eluted TDFs were reamplified, cloned and sequenced. A number of isolated
TDFs had a sequence length of less than 100 bp, and only 104 produced
reliable sequences ranging from 100 bp to more than 700 bp in length. All the
TDF sequences have been submitted to the NCBI GenBank dbEST
(accession numbers: JK263397 to JK263500) (Appendix B). Vector
sequences were trimmed off and sequences smaller than 100 bp were
eliminated. Each sequence was then identified by similarity search using the
basic local alignment search tool (blast) program against the non-redundant
(nr) public sequence databases. Appendix B presents a list of the blastx
results obtained after homology search.
Sequence comparison of the TDF sequences against the nr database
revealed that majority of them (approximately 30.7%) presented no hits or ‘no
significant similarity’ to the proteins or genes available in the database.
These TDFs could be considered novel ESTs. The remaining TDFs could be
assigned putative functions as they were found to be significantly similar to
the proteins/genes in the database. These were then classified into different
categories based on their putative functions and the biological processes
they are associated with, through Gene Ontology (GO) annotation. The
functions of the identified transcripts as reported in other plant species was
also taken into consideration while classifying them into functional categories.
A considerable number of the transcripts (8.65%) obtained from the analysis
were found to be homologous to predicted/hypothetical proteins and were
classified as ‘unknown’. As shown in Figure 2.4, a large group of the
transcripts (24%) was found to be associated with metabolism. The genes
involved in signal transduction constituted the smallest group, comprising 2%
of the TDFs. Genes involved in transport constituted 6.73% of the TDFs while
5.7% of the genes were found to be associated with photosynthesis and
energy. The transcripts implicated in nuclear organization, transcriptional
regulation and cell structure together represented 9.6% of the sequences.
From this study, we have identified that approximately 6.8% of the TDFs are
directly related to defence or biotic stress response. Some of these have
shown homology with proteins like serine/threonine protein kinase (STK), PR
protein chitinase and lipid-associated proteins. Others are associated with
hypersensitive response like, superoxide dismutase and hydrogen peroxide
induced proteins, which are responsible for activation of various defence
signalling pathways. A few transcripts which are not directly induced in
response to pathogen attack but are indirectly involved in pathways
responsible for conferring stress resistance, have also been obtained from
the analysis. These include proteins like hydroxyproline-rich glycoprotein,
proline-rich protein, 12-oxo-phytodienoic acid reductase (OPR), gibberellin-
20-oxidase, lipoxygenase, acyl-CoA-binding protein and calcium ion binding
protein. Nearly 5.7% of the transcripts were found to be associated with
response to abiotic stress. Thus, a considerable overlap between biotic and
abiotic stress responses could be observed from the study.
Figure 2.4. Functional categorization of the TDFs on the basis of their
putative molecular functions and the associated biochemical processes.
2.3.4. Validation of differentially expressed TDFs by qRT-PCR
To validate the results generated from the cDNA-AFLP analysis, an
independent expression study was performed for seven selected TDFs by
qRT-PCR. Based on their sequences, specific primer pairs were designed for
fragments (AA-CAA)6, (TA-CAA)4, (AA-CAT)3, (TT-CAT)8, (TT-CAA)7, (AA-
CA)3 and (AA-CA)5, standardized by semi-quantitative RT-PCR and used for
qRT-PCR analysis. The expression analysis confirmed that, the seven
isolated TDFs were differentially expressed in the tolerant genotypes as
compared to the susceptible genotypes upon infection by the blister blight
pathogen. The software GenePattern was used to show a representation of
normalized gene expression profiles of the TDFs by hierarchical clustering
(Figure 2.5). A graphical representation of the relative expression patterns of
the genes (TDFs) from blister blight infected plant materials evaluated by
qRT-PCR can be seen in Figure 2.6.
Most of the TDFs, but not all, were found to be upregulated in the
tolerant cultivars as compared to the susceptible cultivars after blister blight
infection. The TDFs (TT-CAA)7, (AA-CA)3, (AA-CA)5 and (TT-CAT)8
showing homology with acyl-CoA binding protein, zinc finger family protein,
ubiquitin and proline-rich protein respectively, were considerably upregulated
after infection and revealed very good correlation between cDNA-AFLP and
qRT-PCR expression patterns of the genotypes under control and disease
stress. However, for (AA-CAA)6, (TA-CAA)4 and (AA-CAT)3, the expression
patterns were not found to be in correspondence with cDNA-AFLP
expression patterns. These TDFs, although were contemplated to be
promising candidates for blister blight resistance with regard to their
homology to proteins like OPR, chitinase and STK, were not found to show
any considerable difference in their expression patterns among the tolerant
and susceptible genotypes. This could be in compliance to the fact that
resistance responses to disease stress are incited in all plants irrespective of
their resistance/susceptibility towards the pathogen. Therefore, the same
categories of genes and proteins are likely to get expressed in both tolerant
and susceptible genotypes, at higher, lower or equivalent intensities.
Figure 2.5. Heat map showing relative normalized gene expression profiles of
the selected TDFs generated by hierarchical clustering using GenePattern.
Figure 2.6. Graphical representation of the relative expression patterns of seven isolated TDFs evaluated by qRT-PCR.
Based on the expression pattern analysis, the TDFs (TT-CAA)7, (AA-CA)3
and (TT-CAT)8 were identified as promising candidate genes for blister blight
tolerance, due to high correspondence of their cDNA-AFLP and qRT-PCR
expression patterns and homologies to proteins acyl-CoA binding protein,
ubiquitin and proline-rich protein respectively, which are known to be involved
in defence mechanisms in plants. Thus, these transcripts could be assumed
as putative molecular markers for blister blight tolerance in tea; and to further
establish this hypothesis, a statistical analysis was carried out.
2.3.5. Statistical analysis
Through a statistical clustering analysis, we wanted to determine if
morpho-physiological traits related to disease incidence and its components
were correlated with molecular traits related to gene expression and
transcript accumulation in the infected leaves of tea, which harbour the
fungus. Using a set of morpho-physiological and gene expression data, a
principal component analysis (PCA) was carried out in order to visualize any
correlative relationships that may exist between transcript abundance and
disease incidence in the leaves of the various cultivars considered for this
study (Figure 2.7). PCA was performed for the different variables using all the
cultivars as a reference. The variance of each principal component is
represented by the two axes, and the barycentres explain the significance of
each of the two axes based only on the genetic variability for the traits
observed in each of the cultivars.
When a set of variables is located in the vicinity of a specific
barycentre, it means that the values of these variables are higher. The
variables, in this case the morpho-physiological and gene expression data for
the 5 different cultivars, were observed to be mostly located in the vicinity of
the tolerant germplasms P-312 and P-1258, away from the neighbourhood of
the susceptible cultivars. In particular, the transcripts for acyl-CoA binding
protein and proline-rich protein were found to be positioned in close proximity
to P-1258, which is the most tolerant cultivar among the group. Therefore,
from the results of the PCA analysis and keeping in view, their expression
Figure 2.7. A Principal Component Analysis (PCA) showing the
correlation of the morpho-physiological traits of the cultivars upon
infection, and the normalized expression data of the TDFs in the different
coordinates.
patterns across the different cultivars, the two transcripts could well be
hypothesized as putative molecular markers for the trait under study i.e.
blister blight tolerance. However, further studies have to be carried out in
large populations to investigate their association with the trait in order to
establish these TDFs as markers for the trait. The analysis shows all the
cultivars and their interrelationships based on the variables considered. It
also shows P-1258 as a unique germplasm, as it forms an out-group. Thus,
between the cultivars there is a high variability of the studied traits and this
variability, in all probability, is genetically controlled.
2.4. DISCUSSION
Blister blight is one of the oldest known fungal diseases of tea.
Understanding the complex transcriptional changes occurring in tea in
response to E. vexans is important for efficient management of this
pathogen. Based on the results of the blister blight infection experiment, it
could be inferred that the tea cultivars vary in tolerance or susceptibility to the
disease in terms of disease severity, lesion size and lesion distribution.
Debnath and Paul (1994) have studied blister blight occurrence in various
Darjeeling tea cultivars. Based on disease severity they have grouped the
cultivars as tolerant, less tolerant and least tolerant. They found that P-1258
is highly tolerant with a disease severity percentage of 1% or less, which is in
accordance with the results obtained from the present experimental
observations. Same is the case with the other cultivars with disease severity
following a similar pattern. However, in case of T-78, the disease severity
percentage has been found to be 3.4%, which is higher than that calculated
by them i.e. 2.05%.
In the susceptible cultivars, vigorous blister formation was observed
but in the tolerant cultivars, infection was restricted by tissue death at the site
of infection. This could be attributed to a phenomenon called hypersensitive
response (HR), manifested as an induced early defence response to
pathogen infection. It is characterized by necrotic lesions resulting from
localized cell death at the site of infection (Goodman and Novacky, 1994)
thus restricting further growth and spread of the pathogen into healthy tissues
(Dangl et al., 1996; Greenberg, 1996). The HR mechanism is more
pronounced in a tolerant host which prevents pathogen colonization. On the
other hand, a susceptible host plant is not able to prevent growth of the
pathogen as a result of which it is often severely damaged or even killed by
the infection. Thus, a major difference between resistant and susceptible
plants is the timely recognition of the invading pathogen and the rapid and
effective activation of host defence mechanisms. A resistant plant is capable
of rapidly deploying a wide variety of defence responses that prevent
pathogen colonization. In contrast, a susceptible plant exhibits much weaker
and slower responses that fail to restrict growth or spread of the pathogen.
To elucidate the tea defence responses during interaction with E.
vexans, we carried out a cDNA-AFLP study of the fungus-induced changes
at the transcriptional level to identify genes upregulated in the tolerant
cultivars during a host-pathogen interaction and thereby, identify their
possible utility as markers for blister blight tolerance. In addition to being a
highly reproducible technique, cDNA-AFLP is an unbiased method, which
can be used to reveal altered expression of any gene that carries the suitable
restriction site (Durrant et al., 2000). In this study we identified that, among
the TDFs with known functions, a vast majority were involved in metabolism.
Others were implicated in transport processes, response to biotic/abiotic
stresses, nuclear organization, transcriptional regulation, photosynthesis etc.
Approximately 6.8% of the TDFs have been found to be directly regulated in
response to disease stress or pathogen challenge. This relatively low level of
the tea defence-transcriptome coverage was probably due to the analysis
covering a single time-point. Moreover, the finite number of primer pairs that
can feasibly be used limits the number of transcripts that can be detected,
specially the defence-related ones which are expressed at low levels. In a
few cases, TDFs derived from different bands seemed to represent the same
homolog. In addition, sequencing failed for several TDFs, which could not be
characterized further. Most of the TDFs sequenced could not be assigned
putative functions as they did not show any homology to the genes/proteins
in the databases. Furthermore, a good number of transcripts detected and
isolated were of very short sequence length and could not be analyzed
further. Nevertheless, the study has provided a preview of the genes
associated with tea- E. vexans interaction, which could potentially be
converted to molecular markers through studying their association with the
trait of interest. Besides, the novel ESTs generated from this study could be
investigated further for their probable roles in resistance mechanisms.
Majority of the TDFs identified in the study were found to be involved
in various metabolic processes. These include proteins involved in
photosynthesis (e.g. Ribulose-1, 5-bisphosphate carboxylase/oxygenase
(RubisCO) small subunit, ATP synthase, chloroplast glyceradehyde-3-
phosphate dehydrogenase); metabolism of proteins (e.g. branched-chain
amino acid aminotransferase, eukaryotic translation initiation factor, 40S and
60S ribosomal proteins), carbohydrates (e.g. starch-branching enzyme I,
polygalacturonase) and lipids (e.g. plastid lipid-associated protein, acyl-CoA-
binding protein, lipoxygenase); and those involved in nuclear organization
(e.g. histone, nucleic acid binding protein, small nuclear ribonucleoprotein).
RubisCO small subunit, which has been found to occur twice in the cDNA-
AFLP analysis, is an important photosynthetic enzyme, and is known for its
involvement in heat stress, salt stress, photorespiration, pathogens and other
physiological responses (Whitney et al., 1999). The expression of
photosynthetic genes in plants is constitutive; however, they may be required
to improve the photosynthesis of plants so as to defend themselves from
pathogen attack in an incompatible interaction (Bolton, 2009). Proteins such
as plastid lipid-associated protein (fibrillin) and acyl-CoA-binding protein,
although are involved with lipid metabolism, have a potential role in
conferring resistance to pathogen infection by contributing towards basal
protection and improving plant survival under stress conditions. Acyl-CoA-
binding proteins along with lipid-transfer proteins (LTPs) have been identified
as candidates for lipid transfer within the cell. In addition to their roles in
phospholipid metabolism, these can contribute towards the plant’s resistance
to pathogens (Li et al., 2008; Xiao and Chye, 2011). Another important
enzyme implicated in lipid metabolism as well as in pathogen defence,
lipoxygenase (LOX), has been found to be differentially expressed in the
tolerant cultivars after blister blight infection. LOX initiates the synthesis of a
group of compounds collectively called oxylipins, which are products of fatty
acid oxidation, with diverse functions in the cell. In plants, linolenic and
linoleic acids are the most common substrates for LOX (Siedow, 1991).
LOXs are crucial for lipid peroxidation processes during plant defence
responses to pathogen infection and have been well documented by a
number of studies (Christophe et al., 1996; Rance et al., 1998; Kenton et al.,
1999). The function of LOXs in defence against pathogens is likely to be
related to the synthesis of fatty acid hydroperoxides and of volatile products
with signalling functions (Rusterucci et al., 1999) and antimicrobial activity
(Croft et al., 1993; Weber et al., 1999). Gao et al. (2007) suggested that
oxylipin metabolism mediated by specific LOXs, may be involved in fungal
pathogenesis in maize. In addition, a pyridoxin biosynthesis protein PDX1,
which has been often detected in plants undergoing cellular antioxidant
defence (Graham et al. 2004; Denslow et al., 2005) has also been identified
in the study.
The PR-protein chitinase, is induced by various factors including
fungal (van Kan et al., 1992; Danhash et al., 1993), bacterial (Broekaert and
Peumans, 1988), and viral infections (Vogeli-Lange et al., 1988; Margis
Pinhero et al., 1993), fungal elicitors (Hedrick et al., 1988; Mauch et al.,
1988), treatment with plant hormones (Boller et al., 1983; Shinshi et al.,
1987), abiotic factors (Roby et al., 1986), and environmental stress (Yeh et
al., 2000). Induction of chitinase is often coordinated with the induction of
specific β-1,3-glucanases and other PR proteins (Collinge et al., 1993).
Studies have indicated that chitinases inhibit fungal growth by degrading
chitin, a major structural cell wall polysaccharides in growing hyphae
(Bartnicki-Garcia, 1968). Thus, the degradation of the fungal cell walls by the
host chitinase acts as an active defence mechanism of disease resistance in
plants. The present study describes the identification of a chitinase-like
protein in response to blister blight infection in the tolerant cultivars,
indicating its potential role in conferring resistance to E. vexans.
A well known class of receptor protein kinases, associated with
recognition of pathogen signals, are serine/threonine protein kinases (STKs),
which play a central role in reception of pathogen signals and subsequent
activation of plant defence mechanisms. In the present investigation, we
have encountered the induction of STKs, establishing their contribution
towards disease resistance against E. vexans infection in tea. A cytoplasmic
STK, PTO is known to be involved in resistance to Pseudomonas syringae by
recognition of AvrPTO (Bogdanove and Martin, 2000). In rice, STKs has
been found to be involved in plant-pathogen interaction and defence
responses to bacterial blight (Song et al., 1995). A calcium ion binding
protein has also been identified in the study. Ca2+ is one of the most
important second messengers in plants. Ca2+ signalling has a pivotal role in
disease resistance and symbiosis and an influx in Ca2+ ion has been shown
to be essential for the activation of defence responses such as phytoalexin
biosynthesis, induction of defence-related genes, and hypersensitive cell
death. The Ca2+ binding protein calmodulin, which acts as a Ca2+ sensor, has
been found to be involved in basal defence against necrotrophic pathogens
in tobacco (Takabatake et al., 2007) and soybean (Heo et al., 1999) and
many other plant species through SA-mediated defence signalling pathways
or SA-independent activation of defence responses. Among other
compounds involved in mediating defence signals, 12-oxo-phytodienoic acid
reductase (OPR) is an important member implicated in jasmonic acid (JA)-
mediated signal transduction pathways. In the present study, an OPR
transcript has been observed to be differentially expressed. The OPR genes
are known to participate in the octadecanoid pathway which converts
linolenic acid to the phytohormone JA, which has been shown to play a key
role in response to infections by necrotrophic fungi (Penninckx et al., 1998;
Staswick et al., 1998; Vijayan et al., 1998) and insect attack (Howe et al.,
1996; McConn et al., 1997), as well as abiotic stresses.
Pathogen infection leading to HR and cell death is also associated
with oxidative stress, with the generation of reactive oxygen species (ROS) in
the infected plant cells leading to activation of several defence signal
transduction cascades. Subsequent transcriptional and/or post-translational
activation of transcription factors eventually leads to the induction of plant
defence genes (Zhu et al., 1996). The ROS being destructive to the cellular
components and metabolites are detoxified by enzymes such as superoxide
dismutase, which has been found to be induced by blister blight infection in
the present study. A transcript with strong homology to NADH
oxidoreductase, an enzyme catalyzing the formation of ROS leading to HR
(Papadakis and Roubelakis-Angelakis, 2005), has also been observed to be
upregulated post-infection in the tolerant cultivars. The typical ROS
accumulated in stressed plant cells may be directly toxic to pathogens, and
also contribute to structural reinforcement of cell walls by cross-linking various
extracellular proteins such as (hydroxyl-) proline-rich glycoproteins to the
polysaccharide matrix. Hydroxyproline-rich glycoprotein and proline-rich
protein, involved in cell wall strengthening, have also been identified in this
investigation. These proteins may function both in determining cell-type-
specific wall structure during plant development and by contributing to
defence reactions against physical damage and pathogen infection (Fowler
et al., 1999). These are rapidly insolubilized within the cell wall in response to
physical damage, treatment with fungal elicitors, and pathogen infection
(Kleis-San Francisco and Tierney, 1990; Bradley et al., 1992; Brisson et al.,
1994), indicating their active roles in plant defence reactions.
Another group of transcripts identified in this study, which are of
particular interest to defence mechanism, are those related to gene
expression. Blister blight infection has revealed the upregulation of
transcription factors such as zinc-finger proteins, which have been
documented thrice in the analysis, and NAC domain protein. Transcription
factors are the proteins that regulate gene expression by binding to specific
cis-acting promoter elements, thereby activating or repressing the
transcriptional rates of their target genes (Riechmann et al., 2000; Wray et al,
2003). The zinc-finger proteins are known to play a crucial role in the
activation of the pathogen defence response in plants (Serrano and Guzman,
2004; Oh et al., 2005). The characterized plant C2H2 zinc-finger proteins are
mainly involved in plant growth and development as well as responses to
environmental stresses. A novel blast-inducible RING-H2 type zinc-finger
protein acts as a transcriptional regulator in plant stress response signal
transduction pathways (Meng et al., 2006). The NAC domain proteins are
plant-specific transcriptional factors, the functions of which are very diverse in
plants. These have been implicated in a wide range of plant developmental
processes, as well as in plant abiotic stresses and defence responses to viral
and fungal infection (Ren et al., 2000; Collinge and Boller, 2001; Jensen et
al., 2008; Wang et al., 2009).
A protein concerned with protein degradation, ubiquitin, has also been
well represented in this investigation. The control of protein degradation
through the ubiquitin-proteasome system (UPS) is a central modifier of
signalling in animals and plants, and therefore influences many processes
such as the cell cycle, signal transduction, transcription, and stress
responses including defence. Although, no ubiquitin ligase targets that are
associated with disease resistance have yet been identified in plants, there is
evidence that this well-known protein-modification system may regulate plant
defence against pathogens (Devoto et al., 2003; Sullivan et al., 2003; Zeng et
al., 2006; Dreher and Callis, 2007).
Another protein identified in this study is the ABC transporter, the
function of which is not clearly understood but is thought to be involved in
intracellular binding of cytotoxins (Jasinski et al., 2001). However, PDR-like
ABC transporters have been implicated in plant defence, particularly the
PDR5-like family, where its increased expression has been linked with
increase of anti-fungal protein expression. These proteins may have a role in
defence response by being responsible for the excretion of secondary
compounds from cells (Jasinski et al., 2001).
Apart from proteins related to defence, a number of TDFs have shown
homology to proteins implicated in abiotic stress responses like rhodanese,
thioredoxin and metallothionein, which does not rule out the possibility of a
cross-talk between biotic and abiotic stress responses in plants.
Metallothioneins are ubiquitous proteins that bind metal ions, and their role in
plants affected by stress conditions is not clear. However, as
metallothioneins are also potent scavengers of hydroxyl radicals, it has been
proposed that they could protect cellular constituents from oxidative damage
(Choi et al., 1996). The induction of metallothionein-like proteins by insects,
wounding and pathogen infection has been described in various studies
(Choi et al., 1996; Zhu-Salzman et al., 2004; Degenhardt et al., 2005), and
could also have a potential role in defence induction in tea.
2.5. CONCLUSION
The present investigation based on cDNA-AFLP and expression
profiling provided an overview of the various changes taking place in the tea
transcriptome in defence against the blister blight fungus. From the statistical
analysis carried out in the study, it can be inferred that there is a complex
interaction between the expression of the morpho-physiological and
molecular traits that depends both on the genetic background as well as the
genotype examined. For example, networks of regulation occurring at various
functional levels (gene expression, protein synthesis, post-transcriptional and
translational modifications, enzyme activities, metabolic fluxes, and
metabolite accumulation) may be different in a given genotype with possible
interactions between these various parameters. When a set of traits exhibits
a tissue- or genotype-specific repartition, it is likely that both parameters
influence the expression of these traits in an interactive manner. Therefore,
clustering various physiological and molecular traits in a given genotype can
provide information on the capacity of a specific cultivar to confer disease
resistance both at the physiological and molecular levels. To achieve this, the
selected panel of tea cultivars covering the tolerance capacity of the
genotypes towards the fungal disease was analysed in order to exploit the
genetic variability for future studies on marker-trait association. A
comprehensive analysis of genes so far identified would lead to a better
understanding of the mechanisms involved in defence to biotic stresses and
contribute towards the design of molecular breeding strategies to improve
disease resistance in tea. Furthermore, the TDFs corresponding to acyl-CoA
binding protein, ubiquitin and proline-rich protein could be investigated further
to assess their potentiality as functional molecular markers. Apart from using
them for MAS, these putative markers could well be utilized in breeding
programmes for the development of varieties with improved tolerance/
resistance to the disease through studying their association with the trait in
segregating populations.