cDNAcDNA- ---AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT...

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Chapter II Chapter II Chapter II Chapter II cDNA cDNA cDNA cDNA-AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT PROFILING FOR IDENTIFICATION OF PROFILING FOR IDENTIFICATION OF PROFILING FOR IDENTIFICATION OF PROFILING FOR IDENTIFICATION OF 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 TEA TEA TEA TEA

Transcript of cDNAcDNA- ---AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT...

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Chapter IIChapter IIChapter IIChapter II

cDNAcDNAcDNAcDNA----AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT AFLP BASED TRANSCRIPT

PROFILING FOR IDENTIFICATION OF PROFILING FOR IDENTIFICATION OF PROFILING FOR IDENTIFICATION OF PROFILING FOR IDENTIFICATION OF

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

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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.

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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.

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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

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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

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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.

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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.

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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

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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

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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

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(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

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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.

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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

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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.

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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

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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

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B) MseI-AA X BstYI-CAG

B157i RR17/144i T78c T78i P312c P312i P1258c P1258i

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C) MseI-AA X BstYI-CAT

B157i RR17/144i T78c T78i P312c P312i P1258c P1258i

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D) MseI-AT X BstYI-CAG

B157i RR17/144i T78c T78i P312c P312i P1258c P1258i

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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

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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.

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Figure 2.4. Functional categorization of the TDFs on the basis of their

putative molecular functions and the associated biochemical processes.

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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.

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Figure 2.5. Heat map showing relative normalized gene expression profiles of

the selected TDFs generated by hierarchical clustering using GenePattern.

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Figure 2.6. Graphical representation of the relative expression patterns of seven isolated TDFs evaluated by qRT-PCR.

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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

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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.

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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

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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

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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

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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

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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.

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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,

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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,

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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

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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.