Chapter II Review of literature -...

Review of literature

23

Chapter II Review of literature

2.1 Management of the disease caused by P. colocasiae

Several tolerant cultivars have been registered but their response to varying

environment condition may not guarantee for disease resiastance against Phytophthora

but there several methods for the management of leaf blight of taro have been

demostrated. Some of management practices are discussed here.

2.1.1 Cultural practices

Various cultural methods have been recommended for the control of taro leaf

blight. Removal of infected leaves has been effective during the early stages of disease

development in a number of countries. Wide spacing of plants has been reported to

reduce disease severity but this appears to have a negligible effect when conditions favour

disease development. Other cultural methods that have been recommended include

delaying planting on the same land for a minimum of three weeks, avoiding plantings

close to older infected ones and preventing the carry over of corms or suckers which can

harbour the pathogen from one crop to the next (Jackson, 1999). Preliminary findings

have indicated that fertilizer treatment may also help the plant cope with leaf blight.

Removal and destruction of infected leaves and use of healthy corms and crop rotation

have been recommended as control measures (Mundkur, 1949). However, in a study it

was reported that removal of infected leaves and traditional wide spacing did not help in

reducing the blight disease incidence (Jackson et al., 1980).


24

2.1.2 Biological Control

Role of phylloplane microflora was studied on C. esculenta in relation to P.

colocasiae and it was found that Myrothecium roridum, 3 Streptomyces spp. and 2 bacterial

isolates had antagonistic activity against P. colocasiae in dual culture plates. In vivo, the

bacteria reduced the disease incidence up to 43%; Streptomyces albidoflavus reduced

infection by 90-93% and S. diastaticus by 76%. Among fungi, Botrytis cinerea gave the best

control of 33% reduction of plant infection (Narula and Mehrotra, 1987). Trichoderma

viride, T. harzianum, Gliocladium virens and one more unidentified sterile fungal culture

were found to have potential antagonism against P. colocasiae in vitro (Sawant, 1995). The

mycoparasitic or hyperparasitic activities of these isolates on P. colocasiae were brought

about through several morphological changes like coiling of hyphae, formation of haustoria-

like structures, disorganization of host cell contents and penetration into host hyphae.

Rhizobacteria isolated from Colocasia rhizosphere soil have been reported to have the

ability to completely inhibit the growth of P. colocasiae in vitro. T. viride effectively

inhibited the population of P. colocasiae up to 88.88%, whereas T. harzianum and T.

pseudockei reduced the population of P. colocasiae up to 77.77 and 88.88%, respectively.

Apart from Trichoderma, other fungi and bacteria isolated from rhizosphere soil from

Colocasia fields were tested by the dual plate method for their ability to inhibit P.

colocasiae. Several fungi and bacteria completely inhibited the growth of P. colocasiae

(Misra et al., 2001).

2.1.3 Fungicidal control

Spraying of copper fungicides was found helpful in controlling the P. colocasiae

infection on taro. Copper oxychloride applied at a rate of 4.5 kg per 100 litres of water

per hectare gave good control of the disease in Solomon Islands (Jackson, 1996). Early

trial work in Samoa concentrated on trials of Ridomil MZ, Manzate and phosphorous acid

(Foschek) on pot experiment and showed the superiority of phosphorous acid over


25

Ridomil MZ. Whereas, when experiments were compared with phosphorous acid

formulations (Foschek, Agri-Fos 400 and Foli-R-Fos) no difference was found in terms of

disease control (Adams, 1999). In Samoa, a recommendation for fungicide spraying was

made for Foschek, alternated with Manzate to minimise resistance problems but the costs

were prohibitive for the majority of farmers. Copper fungicides have proved very

effective in successfully controlling the disease in many places like Fiji (Parham, 1949);

India (Mundkur, 1949); Hawaii (Bergquist, 1974) and Solomon islands (Jackson et al.,

1980). Dithane M-45, Polyram, Benlate, Perinox and Dyrene were also found to be very

effective in controlling this disease (Anonymous, 1950; Maheshwari et al., 1999).

Application of copper oxychloride at 14-day intervals adequately controlled the disease

(Jackson and Gollifer, 1975). Besides metalaxyl, captafol and chloroneb were also found

to be effective in controlling P. colocasiae under in vitro and in vivo conditions

(Aggarwal and Mehrotra, 1987). Aggarwal and Mehrotra (1988) observed that besides

controlling P. colocasiae in the field, metalaxyl could inhibit the cellulolytic and

pectinolytic enzymes produced by P. colocasiae. Results on the chemical control of taro

leaf blight with metalaxyl and breeding for disease resistance were also reported.

Aggrawal et al. (1993) reported the effect of potassium iodide, arsenic oxide on the

mycelial growth, sporangial production, pectolytic and cellulolytic enzyme production

and control of P. colocasiae on taro. The effect of fungicides in controlling leaf blight

caused by P. colocasiae in C. esculenta revealed that 0.2% metalaxyl and mancozeb (as

Ridomil MZ-72) was the most effective treatment, followed by 0.2% captafol (as Foltaf),

bordeaux mixture (1% copper sulfate and lime) and 0.25% mancozeb (as Foltaf)

(Bhattacharyya and Saikia, 1996). Metalaxyl with copper (as 0.3% Ridomil) gives

excellent control of taro leaf blight when applied at 2-week intervals using a knapsack

sprayer (Cox and Kasimani, 1990). The efficacy of copper oxychloride, mancozeb,


26

metalaxyl, captafol, ziram and Bordeaux mixture against leaf blight disease of taro var.

antiquorum has been reported (Das, 1997). Sahu et al. (1989) observed that four sprays of

zineb at 15-day intervals starting from the end of July to early August reduced the

incidence of P. colocasiae in C. esculenta. Ghosh and Sitansu (1991) found that spraying

with metalaxyl (Ridomil M272WP) at 3 kg/ha at 15-day intervals were highly effective in

controlling the disease. Cox and Kasimani (1990) found that 5 applications of metalaxyl

at 3-week intervals resulted in an increase of almost 50% in tuber yield. Towards the

prospect of taro leaf blight disease control, field experiments were conducted to observe

the effect of borax in controlling Phytophthora leaf blight using ten taro different

genotypes (Misra et al., 2007).

2.1.4 Resistance variety

Marginal farmers in the developing countries cannot afford the extra costs

required for fungicides and labour involved in leaf removal and spraying. And therefore,

alternative sustainable strategies for the management of the disease are needed like use of

resistant varieties. In Samoa, a programme was initiated to screen and evaluate exotic

taros by observing the high impact of leaf blight on susceptibility of local taro varieties

(Iosefa and Rogers, 1999). In other Colocasia growing countries, varieties having a

reasonable degree of resistance to blight have not been found in good number (Dayrit and

Phillip, 1987; Dey et al., 1993; Anonymous, 1999).

Many cultivars of taro tolerant to leaf blight have been reported from India.

Deshmukh and Chibber (1960) have identified var. „Ahina‟ as resistant to blight where

numbers of sporangia produced and size of infected area were studied. On „Ahina‟, the

numbers of sporangia produced were less as compared to susceptible variety and size of

the infected area increased more slowly in the resistant variety than in susceptible variety.

Later, Paharia and Mathur (1964) screened 20 varieties at Shimla and reported var.

„Poonam Pat‟ as immune, „Sakin V‟ as resistant and another seven as moderately resistant


27

to blight. Choudhury and Mathura Rai (1988) used wild varieties of Colocasia for

resistance breeding and selected the resistant lines from them. Out of 20 varieties tested at

Arunachal Pradesh, five were immune to blight. The aroid species Xanthosoma

sagittifolium and Alocasia macrorrhiza were presumed to be resistant to P. colocasiae

(Ho and Ramsden, 1998). Goswami et al. (1993) tested 50 lines against taro blight and

found that 5 lines were showing resistance to the disease. At CTCRI, Trivandrum (Pillai

et al., 1993) developed resistant taro lines through breeding. The maximum proportion of

resistant genotypes was obtained from variety „c-320‟ self (66%), followed by open

pollinated progeny of „c-12‟ (33.33%), „c-78‟ (30%) and „Nadia local‟ (26.31%). Among

the crosses, the maximum proportion of resistant genotypes were obtained in „G2 × G16‟

(25%) followed by „Pig × G6‟ (23.8%). None of the tolerant parents bred true for resistant

genes. Misra (1988, 1989 and 1990) screened 43 cultivars of Colocasia and cvs. „Jankhri‟

and „Muktakeshi‟ were reported as highly tolerant to blight. Progress of leaf blight in six

selected cultivars showing varying degree of resistance (including „Jankhri‟ and

„Muktakeshi‟) was studied. The appearance of blight was delayed in tolerant cultivars and

its subsequent spread was also slow as compared to susceptible cultivars (Misra and

Singh, 1991; Misra and Chowdhury, 1997).

These resistant varieties showed resistance at state level but it has not been

assured from these studies that when these resistant lines will be exposed to some new

virulent strain of P. colocasiae in different countries or state, then resistance would be

sustained. Moreover some of the resistant variety has poor yield and based on that there is

need to screen for more resistant varieties by using molecular marker and moreover it is

imperative to look for genetic diversity of P. colocasiae so that we may correlate, if, there

is any effect of genet structure among taro variety to cope up with taro blight.


28

Occurrence of some of the wild type resistant variety of taro indicates that there

has been long-term coevolution between the pathogen and native taro species. This

suggests that natural populations of the wild species of taro are likely to contain

resistances that can be used to control the effects of the pathogen on cultivated taro.

Based on this we aimed our study towards developing an understanding of the relative

importance of the different mechanisms (migration; sexual and asexual recombination;

mutation) that contribute to the origin and maintenance of variation in P. colocasiae

populations. In particular we have investigated: (i) identifying resistance to taro leaf

blight in regional local variety of taro by screening a broad range of accessions of native

taro representing populations from a wide range of geographic locations by the use of

biochemical (Isozyme) and molecular marker (RAPD and AFLP); (ii) evolutionary

changes in P. colocasiae in India over the past few years; (iii) the occurrence and origin

of novel genotypes of P. colocasiae; (iv) genetic variability and pathogenicity of P.

colocasiae on taro; and (v) phylogenetic relationships within the P. colocasiae. Results

from these studies would help to reduce the impact of P. colocasiae on taro production

through: (i) identification of potentially valuable sources of naturally occurring resistance

to taro blight; (ii) determination of the genetic control and heritability of such resistance;

(iii) provision of early warning of future problems by assessing the possible occurrence of

additional pathotypes of P. colocasiae that are yet to appear in cultivated taro; (iv) further

development of molecular markers for fingerprinting and screening of resistant variety of

taro.

2.2 Origin and analysis of genetic variations

Spontaneous origin of new variations in an organism is termed as mutation. De

Vries (1905) used the term mutation for new phenotypes that arose abruptly in a stock of

plants. Mutations may affect single locus (point mutations) generating new genes able to


29

produce new individuals (Buss, 1987) or rearrangements of genes by inversions or

translocations or even by recombination at the time of sexual reproduction which give

rise to novel multi-locus gene combinations. A gene may consist of thousand to several

thousand nucleotide pairs which could be one of the reasons for generating multiple

alleles. The multiple alleles can be visualised at isozyme or DNA level and are actually

different variants in base sequence of DNA (Sharma et al., 2008a). In highly inbred

plants, the accumulation of mutant alleles can be as high as 0.5 per generation per

individual (Charlesworth et al., 1990).

The genetic structure of a population depends on allele frequencies at different

loci and on their effects. Generation of genetic variation within a species by mutations is a

continuous process and it may affect overall genetic structure of the species. But there are

some other factors that affect allele frequencies in a random mating population like

genetic drift, gene flow, natural selection. Natural populations are often finite and small in

number, and random genetic drift refers to the chance fluctuations in allele frequencies in

each population. In such populations, the probability of extinction for any mutant allele is

particularly high (Spiess, 1989). In many species, the population is a network of sub-

populations with intermittent gene flow between adjacent sub-populations. Hence overall

genetic variation in a species/population is primarily due to the dynamic balance between

genetic drift and gene flow. Another important factor affecting allele frequency especially

in the case of fitness-related characters is natural selection (Endler, 1986). Selection can

stabilize phenotypic characters in such a way that the genetic variation within a group is

reduced and among groups it is increased. Thus, any genetic variation that arises as a

result of mutation changing allele frequencies will further be under the positive or

negative control of factors such as drift, gene flow and selection, which contribute to the

overall evolutionary processes.


30

Plant diseases are caused by a number of pathogens hence study of population

biology is very imperative. One pathogen lesion on one leaf does not have a significant

economic or ecological impact but thousands of infection events causes significant crop

loss which involve an entire population of parasites and their host plants (Milgroom and

Fry, 1997). Thus it is important to understand the population biology of plant pathogens

in order to develop rational control strategies. Population genetics focuses on the

processes that lead to genetic change, or evolution, in populations over time and space

(McDonald and Linde, 2002). When considering plant pathogens, we assume of plant

disease in natural ecosystems as a co-evolutionary process. Evolution consist of two-steps

i.e. mutation that give rise to genetic variation in populations and selection or genetic drift

act to change allele frequencies in populations (McDonald and Linde, 2002). Co-

evolution happens when a trait of one species evolves in response to a trait of another

species, which trait in turn evolves in response to the trait in the first species (Janzen

1980). A genetic change in one species causes a genetic change in the coevolving species

and this genetic change in turn causes new genetic change in the first species (Mundt,

1990). Co-evolution begins with a plant population affected by a disease and generally is

a complex geographic process, with the same species often coevolving in at least slightly

different ways in different geographic areas (Goodwin, 1997).

In agroecosystems, we are concerned with how a control method (e.g. introduction

of a resistance gene, application of a fungicide, crop rotation, etc.) affects the population

genetics, or evolution, of the targeted pathogen population. To study the population

genetics that affect pathogen population we generally consider mutation, genetic drift,

reproduction and mating system, gene flow and natural selection (Chen et al., 1994).

Study of pathogen populations is very essential to determine whether a new pathogen

population is derived from a pre-existing pathogen population or represents a host-jump


31

or a new species of pathogen. We studied taro and its pathogen P. colocasiae using

biochemical and molecular tools to understand how the genetic structure and its

geographic distribution behave in India.

2.3 Isozyme (Biochemical markers)

In one of the earlier studies, Mallette and Dawson (1949) obtained five purified

tyrosinase preparations from the common mushroom, Psalliota campestris. However,

later Markert and Moller coined the term „isozyme‟ in 1959 to describe the multiple

molecular forms of enzymes that exhibit the same enzymatic specificity. The marker

became essential tools for genetic study of plant poulations in early 1970. Even today

isozyme markers are the best tools to answer many research questions in analysis of

genetic variations. Isozymes are distinguished by many methods like electrophoresis,

chromatography, gel filtration, catalysis, immuno-techniques, and sedimentation. Among

these, electrophoresis is often the choice for studies of heritable variations by geneticists,

systematists, and population biologists (Conkle et al., 1982; Liengsiri et al., 1990; Adams

et al., 1991).

Biochemical marker provide an estimate of gene and genotypic frequencies within

and between populations which can be used to measure population subdivision, genetic

diversity, gene flow, genetic structure of species, and comparisons among species out-

crossing rates, population structure and population divergence, such as in the case of crop

wild relatives. Major advantages of these types of markers consist in assessing co-

dominance, absence of epistatic and pleiotrophic effects, ease of use, and low costs,

however, there are some cons of this techniques like (i) only few isozyme systems per

species (ii) the number of polymorphic enzymatic systems available is limited (iii) Due to

affected extraction methods, stage of plant and choice of plant tissue comparison becomes

different for different species and loci. Isozyme markers are effectively used to study the


32

genetic diversity in many plants (Zanetto et al., 1993; Sabu et al., 2001). Some studies

were conducted in a number of taxa that revealed threatened or otherwise significant

populations for conservation or management of genetic resources (Petit et al., 2001;

Jaradat and Shahid, 2006). Correlation between geographic and genetic distances was

revealed by allozyme electrophoresis in a number of organisms. Significant correlation

determined between genetic distances and geographic distances among the Indonesian

island populations of Cynopterus nusatenggara (Schmitt et al., 1995).

In our study of taro-P. colocasiae interaction, Isozyme variations were used as a

powerful tool to complement and to supplement conventional breeding methods. In our

study we tried to explore the sufficient isozyme markers so that these markers could allow

assessment of different factors on the genetic structure of the populations. The isozyme

marker assisted us to study the factors with important impacts on the co-evolution of taro

and P. colocasiae populations including extent of variability, mutation rates, random

genetic drift, recombination rates, extent of gene flow, and the effects of selection.

Isozyme study was done with 7 isozyme markers esterase (EST, 3.1.1.1), malate

dehydrogenase (MDH, 1.1.1.37), phosphoglucomutase (PGM, 2.7.5.1), hexokinase

(HEX, 2.7.1.1), β-Glucosidase (β-GLU, 3.2.1.21), acid phosphatase (ACP, 3.1.3.2), malic

enzyme (ME, 1.1.1.40) to explore the following factors, e.g. extent of genetic

variability, gene flow, and selection, that appear to be particularly relevant to

coevolution of taro- P. colocasiae systems (Sharma et al., 2008a; Mishra et al., 2010).

2.4 Variation at DNA level

The ability to investigate DNA sequences directly became available to population

biologists only during the late 1970s. Currently, three major DNA-based techniques have

been widely used for analysing the genetic diversity in natural populations. These include

(i) restriction fragment length polymorphism (RFLP; Botstein et al., 1980), (ii)


33

polymerase chain reaction (PCR; Mullis and Faloona, 1987), and its derivatives, termed

random amplified polymorphic DNA (RAPD) (Williams et al., 1990); AP-PCR (Welsh

and McClelland, 1991) and (iii) a hybrid of both the above techniques named

amplification fragment length polymorphism (AFLP; Vos et al., 1995).

2.4.1 RAPD

RAPDs were the first PCR-based molecular markers to be employed in genetic

variation analyses (Welsh and McClelland, 1991). RAPD markers are generated through

the random amplification of genomic DNA using short primers (decamers), separation of

the obtained fragments on agarose gel in the presence of ethidium bromide and

visualization under ultraviolet light. The use of short primers is essential to increase the

probability that, although the sequences are random, they are able to find homologous

sequences suitable for annealing. DNA polymorphisms are then produced by

rearrangements or deletions at or between oligonucleotide primer binding sites in the

genome (Williams et al., 1991). This approach requires no prior knowledge of the

genome analyzed, it can be employed across species using these universal primers. The

major drawback of this method is that the profiling is dependent on reaction conditions

which can vary between laboratories; even a difference of a degree in temperature is

sufficient to produce different patterns. Additionally, as several discrete loci are amplified

by each primer, profiles are not able to distinguish heterozygous from homozygous

individuals (Bardakci, 2001). Variants of RAPD are another developed methodologies i.e

AP-PCR (Welsh and McClelland, 1991) and DNA Amplification Fingerprinting (DAF)

used in DNA fingerprinting.

The RAPD markers have been applied to many organisms including forest trees,

crops, medicinal plants as well as on lower plants for genetic linkage mapping (e.g.,

Carlson et al., 1991; Tulsieram et al., 1992; Grattapaglia and Sederoff, 1994; Nelson et

al., 1994), phylogeny and systematics (Wilde et al., 1992; Joshi and Nguyen, 1993;


34

Caetano-Anolles, 1994) and population genetics (e.g., Schierenbeck et al., 1997; Inglis et

al., 2001; Sharma et al., 2008a). Only disadvantage of these markers is the degree of low

reproducibility due to the sensitivity of RAPD banding patterns to reaction conditions,

and the difficulty in exactly replicating reaction conditions across laboratories, where

different brands of thermo cyclers may be used (Ellsworth et al., 1993; Skroch and

Nienhis, 1995).

As is common with most organisms, plant-pathogenic oomycetes rely on the

processes of mutation and recombination as the ultimate source of genetically based

variation. Within a species, gene flow between populations supplements these processes

as propagules spread from one epidemiological area to another and from one deme to the

next. Studies of a wide variety of fungal and oomycetes pathogens have highlighted the

importance of some of the mechanisms behind these broad groupings as sources of

diversity. In view of that we have conducted RAPD study in P. colocasiae to investigate

the way in which these mechanisms intermesh to generate the overall variation

encountered within this species (Mishra et al., 2010). Beside this, we focused on

understanding the extent of variation and genotype diversity in various populations of taro

(Sharma et al., 2008a, Mishra et al., 2008a).

2.4.2 AFLP

To overcome the limitation of reproducibility associated with RAPD, AFLP

technology was developed by the Dutch company, Keygene which based on the

combination of the main analysis techniques: digestion of DNA through restriction

endonuclease enzymes and PCR technology (Vos et al., 1995). It can be considered an

intermediate between RFLPs and RAPDs methodologies as it combines the power of

RFLP with the flexibility of PCR-based technology. The primer pairs used for AFLP

usually produce 50-100 bands per assay and number of amplicons per AFLP assay is a

function of the number selective nucleotides in the AFLP primer combination, the


35

selective nucleotide motif, GC content, and physical genome size and complexity. AFLP

generates fingerprints of any DNA regardless of its source, and without any prior

knowledge of DNA sequence. Most AFLP fragments correspond to unique positions on

the genome and hence can be exploited as landmarks in genetic and physical mapping.

The technique can be used to distinguish closely related individuals at the sub-species

level and can also be used in genes map (Althoff et al., 2007).

The origins of AFLP polymorphisms are multiple and can be due to: (i) mutations

of the restriction site which create or delete a restriction site; (ii) mutations of sequences

flanking the restriction site, and complementary to the extension of the selective primers,

enabling possible primer annealing; (iii) insertions, duplications or deletions inside

amplification fragments (Vos et al., 1995). AFLP is now increasingly used in a number of

species including many wild plant species (Cervera et al., 1998; Beismann et al., 1997;

Gaiotto et al., 1997). For example, in India, AFLP markers have been used in the

assessment of genetic diversity in 37 neem accessions from different eco-geographic

regions of India and four exotic lines from Thailand (Singh et al., 1999).

The use of DNA marker systems, such as RAPDs (Williams et al., 1990), AFLPs

(Vos et al., 1995), and simple sequence repeats (SSRs) (Akkaya et al., 1992), has

contributed greatly to the development of genetic linkage maps for many important crop

species including cowpea and barley (Fatokun et al., 1993; Waugh et al., 1997). In

combination with the bulked segregant analysis (BSA) method, (Michelmore and Meyers,

1998) the use of RAPDs, AFLPs, and SSRs has made it possible to rapidly identify

molecular markers linked to genes of agronomic importance (Lee, 1995; Young, 1999).

The development and use of molecular marker technologies has also facilitated the

subsequent cloning and characterization of disease, insect, and pest-resistance genes from

a variety of plant species (Hammond-Kosack and Jones, 1997; Meyers et al., 1998).


36

These studies have led to a greater understanding of the basis for plant-pathogen

interactions and the process of plant disease resistance (Ribault and Hoisington, 1998;

Kumar, 1999; Young, 1999). AFLP marker has been used to study the genetic variability

in taro. Moreover we have reported these markers in a marker-assisted selection program

and in the eventual map-based cloning of genes conferring resistance to P. colocasiae in

taro (Sharma et al., 2008b).

2.4.3 Data analyses and estimation of genetic variation

In the literature several statistics have been used to summarize data on genetic

variation, which come under two major concepts: first, there is allelic richness, or the

number of distinct kinds of alleles encountered in a sample of particular size and secondly

on evenness, which is related to the distribution of allelic frequencies (Brown and Weir,

1983). One of the most common measure of genetic diversity has been the equivalent of

Simpson‟s (1949) index of ecological diversity. This measure, or simple transforms of it,

has received various names such as effective number of alleles (Kimura and Crow, 1964),

expected heterozygocity (Hubby and Lewontin, 1966), polymorphic index (Marshall and

Jain, 1969) and gene diversity (Nei, 1973, 1978). Among all these, Nei‟s gene diversity is

the one frequently used since it does not rely on knowledge of genotype frequencies and

can be estimated from allele frequencies in terms of the expected heterozygocities within

and between populations (Brown and Weir, 1983).

Genetic distances are designed to express the genetic differences between any two

populations as a single number (Smith, 1977). In estimates of genetic distances using

allelic frequency differences among populations, Nei‟s standard genetic distance, D (Nei,

1972) has been most frequently used. In isozyme studies, Nei (1974) reported that species

are characterized by genetic distances of 0.1 to 1.0, subspecies and varieties by 0.02 to

0.20 and races by 0.01 to 0.05. The genetic structure of subdivided populations can be

analysed by F-statistics using the correlation between uniting gametes (Wright, 1943,


37

1951). These provide an approximate measure of inbreeding in each subpopulation (FIS)

and in the entire population (FIT), (Weir and Cockerham, 1984; Weir, 1990), and FST

measures the degree of genetic differentiation among populations. The latter is always

positive, between 0 and 1, and as such is used as a measure of genetic distance (Long et

al., 1987; Weir, 1990). Nei‟s distance is appropriate for long-term evolution when

populations diverge because of drift and mutation. The distance is proportional to the time

since divergence in the special case of the infinite alleles mutation model and equilibrium

in the ancestral population. The FST distance is the most appropriate for short-term

evolution, for divergence due only to drift, and no assumptions need to be made about the

ancestral population (Reynolds et al., 1983; Weir, 1990). The genetic distance values can

also be used for reconstruction of phylogenies to establish relationships of species. The

most widely used is unweighted pair group method with arithmetic averages (UPGMA)

(Mondini et al., 2009).

2.5 Pathogen and resistance

Plants, like animals, are subject to attack by bacteria and fungi as well as viruses

and insects; however, unlike animals, plants are sessile organisms making defence an

even greater imperative. To protect themselves, plants have evolved numerous defence

strategies, some of which are preformed (constitutive) while others are only deployed in

response to a challenging pathogen or pest (inducible). To be successful, an attacking

organism must evade, suppress or in some way counter an entire battery of defences that

usually include structural (i.e. morphological) barriers, as well as various secondary

metabolites and antimicrobial agents, such as degradative enzymes and phytoalexins

(Bruce and Pickett, 2007). Understanding the molecular mechanisms underlying plant

diseases continues to be a daunting task. First, the diversity of plant secondary

metabolites among species virtually ensures that each plant-pathogen interaction is to


38

some extent unique. Second, while the animal‟s immune system has specialized attack

cells (i.e. T cells), plants, in an evolutionary sense, seem to have co-opted pre-existing

molecules and biochemical pathways from other functions (i.e. development) to use in the

defence. For example, programmed cell death (PCD) that has important roles in xylem

differentiation and reproductive development is key to the “hypersensitive response

(HR)” which is central to resistance in many plant pathosystems (Mittler and Cheung

2004). Many defence proteins, such as ß-glucanases and chitinases, also have important

roles in development and are induced during senescence or in response to abiotic stresses,

mechanical wounding and herbivory. Such multitasking, coupled with the fact that many

genes encoding these proteins belong to large gene families, often make it difficult to

prove a specific role in resistance or defence (van Loon, 2006; van Ooijen et al., 2007).

Furthermore, much of our understanding is based on a relatively small number of

interactions (egs. Pseudomonas syringae pv. maculicola; Alternaria brassicicola),

involving well-defined gene-for-gene systems that exhibit HR in a single host plant,

Arabidopsis thaliana (Glazebrook et al., 2003; van Wees et al., 2003). We know much

less about the many other types of pathogenic relationships that exist in this and thus its

imperative to decipher disease resistance genes in taro induced as a result of P. colocasiae

attack.

In general, plant defence against disease causing organisms follows three steps:

pathogen recognition, signal transduction and the defence response itself, which may

either, succeed (i.e. resistance) or fail (i.e. susceptibility). This section reviews our current

understanding of genes involved in host-pathogen interactions and recent advances in our

understanding of them.


39

2.6 Pathogen recognition: the role of resistance genes

In their natural environment, plants are continuously exposed to encounters with

potential pathogens and in response have evolved various recognition strategies. All

plants possess a basal recognition strategy involving detection of conserved microbe-

associated molecular patterns (MAMPs) or pathogen-associated molecular patterns

(PAMPs) (He et al., 2007). Pathogenic molecules that elicit this plant basal immunity

include bacterial flagellin and lipopolysaccharides (LPS), fungal chitin as well as

oomycetes glucan. However, pathogens also have been able to the evolve effectors,

sometimes referred to as avirulence factors (avr), that can shut down host pattern

recognition receptors, triggering what is known as “effector-triggered susceptibility”

(Ingle et al., 2006). To combat these effectors, plants, in turn have evolved another more

specific recognition strategy – that of resistance (R) proteins that can recognize these

effectors; plant-pathogen interaction appears to be an arms race in which one species

ultimately dominates over the other (Jones and Dangl, 2006).There are two perspectives

on how R proteins work. In the receptor-ligand (elicitor) model, the R gene product

directly interacts and reacts with the avr gene product (Keen, 1990). For example, there is

a Pto resistance gene in tomato for a corresponding avrPto avirulence gene from

Pseudomonas syringae pv tomato (Tang et al., 1996). An alternate example is the rice

Pita gene interacting with the AVR-Pita gene from the rice blast fungus, Magnaporthe

grisea (Jia et al., 2000). However, other attempts to show direct interaction of cloned R

and avr gene products have failed (Luderer et al., 2002). The lack of more demonstrable

evidence of this model has led to the “guard hypothesis”, which postulates that the

interaction is actually indirect in that the R protein is activated when an interfering protein

(guardee) interacts directly with the avr factor (van der Biezen and Jones, 1998). A

conformational change in the guardee protein resulting from its interaction with the avr


40

factor then activates the R protein to counteract the influence of the avr factor or induce

additional plant defence. This model was supported when the Rpm1 resistance gene in

Arabidopsis was shown to provide resistance to two sequences unrelated effectors,

AvrRpm1 or AvrB, from P. syringae. In this case, another protein RIN4 (the guardee

protein) interacts with these avr proteins and subsequently is hyperphosphorylated

(Mackey et al., 2002). Other evidence can be found in the interaction between Pto, Prf

and AvrPto (Mucyn et al., 2006). The guardee protein is Pto protein kinase, which is

guarded by Prf, a NBS-LRR protein. In any case, both viewpoints strongly support the

importance of these R genes in plant defence. R genes have been characterized over the

years and recent reviews emphasize in detail their molecular structure and biochemical

function (Liu et al., 2007). They can be classified into four classes: TNL, CNL,

RLP/RLK and miscellaneous (van Ooijen et al., 2007).

The first class of R genes encodes proteins belonging to the TNL or TIR-NBS-

LRR class. This class of proteins contains a Toll-like/Interleukin Receptor (TIR) domain,

a Nucleotide Binding Site (NBS) domain and a Leucine-Rich Repeat (LRR) domain. For

example, the tomato Bs4 gene encodes a protein of the TNL class that provides resistance

to Xanthomonas campestris (Schornack et al., 2004). The second class of R proteins is

the CNL class, comprising all the non-TIR NBS-LRR domain proteins. Instead of

possessing a TIR domain, a coiled coil (CC) domain is present leading to the CC-NBS-

LRR acronym; this coiled coil structure is also referred to as the leucine zipper. This class

is represented by the Prf gene in tomato conferring P. syringae resistance. The first two

classes are strictly intracellular; however, the third class comprises transmembrane

proteins with both extracellular and cytoplasmic extensions: the RLP (Receptor-Like

Protein) class. The fourth and final class of R proteins is those that could not be classified

properly according to the above mentioned structural features. Additional research is


41

required to further characterize members of this class in order to fully understand their

function.

A common feature of plant resistance genes is their mode of expression. Most

resistance genes that have been reported have constitutive or basal levels of expression.

An exception to this is the rice Xa1 resistance gene against bacterial blight (Yoshimura et

al., 1998). In this case, resistance gene expression is not detected in uninoculated plants

and is only induced upon bacterial inoculation. There are certain instances, however,

when the level of expression increases over its basal gene expression levels. The rice R

gene Xa3 or Xa26 conferring resistance to X. oryzae pv. oryzae shows gradually

increasing expression from the early seedling stage to adult stage (Cao et al., 2007).

Because the plant cell is a stochastic environment of recognition receptors and

their downstream signalling molecules, it should not be uncommon for crosstalk between

resistance genes to occur. This is also economically sound for the plant because there

would be a conservation of precious cellular and molecular resources during pathogen

defence. For example, the tomato EDS1 is necessary for the function of receptor-like

resistance proteins like Cf-4 and Ve1 and also of resistance proteins in the TNL class (Hu

et al., 2005).

2.7 Signal transduction: the role of defence signalling genes

Pathogenic recognition, then, is interconnected with the signal transduction aspect

of plant defence. The recognition event activates one or more signalling pathways,

sometimes referred to as signalling cascades. One of the first signs that recognition has

occurred is an immediate oxidative burst followed by protein kinase cascade activation

and changing ion fluxes. This leads to the subsequent activation of defence response

genes coding for degradative enzymes and other antimicrobial proteins (Rivas and

Thomas, 2005).


42

Plant defence signal transduction pathways usually involve signalling molecules

(e.g. systemin, ethylene, jasmonic acid [JA] and salicylic acid [SA]), protein kinases

(primarily the MAP kinases), ion channels and/or secondary messengers (e.g. Ca2+

and

diacylglycerol). It also is interesting to note that the genes whose expression are regulated

by the signalling cascade include not only defence genes but also wound-responsive

genes, as there actually is a functional overlap between wound response signalling and

resistance signalling (Durrant et al., 2000). Crosstalk between both abiotic and biotic

stress responses has been reviewed recently (Fujita et al., 2006).

These seemingly universal features of plant defence signalling would suggest

conserved and overlapping pathways among different resistance genes and pathosystems.

Such interplay between these various signal transduction cascades has been reviewed in

detail (Koornneef et al., 2008). This is demonstrated in tomato when Pto binds

transcription factors that possess homology to ethylene-response factors (Gu et al., 2002),

suggesting that ethylene signalling plays a role in Pto-mediated resistance. The

transcription factors actually bind to GCC boxes that are motifs found in the regulatory

elements of the defence-associated PR genes (Chakravarthy et al., 2003). Pto expression

also results in the elevation of MAP kinase activity (e.g. LeMPK2 and LeMPK3),

demonstrating the involvement of the MAP kinase cascades (Pedley and Martin, 2004).

Finally, although not yet demonstrated in tomato, a SA-binding protein SABP3 mediates

the hypersensitive response in Pto/avrPto-expressing tobacco (Pedley and Martin, 2003),

indicating a connection with salicylic acid signalling and thus PR gene activation and

expression. It is less clear whether and which exclusive pathways may be involved with

specific recognition receptors or plant-pathogen interactions.

2.7.1 Mitogen-associated protein (MAP) kinase signalling

The MAP kinase cascade is one of the central and most studied features of plant

defence signalling (Zhang and Klessig, 2001). This pathway also is present in animals,


43

indicating evolutionary conservation of this very efficient mechanism (Nishihama et al.,

1995). In plants, the MAP kinase signal transduction cascades are activated readily when

plants are subjected to both pathogens and pathogenic elicitors. For example, resistant

tomatoes inoculated with X. campestris pv. vesicatoria or P. syringae pv. tomato show

increased mRNA levels of the MAP kinase LeMPK3 (Mayrose et al., 2004). Similarly,

this was observed after treatment with fungal elicitor incubation or mechanical wounding

(Mayrose et al., 2004). Interestingly, the increased levels of transcript correlated with

LeMPK3 kinase activity. As MAP kinases usually play a central role in various signal

transduction cascades, LeMPK3 may be involved widely in the defence response

signalling in plant. Microarray data from various tomato pathosystems also support the

wide spread involvement of MAP kinases (Gibly et al., 2004; Robb et al., 2009).

2.7.2 Jasmonic acid/jasmonate (JA) signalling

Jasmonic acid (JA) is a lipid-derived hormone that plays an important regulatory

role in various features of plant development and defence (Wasternack et al., 2006).

Exogenous application of JA signaling compounds, including jasmonic acid, methyl

jasmonate, as well as their conjugated compounds to the plant cell culture or intact plant

stimulates biosynthesis of secondary metabolites (Tamogami et al., 1997). Induction of

secondary plant metabolite accumulation by the JA signaling pathway is not limited to

certain types of metabolites, but includes a wide variety of plant secondary products

including terpenoids, flavonoids, alkaloids, and phenylpropanoids plus many other types

of secondary metabolites in most plants. Therefore, the JA signaling pathway is generally

regarded as an integral signal for biosynthesis of many secondary plant products. Also

pathogen attack stimulate endogenous JA biosynthesis in plants, the JA signaling pathway

is regarded as a transducer or mediator for signaling, leading to accumulation of

secondary plant metabolites (Mueller et al., 1993).


44

2.7.3 Ethylene signalling

Working synergistically with jasmonate is the hormone ethylene, which has been

shown to act during the plant defence response (Xu et al., 1994). Its roles in plant

development are well-established (Chang et al., 2008). Its role in defence can be said to

be dual since it has been implicated as both a signal molecule during plant resistance

(Boller, 1991) and a virulence factor that can lead to pathogenesis, symptom expression

and plant susceptibility (Lund et al., 1998; Chagué et al., 2006). Therefore, ethylene is

thought to have differential effects during plant defence in different pathosystems (van

Loon et al., 2006). Regulation and control of events during ethylene signalling mostly

happen at the level of ethylene biosynthesis (de Paepe and van der Straeten, 2005). A

model has been proposed in which these receptors function as negative regulatory factors

of downstream ethylene-responsive defence genes and responses (Klee, 2002). This and

the fact that ethylene biosynthesis shows interplay with certain MAP kinase cascades

(Kim et al., 2003) demonstrates the undeniable role of ethylene during plant defence.

2.7.4 Salicylic acid (SA) signalling

Salicylic acid (SA) plays an important signalling role in the activation of plant

defence responses following pathogenic invasion, including both systemic (termed

systemic acquired resistance) and localized responses usually characterized by HR

(Dempsey et al., 1999). Several potential components of the SA signalling pathway

initially were identified and cloned in tobacco including the bZIP transcription factors

(Zhou et al., 2000). The bZIP transcription factors bind to SA-responsive elements in the

promoters of defence genes, primarily those of pathogenesis-related or PR genes (Klessig

et al., 2000). This, coupled with increasing genetic and biochemical evidence showing

crosstalk between SA-, ethylene- and JA-associated defence pathways, underlie the

crucial significance of salicylic acid signalling in plant resistance (Pedley and Martin,

2003).


45

2.8 Defence response: the role of plant defence genes

R protein activation after pathogen detection alerts a battery of signalling cascades

ranging from MAP kinases to SA. The end result of these pathways is a sort of

transcriptional reprogramming (Caplan et al., 2008) that results in a multifaceted defence

response. This can involve the up or down regulation of hundreds of genes, some of

which “target the pathogen”.

In addition to programmed cell death, tissue repair, strengthening of structural

defences and production of antimicrobial phytoalexins, various novel proteins are induced

during pathogen attack, known collectively as pathogenesis-related (PR) proteins. It

should be stressed that many of these genes are involved in secondary metabolism, such

as shikimate and phenylpropranoid pathways (Somssich and Hahlbrock, 1998).

Accumulation of the pathogenesis-related proteins represents the major quantitative

change in protein composition that occurs at low levels in healthy plant parts that, upon

pathogen attack are induced both locally and systematically (van Loon, 1997). There is

evidence that specific sequences in the promoter regions of these genes are important for

the induction. The proteins induced have been grouped into 17 PR classes, according to

serology and homology (Table 2.1) (van Loon et al., 2006), though not all are induced in

all interactions nor in all plant species.

Within one family several members may share similar biological activities but

differ substantially in other properties such as substrate specificity, physicochemical

properties or subcellular localization. The inducible pathogenesis-related proteins are

mostly acidic proteins that are secreted into the intercellular space (van Loon, 1997). In

addition, basic pathogen-related proteins occur at relatively low levels in the vacuole.

The biochemical role for many of these proteins has been determined. For

example, the Chitinase (PR-3, PR-4, PR-8, PR-11), which are classified according to their


46

specific activities on different substrates, are presumed to hydrolyze chitin in fungal cell

walls (Neuhaus, 1999). There is experimental evidence that supports the role for induced

chitinase enzymes in degrading chitin in exposed hyphal tips of fungi (de A Gerhardt et

al., 1997). This will interfere with fungal growth and also release small oligosaccharide

elicitors, which may be involved in inducing and/or amplifying other plant defense

responses (de A Gerhardt et al., 1997). Glucanases (PR-2), proteinases (PR-7) and

RNases (PR-10) presumably have similar roles as hydrolytic enzymes and have activity

against bacteria, oomycetes and viruses as well (Park et al., 2005). Glucanases may have

an alternative role in viral infection through removal of virus-induced callose plugs in the

plasmodesmata. Some PR-1 proteins have been shown to interact with the plasma

membrane and inhibit the growth of oomycetes (Niederman et al., 1995), although the

exact biochemical role has yet to be elucidated. Further experiments with these

hydrolytic-enzyme-type PR proteins have shown that compartmentalization of the protein

is important and that if basic isozymes are targeted to the vacuoles, they are more

effective than acidic isozymes and untargeted enzymes. This suggests that these proteins

are more likely to be secondary line of defense against invading pathogens, with role in

degrading the invading organisms once it has already been contained.

Some PR proteins have putative roles in combating pathogenicity factors. Protease

inhibitor (PR-6) is produced that inhibit insect and microbial protease enzymes.

Polygalacturonase inhibitor proteins (PGIPs), although not classified as PR proteins, are

induced in the interactions such as between bean and Colletotrichum lindemuthianum,

where it has been shown that they accumulate more rapidly and intensely in incompatible

compared with compatible interactions. PGIPs are widespread in dicotyledenous plants in

pectin-rich-monocotyledenous plants such as onions and leeks. Since polygalacturonases

(PG) enzymes are used by many cell-wall-softening pathogens, it is believed that PGIPs


47

with specific recognition capabilities may be produced to counter these, and that this may

be particularly important in defense in fruit. For examples, PGIPs purified from pear fruit

were shown to inhibit the B. cinera-encode PG activity, but not that of the endogenous

fruits PGs required for fruit ripening and softening. Furthermore levels of PGIP mRNAs

were much higher in fruit compared to flowers and leaves. The structural similarities

between PGIPs and certain classes of plant disease resistance genes, in that they comprise

a leucine-rich repeat motif and a signal peptide for translocation to the endoplasmic

reticulum, has led to speculation that they may also have role in eliciting further

downstream defense responses in plants, or they may be an evolution link between PGIPs

and resistance genes.

The roles of other PR-proteins are more diverse. The PR-9 family is peroxidases,

involved in indirect antimicrobial activity by catalyzing oxidative cross linking of protein

and phenolics in the plant cell wall, leading to reinforcement of physical barrier

(Odjakova and Hadjiivanova, 2001). The PR-5 family of thaumatin-like proteins has

homology to permatins that permeabilise fungal membrane, whilst the PR-12 type

defensins and PR-13-type thionins are classified because of their homologies to similar

antimicrobial compounds present in other organisms such as the insect and mammalian

defensins (Fritig et al., 1998). These peptides may exert antifungal activity by altering

fungal membrane permeability and (or) inhibiting macromolecule biosynthesis (Broekaert

et al. 1997).

The role of PR-14 family lipid transfer proteins (LTPs) in plant defense is not

clearly understood but LTPs have been implicated in plant defense against viral, bacterial,

and fungal plant pathogens (Sohal et al., 1999a). Certain LTPs appear to be involved in

the formation of cutin and suberin layers in the plant epidermis, thereby strengthening

structural barriers in organs against mechanical disruption and pathogen attack


48

(Guiderdoni et al., 2002). Elevated LTP transcript or protein levels have been observed in

SAR signaling in Arabidopsis (Maldonado et al., 2002). Additionally, elevated transcripts

of genes involved in fatty acid metabolism and transport such as lipases and lipid transfer

proteins, may be important in priming the synthesis of plant defense-signaling molecules

such as JA and other oxylipins (Graham and Eastmond, 2002).

The PR-15 family of proteins are oxidoreductases, specifically oxalate oxidases.

One of the products of their activity is hydrogen peroxide, which, in turn, contributes to

oxidative stress. The genes that encode these proteins were first described in barley in

which they form a small multigene family (Zhou et al., 1998). Increased gene expression

in leaves infected by the powdery mildew fungus was associated with HR (Zhou et al.,

1998). Corresponding genes also have been reported in tomato and suppression of

expression followed by reduction in levels of oxalate oxidase proteins in the roots when

stressed by aluminum toxicity, apparently contributes to tolerance (Zhou et al., 2009).

2.9 Gene expression studies in taro pathosystems

Historically, the cytology and biochemistry of plant host-pathogen interactions

have fascinated plant pathologists for more than a hundred years (Ward, 1902) and of

course, farmers and crop breeders have selected for healthier and more resistant plants for

centuries. Modern understanding of plant pathogen interactions really began with the

seminal work by Flor on the gene-for-gene hypothesis (Flor, 1971), which stated that for

each host R gene a corresponding Avr gene exists in the pathogen and that successful

resistance requires an interaction between the protein products of these two genes that

initiates the host‟s defensive responses.

During the last two decades, advances in recombinant DNA technologies, as well

as global approaches to studying gene expression, at the mRNA (transcriptome) and

protein (proteome) levels, in both host and pathogen, have permitted explorations into the


49

molecular mechanisms underlying plant diseases. Studies of changes in the host

transcriptome (Wise et al., 2007) or proteome (Mehta et al., 2008) during infection by a

range of pathogens suggest hundreds and perhaps as many as a thousand plant genes are

up or down regulated during the response. These include genes involved in pathogen

recognition (van Ooijen et al., 2007), signal transduction (Beckers and Speol, 2006) and

the actual defence response (Fritig et al., 1998), as well as genes involved in the

redirection and recruitment of energy (Bolton, 2009). Plant-pathogen interactions are

indeed a battleground wherein plants deploy defence strategies ranging from basal

MAMP recognition to very sophisticated R gene mediated immunity, while pathogens

continually evolve effectors to suppress the host resistance response. The defence

response usually begins with recognition of the pathogenic invaders by receptors coded

for by R genes, of which a number have already been cloned and expressed in tomato.

Activation of these R protein receptors leads to establishment of the immediate oxidative

burst and various signalling cascades. The interplay of these pathways ultimately results

in expression and induction of genes coding for defence proteins, ranging from structural

proteins to PR proteins (Cramer et al., 1993). All the while, events during plant defence

are constantly utilizing resources provided by reprogrammed primary metabolism.

Taro has been used a model plant in studying defence. There is research on its

cytology and biochemistry, as well as more targeted and specific molecular biological

investigations. Still, the molecular mechanisms leading to resistance, susceptibility and

tolerance in taro remain undiscovered. The breadth of data generated by using the novel

suppression subtractive hybridization (SSH) approach would assist researcher for

producing disease-resistant taro crop plants interpretation as well as provide holistic view

of plant defence.


50

2.10 Suppression Subtractive Hybridization (SSH) approach and identification of

defense related genes

Identification of differentially expressed genes can lead to greater insights into the

molecular mechanism underlying diseases or plant pathogen interaction. To isolate those

target genes with differentially expression patterns, a variety of methods, such as

subtractive hybridization (Lamer and Palmer, 1984), mRNA differentially display

polymerase chain reaction (DD PCR) (Liang et al., 1992) representational difference

analysis (Hubank and Schatz, 1994), microarray (Chu et al., 1998) and suppression

subtractive hybridization (SSH) (Diatchenko et al., 1996), have been developed. Until

now, so many methods are available; each one has its advantages and disadvantages.

Generally speaking, most of the above methods were initially very labor intensive.

Commercial gene chip, being an automated high throughput method, can provide

quantities of information on differentially expressed genes through relatively simple

procedures. However, microarray technology and its associated equipment are very

expensive and beyond the reach of many academic laboratories. Furthermore, the lack of

genomic sequences to serve templates for probe design restricts their use to some certain

organisms although few reports have described that a variational technology (shotgun

microarrays) was employed to study the expressional changes during the parasite life

cycle of Plasmodium (Hayward et al., 2000). As a result, alternative methods are required

to identify novel genes or to study nonmodel organisms, such as varieties of

microorganisms.

SSH was firstly developed in Clontech‟s laboratories to find out the genes that are

differentially expressed in two samples at the mRNA level in eukaryotes (Diatchenko et

al., 1996). Compared with microarray, this method can be performed in the absence of

sequence information, and meet the requirements for finding novel genes in the nonmodel

organisms such as specific microbes and agricultural crops.


51

As is the case for traditional subtractive hybridization methods, the cDNA

population from which up-regulated (target) sequences are sought is termed the tester,

and the cDNA population for comparison is termed the driver. Diatchenko et al. (1996)

have described the procedure of SSH in details, and this method is generally divided into

six steps, including: (1) synthesis of tester/driver cDNAs; (2) digestion by a four base

cutting restrictive enzyme; (3) separation of the tester cDNA into two samples, followed

by the step of two different suppression–adapter ligations; (4) two successive subtractive

hybridization; (5) PCR amplification of target sequences; and (6) construction of the

subtracted library. The schematic representation of SSH is shown in Fig. 2.2 and 2.3. It

should be emphasized that two sequential hybridizations are typically performed in each

procedure of SSH to guarantee the enrichment and normalization of target fragments with

differential expressions. In the first hybridization step, two tester samples linked with

adapters 1 and 2R were mixed with a large excess of drivers and denatured separately.

They are then subjected to limited renaturation to generate types (a), (b), (c), and (d)

molecules in each sample. The concentration of high- and low-abundance sequences is

equalized among the type (a) molecules because reannealing is faster for the more

abundant molecules. Consequently, type (a) molecules are significantly enriched for

differentially expressed sequences. During the second hybridization, the two primary

hybridization samples without denaturing are mixed together, and then the freshly

denatured driver is synchronously added. The second hybridization should be carried out

over a longer period to ensure that all complementary cDNAs became double stranded.

When the reaction is completed, only the remaining equalized and subtracted ss tester

cDNAs can form type (e) hybrids, which represent the differentially expressed genes

between the tester and driver. Summarizing the entire population of molecules after two

hybridizations, five kinds of products exist in the mixture, among which (e) molecules


52

having two different adapter sequences at their 5′-ends that will allow exponential

amplification in the following PCR cycles. No PCR reactions can be expected in the other

two molecules of type (a) and (d) molecules because they have no primer binding sites.

Double-strand molecules of type (c), with only one adapter at one end, can only be

amplified at a linear rate. Type (b) molecules contain the same LITRs on both ends and

thus form stable “panhandle-like” structures after each denaturation–annealing PCR step.

This resulting “panhandle-like” structure can prevent amplification in PCR reaction

because intermolecular annealing of longer adapter sequences is both highly favored and

more stable than intermolecular annealing of the much shorter PCR primers, which is the

so called suppression PCR effect. So after these two successive nested PCR, those up-

regulated genes in testers can be obtained and used for constructing a subtracted library.

Working on the same theory, reverse SSH (switching the samples used as tester and

driver) can find out the down regulated genes in former testers.

The first application of SSH in the study of plant–microorganism interactions was

for the isolation of potato genes that are up-regulated in the HR to P. infestans (Birch et

al., 1999). Tester cDNA was prepared from potato leaf material 24 h postinoculation (hpi)

with an isolate of P. infestans that elicits the HR. The driver cDNA was prepared from a

compatible (susceptible) interaction with P. infestans, also at 24 hpi. Following SSH,

cloned amplification products, arrayed in 384 well microtitre plates and spotted onto

nylon membranes, were screened by hybridization to both tester and driver cDNAs.

Clones hybridizing only to the former probe were sequenced and were shown to be

specifically induced in the HR to P. infestans. PCR with primers designed to anneal to the

cDNA sequences obtained by this SSH confirmed that they were derived solely from the

plant. Among the HR-associated sequences were cDNAs that showed similarity to genes

implicated in apoptosis in animals, providing evidence that programmed cell death in


53

plants and animals might be related. SSH thus provides a „targeted EST‟ strategy that

allows those cDNAs that are up- or down-regulated in an interaction transcriptome to be

rapidly identified, following subtraction of constitutively expressed cDNAs.

Based on this, initial global characterization of defence gene expression in the

taro-P. colocasiae interaction was undertaken using the commercially available SSH.

Because this initial survey yielded interesting patterns of gene expression that correlated

with the observed biology, this current study was aimed at determining the expression of

selected interesting defence genes along the time course of P. colocaisae infection.

Additionally, this study was undertaken to compare and confirm the expression profiles

that can be generated through Northern blot analysis (Sharma et al., 2009).

2.11 Future Issues

Achievements today in plant biotechnology have already surpassed all previous

expectations, and the future is even more promising. The technology evolves toward the

use of tissue-specific or pathogen-inducible promoters, the expression of engineered traits

that are effective against a broad range of pathogens, and the utilization of synthetically

derived peptides and of R genes, the impact on disease management will be enhanced.

Transgenic plants with enhanced disease resistance can become a valuable component of

a disease management program in the future.

Production of improved plant varieties via genetic transformation offers an

attractive alternative to conventional breeding. Transformation of some agronomically

important monocotyledonous crops such as sugar cane (Bower and Birch, 1992), banana

(Becker et al., 2000; Khanna et al., 2004), maize (O'Kennedy et al., 2001) and wheat

(Jones and Simko, 2005) has been successfully achieved using both Agrobacterium

tumefaciens and microprojectile bombardment gene transfer methods. Genetic

transformation of C. esculenta var. esculenta, for disease resistance however has been


54

largely neglected possibly due to the difficulties in developing an efficient regeneration

system or the lack of focus on a crop of low significance to developed nations where a

large portion of funding and expertise resides.

Fukino et al. (2000) reported transformation in C. esculenta var. antiquorum

callus by particle bombardment where 96 bombardments were conducted and only two

putative transgenic plants were analyzed. Transformation in C. esculenta var. esculenta

via microprojectile bombardment (He et al., 2004) and Agrobacterium tumefaciens (He et

al., 2008) has also been reported. Another important aspect of transgenesis is the relative

activity and tissue specificity of promoters needed to control transgene expression.

Transient activity of maize polyubiquitin-1 (Ubi-1) promoter, Cauliflower mosaic virus

(CaMV 35S) and Taro bacilliform virus (TaBV-600) promoters has been examined in

both bombarded leaves (Yang et al., 2003b) and embryogenic suspension cultures (Deo,

2008) of taro.

Although it is now possible to confer many different traits through transgenics, the

highest priorities for taro would be resistance to taro leaf blight and taro beetle, which are

considered the most serious threats to production. Identifying a suitable promoter driving

these genes in taro leaves may be an issue requiring considerable experimentation on

gene expression pattern and stability in transgenic taro. Although a preliminary promoter

study has been undertaken recently (Deo, 2008), a greater range of promoters in stably

transformed plants needs to be assessed in the field.

Moreover the patterns of gene defence gene expression in the taro-P. colocasiae

pathosystem could then be used as an accurate indicator of a „defence readout‟ when

performing subsequent functional assays like gene silencing, over-expression and mis-

expression studies. The identification of defence gene expression in the course of P.

colocasiae infection, by this research provided an example to further understand the


55

molecular mechanism of how the taro plant responds to this pathogen. Understanding the

molecular biology of plant defence could be very helpful in the development of new

concepts for disease resistance and crop protection. At a more fundamental level, it might

contribute to a better understanding of factors and pathways regulating development of

various pathosystems in plants. It is indeed becoming important that particular attention

be directed into how various pathways interact rather than just expression of single genes

as this provides a better global outlook about the events in the plant itself. As plant-

pathogen interactions are not statically linear but a stochastic labyrinth of signalling

networks, a new dawn of research into plant-pathogen interactomics becomes apparent

and imperative (Paul et al., 2009). Nevertheless, detailed functional analysis of specific

genes involved in defence must be continued to avoid superficial interpretations. An

increasing number of studies slowly have started to look into the roles of different players

in the plant defence response, with the aid of various methods like RNAi silencing. Future

studies could be geared towards silencing and overexpression of P. colocasiae resistance

genes in taro and see how this affects defence gene expression. This functional genomics

era indeed raises great excitement for plant biology researchers in deciphering the still

countless mysteries associated with taro- P. colocasiae interaction.


56

Table 2.1 Recognised family of Pathogenesis-related protein (PRs) families in plants

and their putative functions (cited from van Loon et al., 2006).

Protein

family

Reporter protein

activity

Targeted

pathogen sites or

components

Type member Gene

symbols

PR-1 Pathogenesis-related

protein 1 precursor

Fungal membrane Tobacco PR-

1a

Ypr1

PR-2 1,3-β-glucanase Cell wall glucan I,

II, IV, V, VI, VII

Tobacco PR-2 Ypr2,

[Gns2

(„Glb‟)]

PR-3 Endochitinase Cell wall chitin I,

II

Tobacco P,Q Ypr3, Chia

PR-4 Endochitinase Cell wall chitin Tobacco R Ypr4, Chid

PR-5 Osmotin Fungal

membrane,

Thaumatin like

Tobacco S Ypr5

PR-6 Protease inhibitor Proteinase

Tomato

Inhibitor I

Ypr6, Pis

(‘Pin’)

PR-7 Endoprotease Endoproteinase

Tomato P69 Ypr7

PR-8 Endochitinase Cell wall chitin III Cucumber

chitinase

Ypr8, Chib

PR-9 Peroxidase Peroxidase

Tobacco

“lignin-

forming

peroxidase”

Ypr9, Prx

PR-10 Ribonuclease Pathogen-RNA, Parsley “PR-1” Ypr10


57

Ribonuclease-like

PR-11 Endochitinase Cell wall chitin, I Tobacco

“Class V”

chitinase

Ypr11,

Chic

PR-12 Defensin Fungal

membrane,

defensin

Radish Rs-

AFP3

Ypr12

PR-13 Thionin Fungal

membrane,

Thionin

Arabidopsis

THI2.1

Ypr13, Thi

PR-14 Lipid-transfer protein Lipid -transfer

protein

Barley LTP4 Ypr14, Ltp

PR-15 Oxalate oxidase Oxalate oxidase Barley OxOa

(germin)

Ypr15

PR-16 Oxalate- oxidase like Oxalate- oxidase

like

Barley OxOLP Ypr16

PR-17 Unknown Unknown Tobacco

PRp27

Ypr17


58

Figure 2.1. Major components of the signal-transduction chain from elicitor

perception to gene activation. Recognition of the elicitor by its plasma membrane

receptor stimulates transient influxes of H+ and Ca+, and effluxes of K+ and Cl–.

These ion fluxes are prerequisite for the activation of specific MAP (mitogen-

activated protein) kinases and for the generation of reactive oxygen intermediates

(the oxidative burst). Phosphorylation and dephosphorylation of some proteins is

also observed. Binding of the elicitor stimulates also the generation of jasmonic acid

via a membrane associated phospholipase. (Adapted from Odjakova and

Hadjiivanova, 2001).


59

Figure 2.2 Overview of the PCR-Select procedure. The cDNA in which specific

transcripts are to be found is referred to as tester (UL-56) and the reference cDNA is

referred to as driver (Muktakeshi). Adapted from www.clontech.com.


60

Figure 2.3. Schematic diagram of PCR-Select bacterial genome subtraction. Type e

molecules are formed only if the sequence is present in the tester (UL-56) DNA, but

absent in the driver DNA (Muktakeshi). Solid lines represent the Rsa I-digested

DNAs. Solid boxes represent the outer part of the Adaptor 1 and 2R overhanging

strands and corresponding PCR Primer 1 sequence. Clear boxes represent the inner

part of Adaptor 1 and the corresponding Nested Primer 1 sequence. Blue boxes

represent the inner part of Adaptor 2R and the corresponding Nested Primer 2R

sequence.

Chapter II Review of literature -...

Documents

Transcript of Chapter II Review of literature -...