7

CHAPTER 2: REVIEW OF LITERATURE

2.1. The family-Zingiberaceae

India has one of the richest ethno botanical traditions in the world with

more than 7000 species of plants found in different agro-ecosystems and

used by various indigenous systems of medicine and industries. Over 95%

of the plants used by the herbal or pharmaceutical industry are collected

from wild sources. Zingiberaceae family constitutes a vital group of

rhizomatous medicinal and aromatic plants. Generally, the rhizomes and

fruits are aromatic, tonic and stimulant; occasionally they are nutritive.

Some of the zingiberaceous taxa are used as food as they contain starch in

large quantities while others are used as ornamentals, cultivated for their

showy flowers. The family comprises of 52 genera and about 1400 species

(Buru and Smith 1972). Plants of this family are found in the tropics of

Africa, Asia and Americas with the greatest number in South-east Asia. In

India, the family is represented by 178 species under 22 genera (Jain and

Prakash 1995) with greater species concentration in Northeastern and the

peninsular region. The important genera of Zingiberaceae are Curcuma,

Kaempferia, Hedychium, Amomum, Zingiber, Alpinia, Elettaria and

Costus. Members of this family are small to large perennial plants with

creeping horizontal or tuberous rhizomes. An important distinguishing

characteristic is the presence of essential oils in their tissues. Plants are

8

small to large herbaceous in nature and consists of distichous leaves with

basal sheaths that overlap to form a pseudo stem. The plants are either self-

supporting or epiphytic. Flowers are hermaphroditic. In some species,

leaves usually do not have an odor (Zingiber sps.) in contrast to other

(Curcuma sps.). The plants are perennial, medium-sized herbs with stout

rhizomes. Most of the species produce the inflorescence on a separate shoot

directly from the rhizome, at the tips of a short or long peduncle. The

duration of the flowers is very short and differs from species to species.

Of the several genera belonging of family Zingiberaceae, the genus

Curcuma is a very important one consisting of about 110 species

distributed in tropical Asia and the South-pacific region (Ravindran et al.

2007). The greatest diversity of the genus occurs in India, Myanmar and

Thailand and extends to Korea, China, Australia, South Pacific islands,

East and West African nations, Malagasy, Caribbean islands and Central

America. The genus Curcuma was named by Linnaeus (1753). The generic

epithet is derived from the Arabic word karkum, meaning yellow, referring

to the yellow color of the rhizome, and Curcuma is the latinized version

(Purseglove 1981; Sirirugsa 1999). The genus Curcuma comprises several

species of which Curcuma longa is the cultivated one having a lucrative

export market worldwide while some of its wild relatives like Curcuma

zedoaria, Curcuma angustifolia, Curcuma aromatica etc. are medicinal in

nature and widely used in various pharmaceutical industries.

9

2.2. Importance of Curcuma longa L.

Curcuma longa L. popularly known as the turmeric, is held sacred from

time immemorial. Turmeric is a native of Southeast Asia. It has been

cultivated most extensively in India followed by Bangladesh, China,

Thailand, Cambodia, Malaysia, Indonesia and Philippines (Ravindran et al.

2007). It is a perennial plant (Figure 2.1) having a short stem with large

oblong leaves and bears ovate or oblong rhizomes, which are often

branched and brownish-yellow in colour. India ranks first in production,

consumption and export of turmeric. The annual production is about

635,950 tonnes from an area of 175,190 hactares as per 2002-2003 data,

which has increased by another 7.6% in the recent times (Ravindran et al.

2007). World demand for turmeric is everincreasing.Turmeric is a

medicinal plant extensively used in Ayurveda, Unani and Siddha medicine

as home remedy for various diseases (Ammon and Wahl 1991). Turmeric

power is traditionally used in Indian medicine for the treatment of biliary

disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorders,

rheumatism and sinusitis (Ammon and Wahl 1991). Duke (2003) reported

turmeric as the natural Cox-2 inhibitors. Recent researches on turmeric are

focused on its anti-oxidant, hepato-protective, anti-inflammatory and anti-

microbial properties in addition to its use in cardiovascular and

gastrointestinal disorders (Ravindran 2007). Curcumin the coloring matter

10

Figure 2.1: Curcuma longa L.

11

of turmeric, is used as a safe food color in cheese, spices, mustard, cereal

products, pickles, soups etc in several countries.

2.2.1. Distribution of Curcuma longa L.

Curcuma longa L. distributed in tropical Asia and the South-pacific

region (Ravindran et al. 2007). The greatest diversity of the genus occurs in

India, Myanmar and Thailand and extends to Korea, China, Australia,

South Pacific islands, East and West African nations, Malagasy, Caribbean

islands and Central America.

2.3. Importance of Curcuma zedoaria (Berg.) Rosc.

The rhizome of C. zedoaria is used to increase appetite and also as a tonic

particularly prescribed to ladies after childbirth. In case of cold, a decoction

of zedoary, long pepper (Piper longum), cinnamon (Cinnamomum verum),

and honey is given. In Ayurveda it is an ingredient of “Braticityadi

kwatha”, used in high fever (Thakur et al. 1989). Root is useful in

flatulence and dyspepsia, and as a corrector of purgatives. Fresh root

checks leucorrhoea and gonorrhoeal discharges. Root powder is a good

substitute for many foreign foods for infants. The juice from the tubers is

given to children for worms. Juice of the leaves is given in dropsy

(Nadkarni 1982). It is an odoriferous ingredient of the cosmetics used for

the cure of chronic skin diseases caused by impure or deranged blood

(Nadkarni 1998). Decoction of fresh rhizomes is used for blood

purification.

12

2.3.1. Distribution of C. zedoaria (Berg.) Rosc.

The round zedoary is mostly found in India and South-East. Asia. Curcuma

zedoaria has 4 -6 leaves with 20-60cm long lamina. The leaf lamina is

oblong-lanceolate, finely acuminate and glabrous on both the surfaces.

Flower stalk is 20-25cm long, emerging before the leaves (Figure 2.2).

Flowers are yellow, while the flowering bract is green tinged with red.

Calyx is 8mm long, corolla tube is twice as long as the calyx. Capsule is

ovoid, trigonous, thin smooth and bursting irregularly.

2.4. Importance of Curcuma aromatica Salisb.

Rhizomes are used in combination with astringents and aromatics for

bruises, sprains, hiccough, bronchitis, cough, leucoderma and skin

eruptions (Warrier et al. 1994). The rhizomes have an agreeable fragrant

smell and yield a yellow colouring matter like turmeric and the fresh root

has a camphoraceous odour. The dried rhizome of curcuma aromatia used

as a carminative and aromatic adjunctant to other medicines (Nadkarni,

1998). Asolkar et al. (1992) reported that the oil is used for treatment of

early stage of cervix cancer. Rhizome is an antidote for snakebite and

carminative (Husain et al. 1992). Essential oil from rhizomes showed

antifungal and antimicrobial activity. Oil also showed inhibitory effect on

sarcoma in mice (Asolkar et al. 1992).

13

2.4.1. Distribution of Curcuma aromatica Salisb.

Curcuma aromatica is found wild throughout India and cultivated in

Bengal and Kerala. It is a perennial tuberous herb with annulate, aromatic

yellow rhizome which is internally orange-red in colour. Leaves are elliptic

or lanceolate-oblong, caudate-acuminate, 30-60cm long, petioles as long or

even longer, bracts ovate, recurved, more or less tinged with red or pink.

Flowers (Figure 2.3) are pink, lip yellow, obovate, deflexed, sub-entire or

obscurely three lobed.

2.5 Importance of Curcuma angustifolia Roxb.

This species of plant is of great nutritional value, especially as a source of

starch for Indian foods and medicines. The rhizomes of Curcuma

angustifolia are typically ground into flour, which can then be mixed

together with milk or water to form a nutritious meal. This flour was a

common commercial crop in the 1800s (Ravindran et al. 2007). Most

importantly, the West has begun to notice its potential as a source of

nutrition and as a non-irritating diet for patients suffering from specific

chronic ailments, recovering from fevers, or experiencing irritations of the

gastrointestinal tract, the lungs, or the excretory system. A drink including

C. angustifolia as an ingredient is also used as a replacement of breast-

milk, or as a nutritional supplement for babies a short while after weaning

(Doble 2012). It is used as a primary ingredient in cakes, fruit preserves,

14

Figure 2.2: Curcuma zedoaria (Berg.) Rosc.

15

biscuits, and puddings (Doble 2012). It is used to heal peptic ulcers and in

treatments of dysentery, diarrhea, and colitis.

It is often employed as an herbal tonic for patients suffering from

tuberculosis. It is also used to sooth coughs and is used to treat bronchitis.

Essential oils from C. angustifolia have been extracted and are used in

antifungal medications. Compounds in the leaves of this plant have also

been shown to have potential as antibacterial agents.

2.5.1. Distribution of Curcuma angustifolia

Curcuma angustifolia is most commonly found growing wild in India,

especially in the northeast and western coastal plains and hills, including

the the states of Maharashtra, Madhya Pradesh, Andhra Pradesh, Himachal

Pradesh, Odisha, Chhattisgarh, Tamil Nadu, and Kerala. It is a perennial

and a flowering plant, with modest and small spiked inflorescences (Figure

2.4) of three or four yellow, funnel-shaped flowers within tufts of pink

terminal bracts (coma bracts) (Ravindran 2012). The bracts are boat-shaped

and encase the entire perianth of the flower. Flowers are usually seen at the

beginning of the rainy season from July to August, before the leaves have

had the chance to fully develop, and they continue to flower even after the

leaves have fully developed. Leaves are typically simple, green, glabrous,

and lance late, with margins that are entire. Leaves may grow to about 36–

37 cm length and 8–10 cm in width. Of great significance to C. angustifolia

16

Figure 2.3: Curcuma aromatica Salisb.

17

is its strong rhizome, which can grow to be up to 1.5 meters in length. The

plant typically grows to be from three to four feet in height.

2.6. Pathogens affecting turmeric production

Turmeric (Curcuma longa L.) is completely sterile and propagated

exclusively by vegetative means using rhizomes. The lack of genetic

diversity due to exclusive vegetative propagation has made them vulnerable

to a range of fungal and bacterial pathogens that causes significant losses.

At the global level, the most serious constraint to turmeric production is

rhizome rot disease caused by the fungal pathogen Pythium

aphanidermatum and Pythium graminicolum. Pythium is an oomycete in

the order Peronosporales. The hyphae are hyaline and the mycelium has no

cross walls. To differentiate P. aphanidermatum from other Pythium

species requires examination under a microscope of the sporangia, oogonia

and antheridia. Sporangia are the asexual spores and in the case of P.

aphanidermatum, they are inflated. Selvan et al. (2002) reported that

Pythium aphanidermatum is the major organism in India causing rhizome

rot in turmeric. It was first reported from Andhra Pradesh and Tamil Nadu

and losses to the tune of 50% were reported from Telangana areas of

Andhra Pradesh (Rao and Rao 1988). Pythium aphanidermatum is a soil-

borne, necrotrophic oomycete, which shows both above ground and below

ground infections on turmeric plant. The diseased plant shows progressive

18

Figure 2.4: Curcuma .angustifolia Roxb.

19

yellowing of leaves, which proceeds first along the margins and later

covers the entire leaf, causing it to dry up.The base of the aerial shoots

shows water-soaked soft lesions. Infection gradually passes to the

rhizomes, which begin to rot and become soft. This results in complete

decaying of the inner tissue and finally the plant dies (Figure 2.5). Dipping

seed rhizomes in Ridomil (0.25%) for 40 min, followed by a soil drench or

treatment with Carbendazim at the first appearance of symptoms can result

in considerable control of rhizome rot (Rathaiah 1982; Shankaraiah et al.

1991). This method of control, however, is unsatisfactory as it provides

very little protection against Pythium, so that rot management fully depends

on cultivation practices.

2.7. Developing disease resistance through biotechnology

Developing disease resistance involves the identification of resistance

genes in traditional cultivars or wild species and the incorporation of this

resistance into commercially acceptable varieties. This can be done through

conventional breeding using hybridization techniques or through molecular

biotechnology using genetic transformation (Hammond-Kosack and Jones

2000). Traditional breeding approaches are particularly difficult in

rhizomatous turmeric as all of their cultivated varieties are sterile in nature

and exhibit obligatory vegetative mode of propagation. In turmeric, natural

20

Figure 2.5: The rhizome rot disease caused by Pythium aphanidermatum.

A) Infected rhizomes; B) Progressive yellowing of the diseased plant; C)

Infected dried plant.

21

seed set is absent due to its sterility and stigmatic incompatibility

(Damayanthi et al. 2003). Therefore the only opportunity for plant breeders

is to opt genetic transformation to overcome the constraints imposed by the

sterility of these rhizomatous plants by transferring specific resistance traits

without compromising on other important traits of interest. Protocols have

been developed in turmeric for plant regeneration from axillary buds

(Balachandran et al. 1990; Tyagi et al. 2004; Mohanty et al. 2010; Shirin

et al. 2000; Rahman et al. 2005; Parida et al. 2010) that makes the genetic

transformation of the whole plant possible. Reports of the genetic

transformation in turmeric using biolistics (Shirgurkar et al. 2006) or

Agrobacterium (Mahadtanapuk et al. 2006) show that it is possible to

express foreign proteins in turmeric such as β-glucoronidase (GUS),

neomycin phosphotransferase-II (NPT-II), phosphinothricin acetyl

transferase (PAT), 1-aminocyclopropane-1-carboxylic acid synthase (ACC)

and others (Shirgurkar et al. 2006; Mahadtanapuk et al. 2006).

Furthermore, different constitutive or tissue specific promoters such as

Cauliflower Mosaic Virus 35S (CaMV35S) and maize ubiquitin have been

tested in turmeric with great success (Shirgurkar et al. 2006; Mahadtanapuk

et al. 2006). Inspite of availability of transformation technology in

turmeric, to date, no resistance genes to the most destructive turmeric

diseases have been cloned and transferred to susceptible turmeric cultivars

for developing disease resistance.

22

2.8. Plant disease resistance genes

Flor (1971) had developed a gene-for-gene interaction model which shows

that the induction of the plant defense response is initiated by the plant‟s

recognition of specific molecules called elicitors produced by the

pathogens. These elicitors are encoded directly or indirectly by avirulence

alleles (avr) of the pathogens. Resistance alleles (R) are thought to encode

receptors for these elicitors. Several evidences indicate that high-affinity

receptors for pathogen-derived signal functions either at the plant cell

surface or inside by conversion of an extracellular signal into an

intracellular signal. Upon recognition of the signal, initiation of an

intracellular signal transduction cascade occurs, triggering activation of the

defense arsenal of the challenged host plant cell (McDowell and

Woffenden 2003; Staskawicz et al.1995). Most R-gene-triggered

resistance is associated with a rapid defense response, termed as

hypersensitive response (HR) resulting in a localized cell and tissue death

at the site of infection, preventing the further spread of the infection

(Hammond-Kosack and Jones 1997). However, the local response however

results in a non-specific systemic acquired resistance (SAR) throughout the

plant. This multi-component response largely depends upon cellular

resources and metabolic reallocation. In absence of an immune system in

plants, they maintain a constant observation over the infecting pathogens

23

by expressing large numbers of R-genes whose product responds to the

products of AvR-genes. These interactions are of two types- direct and

indirect. The direct interaction suggests that the pathogen Avr effectors

interact with plant R proteins directly to trigger R-gene-mediated resistance

signaling. Rice R-gene Pi-ta directly interacts with AVR-pita from rice

blast fungus Magnaporthe grisea while the susceptible allele of Pi-ta

shows no interaction (Jia et al. 2000). However, only a few evidences

reports direct R-Avr interaction. The indirect interaction is otherwise call as

the „guard hypothesis‟ (Van der Biezen and Jones 1998). Here, R-protein

activates resistance when they interact with another plant protein (usually a

guardee) that is targeted and modified by the pathogen in order to create a

favorable environment. In other words, the Avr protein interacts with the R-

protein indirectly through a host protein as well as a molecular chaperon to

form an R-Avr complex to induce resistance. Rather than acting as passive

security guards that idly wait for specific signals from an invader, R

proteins actively and continuously monitor key physiological processes that

are targeted by pathogens. Jones and Dangl (2006) validated the guard

hypothesis using the AvrPto-Prf model from tomato. The Prf is the real R-

gene that encodes a NBS-LLR protein to guard Pto, a component of R-Avr

complex as a target of AvrPto. There are large number of plant resistance

genes or elicitor receptors which have been isolated and cloned based on

positional or map-based cloning and transposon tagging, (Sharma et al.

24

2009), in barley (Mammadov et al. 2006), in pea (Corinne E et al. 2007), in

rice (Chen et al. 2003), rice Xa21(Song et al. 1995) etc. Hammond-Kosack

& Jones (1997) had grouped the isolated R- genes into five basic classes.

The class-I is represented by maize HM1 gene which encode a reductase

that detoxify HC toxin of the fungus Cochliobolous carbonum. The classes

II are represented by majority of functionally known R-genes (RPS2,

RPM1, N, L6 etc) and encode cytoplasmic receptor like proteins that

contain a leucine rich repeat (LRR) domain and nucleotide binding site

(NBS). Class III includes Pto gene from tomato which do not have NBS-

LRR domain but encodes a protein having serine-threonine protein kinase

domain. Class IV includes the Xa21 gene of rice, which encodes an extra-

cytoplasmic LRR domain and an intra-cellular serine/threonine kinase

domain while class V represents Cf genes of tomato that encodes

transmembrane receptors with an extracellular LRR domain and an

intracellular serine –threonine kinase. Recently, cloning of more R- genes

has added new structures and motifs to the R-proteins. HSl pro1

gene from

beets considered till now to have a transmembrane-LRR domain poorly fit

the LRR consensus and has minimal similarity to other R-genes (Cai et al.

1997). The Mlo gene in barley, resistant to powdery mildew fungus

Blumeria codes for a simple putative membrane protein whose function

may be negative regulator of certain defense responses. The tomato Ve1

and Ve2 proteins contain a transmembrane LLR attached with a PEST

25

domain for protein degradation and a short protein motif for endocytosis

(Kawchuk et al. 2001). The Arabidopsis RPW8 gene encodes a protein

containing a transmembrane domain (TM) attached with a coiled-coil

domain (CC) (Xiao et al. 2001). RRS1-R-gene from Arabidopsis that

confers resistance to Ralstonia solanacearum encodes a TIR-NBS-LRR

protein with an additional transcriptional factor WRKY domain towards its

C-terminus and a zinc finger towards the, N-terminus which has a

definitive role in defense response (Deslandes et al. 2002). Rpg1 gene from

barley also represents class III R-genes but has two intracellular serine-

threonine protein kinases (Brueggmann et al. 2002). More recently

identified Pi-d2 gene in rice conferring resistance to rice blast fungus

encodes a protein with intracellular serine-threonine kinase attached with

an extracellular binding-lectin (B-lectin) (Chen et al. 2006). In addition, the

three cloned rice bacterial blight resistance genes Xa5, Xa13 and Xa27

possesses completely new and different structural motifs and do not show

similarity to any of the five known R-gene types.

2.8.1. NBS-LRR disease resistance genes

The NBS-LRR class of disease resistance genes represents the largest class

of R-genes with more than 50 cloned genes from different plant species as

listed by Joshi and Nayak (2011) (Table 2.1). They are basically located in

the cytoplasm and develop resistance response against a variety of

pathogens (Hulbert et al. 2001). Traut (1994) reported that this class of

26

gene encodes proteins with a variable N- terminal domain of approximately

200 amino acids, connected by a predicted NBS domain of approximately

300 amino acids and a more variable C-terminal tandem array of

approximately 10 to 40 short LRR motifs (Jones and Jones 1997). Further,

the NBS-LRR R-genes are categorized into three subgroups based on the

motif within their N-terminus: TIR group, coil-coiled (CC) or leucine

zipper (LZ) group and non-motif group (Joshi and Nayak 2011).

27

Table 2.1: NBS-LRR disease resistance genes in plants

Gene Host Pathogen Protein

type

Reference

RPS2 Arabidopsis Pseudomonas

syringae

CC-

NBS-

LRR

Bent et al.1994

RPS5 Arabidopsis Pseudomonas

syringae

LZ-NBS-

LRR

Warren et al.1998

RPM1 Arabidopsis Pseudomonas

syringae

CC-

NBS-

LRR

Boyes et al.1998

RPP8/HRT Arabidopsis Peronospora

parasiticia

CC-

NBS-

LRR

McDowell et

al.1998 RPP13 Arabidopsis Peronospora

parasiticia

CC-

NBS-

LRR

Bittner-Eddy et

al.2000 RCY1 Arabidopsis Cucumber

mosaic virus

CC-

NBS-

LRR

Hideki et al.2002

RPP/HRT Arabidopsis Turnip crinkle

virus

CC-

NBS-

LRR

Cooley et al.2000

RPM1 Tomato Pseudomonas

syringae

CC-

NBS-

LRR

Grant et al.1995

Prf Tomato Pseudomonas

syringae

CC-

NBS-

LRR

Salmeron et

al.1996 I2 Tomato Fusarium

oxysporum

CC-

NBS-

LRR

Ori et al.1997

Mi-1 Tomato Meloidogyne

javanica

CC-

NBS-

LRR

Milligan et

al.1998 Mi-9 Tomato Meloidogyne

javanica

CC-

NBS-

LRR

Jablonska et

al.2007 Sw-5/Mi Tomato Tospovirus CC-

NBS-

LRR

Brommonschenkel

et al.2000 Rx2 Potato PVX (Potato X

virus)

CC-

NBS-

LRR

Bendahmane et

al.1999 Gpa2/Rx1 Potato Globodera

pallida/PVX

CC-

NBS-

LRR

Vossen et al.2000

R1 Potato Phutophthora

infestans

CC-

NBS-

LRR

Ballvora et

al.2002 Mla1 Barely Blumeria

graminis

CC-

NBS-

LRR

Zhou et al.2001

Mla6 Barely Blumeria

graminis

CC-

NBS-

LRR

Haltermann et al.

2001 Mla12 Barely Blumeria

graminis

CC-

NBS-

LRR

Shen et al.2003

Mla13 Barely Blumeria

graminis

CC-

NBS-

LRR

Haltermann and

Wise 2004

28

Table 2. 1(continued)

Gene Host Pathogen Protein

type

Reference

Pib Rice Magnaporthe grisea CC-NBS-

LRR

Wang et l 1999

Pi-ta Rice Magnaporthe grisea CC-NBS-

LRR

Bryan et

al.2000 Pi36 Rice Magnaporthe grisea CC-NBS-

LRR

Liu et al.2007

Xa1 Rice Xanthomona oryzae CC-NBS-

LRR

Yoshimura et

al.1998 Rp1 Maize Puccinia sorghi CC-NBS-

LRR

Collins et

al.1999 Dm3 Lettuce Bremia lactucae CC-NBS-

LRR

Meyers et

al.1998 Bs2 Pepper Xanthomonas

campestris

CC-NBS-

LRR

Tai et al.1999

Pm3b Wheat Blumeria graminis CC-NBS-

LRR

Yahiaoui et

al.2004 Lr10 Wheat Puccinia triticina CC-NBS-

LRR

Feuillet et

al.2003 Pl8 Sunflower Plasmopara

halstedii

CC-NBS-

LRR

Radwan et

al.2005 RB Potato Phytophthora

infestens

CC-NBS-

LRR

Song et

al.2003 Rpi-

blb1

Potato Phytophthora

infestens

CC-NBS-

LRR

Vossen et

al.2003 Fom-2 Melon Fusarium

oxysporum

CC-NBS-

LRR

Joobuer et

al.2004 Rpg1-b Soybean Pseudomonas

syringae

CC-NBS-

LRR

Ashfield et

al.2004 R3a Potato Phytophthora

infestens

CC-NBS-

LRR

Huang et

al.2005 Rxo1 Maize Xanthomonas oryzae CC-NBS-

LRR

Zhao et

al.2005 RPS4 Arabidopsis Pseudomonas

syringae

TIR-NBS-

LRR

Gassmann et

al.1999 RPP1 Arabidopsis Peronospora

parasiticia

TIR-NBS-

LRR

Botella et

al.1998 Rpp10 Arabidopsis Peronospora

parasiticia

TIR-NBS-

LRR

Botella et

al.1998 Rpp14 Arabidopsis Peronospora

parasiticia

TIR-NBS-

LRR

Botella et

al.1998

29

Table 2.1(continued)

Gene Host Pathogen Protein

type

Reference

RPP4 Arabidopsis Peronospora

parasiticia

TIR-NBS-

LRR

Van der Beizen et

al.2002 RPP5 Arabidopsis Peronospora

parasiticia

TIR-NBS-

LRR

Parker et al.1997

SSI4 Arabidopsis P. syringae pv.

maculicola

TIR-NBS-

LRR

Shirano et al.2002

RLM Arabidopsis Leptosphaeria

maculans

TIR-NBS-

LRR

Staal et al.2006

L6 Flux Melampsora lini TIR-NBS-

LRR

Lawrence et

al.1995 L Flux Melampsora lini TIR-NBS-

LRR

Ellis et al.1999

M Flux Melampsora lini TIR-NBS-

LRR

Anderson et

al.1997 P Flux Melampsora lini TIR-NBS-

LRR

Dodds et al.2001

P2 Flux Melampsora lini TIR-NBS-

LRR

Dodds et al.2001

Bs4 Tomato Xanthomonas

campestris

TIR-NBS-

LRR

Schornack et

al.2004 N Tobacco Tobacco mosaic

virus

TIR-NBS-

LRR

Witham et al.1994

RRS1-R Arabidopsis Ralstonia

solanascearum

WRKY-

TIR-NBS-

LRR

Deslandes et

al.2003 Pi-9 Rice Magnaporthe grisea NBS-LRR Qu et al.2006

Pi2 Rice Magnaporthe grisea NBS-LRR Zhou et al.2006

Piz-t Rice Magnaporthe grisea NBS-LRR Zhou et al.2006

Cre3 Wheat Heterodera avenuae NBS-LRR Lagudah et al.1997

Cre1 Wheat Heterodera avenuae NBS-LRR Majnik et al.2003

I2C Tomato Fusarium

oxysporum

NBS-LRR Ori et al.1997

Hero Tomato Globodera

rostochiensis

NBS-LRR Ernst et al.2002

30

2.8.2. TIR and non-TIR domains

Pan et al. (2000) has classified the NBS-LRR class of R-genes into two

distinct subclasses such as TIR and non TIR domains based on the presence

or absence of an N-terminal motif with homology to the cytoplasmic

domains of the Drosophila Toll protein and the mammalian interleukin-1

receptor (TIR). Recently, these two classes are refered to as „CNL‟ and

„TNL‟ proteins respectively (McHale et al. 2006; Meyers et al. 2005).

These types of domains are believed to be involved in signaling and not

ligand binding as they are found to have similarity with cytoplasmic

signaling domains. Xu et al. 2000 reported that TIR domain interactions

between receptors and adaptors play a critical role in activating conserved

cellular signal transduction pathways in response to bacterial

lipopolysaccharides, microbial and viral pathogens and growth factors.

Considering the similarity in sequence and related functions of the

Drosophila, mammals and plant R-genes, the plant proteins will function in

an analogous manner. Moreover Hammond-Kosack and Jones (1997)

reported that the presence of TIR domains in plant R-genes regulate the

production of activated oxygen resulting in an oxidative burst during gene-

for-gene defense responses. The TIR type NBS-LRR-genes are found

extensively in dicots and rare or absent in monocots (Bai et al. 2002; Myers

et al. 2003). Pan et al. (2000) and Hulbert et al. (2001) reported that the

non-TIR subclass is characterized by the presence of a coiled-coil structure

31

(CC) or leucine zipper (LZ) structure. The coiled-coil structure consist of

two or more alpha helices with a super helical twist and represent a heptad

repeat sequence (abcdefg)n where „a‟ and „d‟ are hydrophobic residues

while the residues at the „e‟ and „g‟ positions are charged and polar (Fluhr

et al. 2001). Most of the NBS-LRR-genes in dicots and monocots including

cereals contain the coiled-coil structure. It is believed that the CC or LZ

domain facilitates the formation of a coiled-coil structure to promote

oligodemerization with a wide variety of proteins including structural

proteins and transcription factors or specific interactions with other proteins

(Nooren et al. 1999; Burkhard et al. 2001). R-gene products can exist as

monomers before infection and then undergo demerization upon activation

or they can even exist initially as dimer before pathogen challenge and

break into monomers after activation. However, its actual role in R-gene

function is yet not known (Joshi and Nayak 2011) known. It is still unclear

whether any protein can indeed interact with R-gene products through

leucine zipper regions or R-gene products can really undergo demerization

through LZ regions. Recently, it has been found that the LZ domain of

RPM1 gene interacts with RPM1 interacting protein RIN4 to negatively

regulate resistance to Pseudomonas syringe (Mackey et al. 2002; Axtell

and Staskawicz 2003).

32

2.8.3. Nucleotide binding site (NBS) domain

Majority of resistance (R) genes have characterstic amino acid sequences

having strong similarity to nucleotide binding sites (NBSs). These NBS

domain occurs in diverse proteins in additions to R-genes with ATP or

GTP binding activity such as ATP synthase b subunits, ras proteins,

ribosomal elongation factors, adenylate kinase etc (Traut 1994). Van der

Beizen et al. (1998) Arvind et al. (1999) reported that the NBS domains of

R-genes are highly homologous to NBS regions of apoptosis related genes

like CED4 from Caenorhbditis elegans and APAF-1, FLASH and Nod1

from humans which facilitates NTP binding. As such the NBS domain

plays a significant role in plant defense signaling. The presence of a NBS

domain in R-genes suggests possible activation of a kinase or G proteins in

the resistance response. Tameling et al. (2002) reported that the tomato I-2

and Mi-1 genes are found to have ATP binding ability at the P-loop site of

the kinase 1a domain. Nucleotide triphosphate binding is believed to be

essential for the functioning of R-gene products because site-specific

mutations in the NBS regions has resulted in loss or negative effect of

function in different R-genes such as RPS2 of Arabidopsis and N gene of

Tobacco (Bent 1996 and Katagiri et al. 2007).

It has been possible to distinguish the TIR and non-TIR subclasses of NBS-

LRR R-genes by the motifs found within the NBS domain. The last amino

acid residue in the NBS kinase-2 motif is generally an aspartic acid (D) or

33

aspartate (N) in the TIR subclass and tryptophan (W) in case of non-TIR

subclass (Myers et al. 1999). Pan et al (2000) reported that the TIR-NBS

types are largely distributed in dicots while the non-TIR-NBS types are

often found in both monocots and dicots. The conserved P-loop

(GVGKTT) and GLPLA hydrophobic motifs of NBS domain are often

used in rapid isolation of the NBS-LRR-genes or resistance gene

candidates (RGCs) from different plant species by using a polymerase

chain reaction (PCR)-based approach with degenerate oligonucleotide

primers (Joshi and Nayak 2011). This strategy has been widely employed

in different plant species such as soybean (Kanazin et al. 1996; Yu et al.

1996), barley (Leister et al. 1999; Seah et al. 1998), rice (Mago et al.

1999), tomato (Pan et al. 2000), wheat (Huang et al. 2003), sugarcane

(Rossi et al. 2003; McIntyre et al. 2005), , Maize (Xiao et al. 2006) and

other plant species (Liu et al. 2007). It has been revealed by genetic

mapping and phylogenetic analysis that many of the RGCs either co

segregate with or are closely linked to known disease resistance loci

(Kanazin et al. 1996; Leister et al. 1996; Mago et al. 1999; Pan et al. 2000)

suggesting that these NBS sequences may form part of the R-genes.

Therefore the NBS domain is often used in the identification and

classification of R-genes became of its ability to recognize R-gene

sequences in the available nucleotide databases, to exhibit great alignment

34

with R-gene sequences at the conserved motifs and to classify NBS-LRR-

genes as TIR or non-TIR types (Bai et al. 2002).

2.8.4. Leucine-Rich Repeats (LRRs)

The leucine rich repeats are tandemly arranged in the C-terminus of the

NBS-LRR R-genes, consisting of 20-29 amino acids residues with a

conserved 11 residue sequence (LxxLxLxx(N/C/T)xL) (where x is any

residue and L can be replaces with valine or isoleucine) (Bostjan and

Andrey 2001). Kobe and Deisenhofer (1994) had given the structure of a

distinct LRR. Yoder et al. (1993) reported that the porcine RNase inhibitor

contains LRR of 29 amino acids while it is believed that a LRR domain

with an average length of 24 amino acids will resemble a β-helical array.

Torii (2004) reported that as many as seven distinct LLR subfamilies,

which provide structural framework to protein interactions in various

cellular processes. The glycosylation pattern within the LRR domain is

directly involved in ligand binding as shown by Zhang et al (1995). Dixon

et al. (1996) reported that LRRs directly facilitates the interaction of R-

gene products with proteins involved in defense signal response and by

default accepted as the recognition domain because all the other motifs

exhibit signaling capacity. Related evidences also suggest that LLRs are

major determinant of resistance specificity (Hulbert 2001). It has been

shown by Jia et al.( 2000) that the resistance specificity of Pi-ta gene

against rice blast varies with a single amino acid difference in the LRR

35

domain. Similarly, difference in six amino acid between the flax rust

resistance genes P and P2 within the β-strand of four LRR units determines

resistance specificity as reported by Dodds et al. (2001). Recent review of

Joshi and Nayak (2013) reveals that the LRR domain can also interact with

other domain of R proteins to block the activation of resistance signaling.

Ade et al. (2007) have shown that the interaction of LRR domain with the

NBS domain result in inactivation of defense signaling of RPS5 gene in

transgenic tobacco. However, this observation needs to be validated in

other type of R-genes through further investigations.

2.8.5. Organization of NBS-LRR R-genes in the plant genome

The complete set of plant R-gene-related NBS–encoding genes were

characterised in the Col-0 Arabidopsis genome by Myers et al. (2003).

More than 160 NBS-LRR encoding genes have been identified which

included the 11 cloned R-genes or the closest Col-0 homologs to R-genes

cloned from other Arabidopsis ecotypes. Additional 58 Arabidopsis genes

which were identified encoded TIR motifs but no LLRs. Bai et al. (2002)

detected 250 predicted full length NBS-LRR-genes and 560 NBS

sequences in rice which do not accurately reflect those in any given rice

genome, mainly because they are based on the analysis of incomplete

databases that contain sequences from japonica rice and indica rice. Zhou

et al. (2004) surveyed the NBS-encoding genes in the complete genome

sequence of one japonica cultivar, Oryza sativa L. var. Nipponbare and

36

found 535 NBS-coding sequences, including 480 non-TIR (Toll/IL-1

receptor) NBS-LRR R-genes. A few genes with the TIR have been

identified in rice without encoding any LRR domain, and were otherwise

divergent from NBS-LRR-genes (Bai et al. 2002). Recently, the diversity

and distribution of NBS-LRR-genes in Populus trichocarpa draft genome

sequence have been examined by Kohler et al. (2008). Mun et al. (2009)

used the genome sequence of Brassica rapa to identify NBS encoding

genes in the Brassica genome. Thus, it appears that many R-genes are

tightly linked in clusters within plant genomes. This phenomena was

reviewed by Hulbert et al. (2001) who noted that genetically linked alleles

or clusters of genes have greater possibility for recombination than simple

loci composed of single genes. Such clusters of R-genes may also

contribute to generation of novel resistance alleles through recombination

and gene conversion (Joshi and Nayak 2013).

2.8.6. Evolutionary characterization of NBS-LRRs : As reviewed by

Joshi and Nayak (2013) the complete genome sequence analysis and EST

development of model dicot, monocot and tree plants has revealed the

genomic organization of NBS-LRR R-genes and has paved ways for their

evolutionary analyses (Meyers et al. 2003; Zhou et al. 2004; Kohler et al.

2008). Extensive global sequencing projects and PCR-based surveys

confirm that all plants maintain large and diverse NBS-LRR families

involved in pathogen surveillance or other unknown functions. This implies

37

that NBS-LRR super family scattered in a diversified manner. The rice

genomic sequences contain more than 500 NBS coding sequences all of

which encode for CNL (Coiled Coil NBS-LRR) R-genes (Bai et al. 2002;

Zhou et al. 2004). In contrast, of the 149 NBS-LRR-genes and 58 shorter

related genes in Arabidopsis, two third are TNLs (TIR-NBS-LRRs) and

one third encodes CNLs whereas Populus trichocarpa contains 60 % of

CNLs and 40 % of TNLs. Although TNL genes out number CNL genes by

nearly two to one in the Arabidopsis genome, several lines of evidence

suggested that the CNL genes may be the more ancient group (Joshi et al.

2013). There is a greater degree of diversity among CNL proteins than

TNLs across different plant species (Cannon et al. 2002). The study of

phylogenetic analysis of CNLs comprises four distinct lineages some of

which exists prior to angiosperm or gymnosperm divergence. Cannon et al.

(2002) reported that the branch of the CNL tree is longer and the intron

positions are less conserved than TNLs.

TNLs are largely over expressed in dicot genomes as compared to CNLs.

Meyers et al (2003) reported that Arabidopsis itself consist of double the

number of TNLs than CNLs within its genome. The presence of TNLs in

pine and moss is further indicating of the fact that that this subfamily of

NBS-LRRs have also evolved prior to the angiosperm–gymnosperm split.

According to Pan et al. 2000, the evolution of TNLs and CNLs involved

two stages. Stage I contains both CNLs and TNLs which evolved during

38

the divergence of angiosperm and gymnosperm about 200 million years

ago. Stage II was dominated by gene duplication and diversification

characterizing the evolution of TNLs and CNLs. Although, Pan et al. 2000

reported that TNLs were completely absent in cereals, this could be

possibly applicable for the entire monocots in general. Till now, Triticum-

Thinopyrum are the only reported monocot containing TNLs (Jiang et al.

2005). Non existence of TIR-NBS sequences has not only been reported in

cereal monocots (Order-Poales), but also in four other monocot orders

namely Zingiberales, Arecales, Asparagales and Alismatales (Tarr &

Alexander 2009). Two TNL type sequences which were isolated from

Agrostis species never had true ORF with NBS domain to qualify them as

monocot TNLs (Budak et al. 2006). Thus, concluded that, although TIR-

NBS-LRRs were present in early land plants, they either never developed

or have been significantly reduced or lost in monocotyledonous plants

(Joshi and Nayak et al. 2013). TIR-NBS sequences are rarely found in

magnolias as well making it even more unclear whether TNLs were lost

before the divergence of monocots and magnolias or degenerated

independently in both lineages. Thus a detail study and characterisation is

required for NBS family of monocots, magnolias and other basal

angiosperms to further validate the evolutionary structure of plant

resistance genes. The CNL of NBS-LRR groups are also unevenly

distributed in dicot taxa. Cannon et al. (2002) reported 18 sequence

39

representations from a CNL lineage in Arabidopsis but only 4 in soybean

and Medicago truncatula while another lineage represented 42 sequences

in soybean and Medicago truncatula but only two in Arabidopsis. This

phenomena has been considered as „birth and death hypothesis‟ as per Nei

and Rooney (2005) according to which many NBS-LRR lineages has been

lost and supplemented with new lineages in the recent times whereas some

lineages has been able to retain themselves for a long time period. So a

single plant genome could not explain the variability pattern of NBS-LRR.

Only a thorough comparison of NBS-LRR sequences from different

monocot, dicot and gymnosperms may possibly provide a universally

acceptable model to study evolutionary dynamics of NBS-LRR-genes

(Joshi and Nayak et al 2013). Besides CNLs and TNLs, there are other

modified R-gene families such as TX (TIR-X) and TN (TIRNBS) add up

new twist to the NBS-LRR evolutionary pattern. The LRR domain is

lacking in TIR-NBS proteins while the TX protein lacks both the

characteristics NBS and LRR domains found in an R-gene. TX and TN

proteins are reportedly expressive in pines and grasses. According to

Meyers et al. (2002) two TN proteins has been reported to be conserved in

both Arabidopsis and rice suggesting these are the ancient group of NBS-

LRR protein families. TX and TN-like sequences have been found in

cereals but no TNL genes have been identified in cereal genomes (Bai et al.

2002; Meyers et al. 2002). The presence of TNL genes in coniferous

40

genomes indicates the possibility of loss of these genes in grasses during

evolution (Bai et al. 2002). Further, the situation has been complicated with

recent identification and characterization of a fusion product of TN and

TNL proteins in Arabidopsis for resistance against Peronospora parasitica.

Thus, a detail study of TNL and CNL genes in different plant families is

needed to interpret the evolutionary pattern leading to the R-gene

diversification.

2.8.7. NBS-LRR signal transduction

The basis of the gene-for-gene model is the physical interaction between R

protein and pathogen effector which results in the plant defense responses

and eventually leads to resistance.The Pto protein kinase of tomato

interacts directly with bacterial effector AvrPto having serine/threonine

kinase domain at residue 204. A second bacterial effector AvrPtoB having

intrinsic E3 ubiquitin ligase activity also being interacts by tomato Pto

protein kinase. NBS-LRRs genes also exibit similar type of direct

interaction. The Arabidopsis RRS1-R protein interacts with bacterial type

III effector Pop2 (Deslandes et al. 2003) rice Pi-ta interacts with AVR-Pita

(a predicted secreted metalloprotease) from the Ascomycete rice blast

fungus Magnaporthe grisea (Jia et al. 2000), whilst the flax L5, L6 and L7

proteins interact in yeast with the corresponding AvrL567 protein variants

from the Basidiomycete flax rust fungus Melampsora lini (Dodds et al.

2006). However, existence of alternative recognition targets or multiprotein

41

recognition complex cann‟t be avoided as there is very poor information

about direct R-Avr interactions in plants. Dangl and Jones (2001) predicts

that an effector protein interacts with a host target, which is itself

recognized by more than one R-protein. Mackey et al. (2002) reported that

qRIN4 protein of Arabidopsis is an example of a host target for type III

bacterial effectors, which is recognized by at least two CNL R-proteins.

Structurally unrelated bacterial effectors AvrRpm1 and AvrB are being

targeted by 211-amino acid acetylated protein RIN4. Then RPM1 R-

protein has been activated after induction of phosphorylation of RIN4 by

both the effectors. AvrRpt2 which is a third effector is recognized by RIN4

inside the plant cell that cleaves RIN4 at two sites. The NBS-LRR protein,

RPS2 is activated by cleavage of RIN4. Resistance created after activation

of hypersensitive response (HR) on pathogen inoculation known as

systemic acquired response (SAR). As a result accumulation of salicylic

acid (SA) throughout the plant expresses set of defense genes. SAR

phenomena add up more resistance to further attack of other pathogens.

(Glazebrook 2001). Jasmonic acid (JA) and ethylene (ET) are signaling

molecules which required for activation of some signal transduction

pathway mediated disease response. Further dissection of local and

systemic signaling networks is possible due to discovery of new genes and

mutants and begins to highlight the complex interplay between defense

molecules such as salicylic acid (SA), nitric oxide, reactive oxygen

42

intermediates (ROI), jasmonic acid (JA) and ethylene (ET) (Thomma et al.

2001). Two mutants‟ NDR1 and EDS1 have been detected by Feys and

Parker (2000) which suppresses race specific resistance to strains of the

bacterium Pseudomonas syringae. Independently EDS1 and NDR1 required

for the function of different NBS-LRR-genes which encodes a lipase like

protein a membrane associate protein respectivelly. TIR-NBS-LRR R-

genes, is suppressed by EDS1 whereas NDR1 suppresses a subset of non-

TIR-NBS-LRR R proteins. According to recent reports R-gene-mediated

resistance is regulated by the cytoplasmically localized signaling complex

SGT1/RAR1/HSP90 in plant as diverse as Arabidopsis, barley and tobacco

(Azevedo et al. 2002; Austin et al. 2002; Muskett et al. 2002; Tor et al.

2002). Proteins present in this complex work together to stabilize various

NBS-LRR R proteins. RAR1 encodes a small zinc-binding protein that

interacts with SGT1 in barley and tobacco extracts. The function of Skp1-

Cullin-F-box protein (SCF) E3 ubiquitin ligase complex is depend on

SGT1 component which targets proteins for degradation by the 26S

proteasome. The ubiquitin-proteasome pathway plays a major role in R-

gene-triggered resistance. Components of mitogen-activated protein kinase

(MAPK) cascades are other important defence regulation that constitute

functionally conserved eukaryotic signal systems in response to various

environmental stresses. Frye et al. (2001) reported that the MAPK kinase,

EDR1, negatively regulates SA-inducible defences whereas Petersen et al.

43

(2000) reported that MAPK 4 appears to differentially regulate SA and JA

signals. These reports indicate strong implication of MAPK modules in

molecular communication between different plant defense pathways. The

ankyrin repeat protein, NPR1, initially identified as an SA response

regulator is another important feature of the systemic signalling which is

required for both SAR and ISR. Kinkema et al. (2000) reported that the

addition of SA to Arabidopsis seedlings promotes movement of NPR1 to

the nucleus where it is able to bind several TGA (TGACG DNA motif)

class transcription factors, conferring a possible direct route to defense

gene induction (Fan and Dong 2002). Lipid derived molecules may also

have role towards disease resistance (Maldonado et al. 2002) came to the

picture after identification of an apoplastic lipid transfer protein, DIR1

which is an inducer of defence signalling in SAR.

2.8.8. Cloning and characterization of NBS-LRR resistance gene

candidates

Conservation of several structural motifs encoded by plant R-genes has

prompted the development of homology-based approaches aimed at

identification of structurally related sequences termed as resistance gene

candidates/analogues (RGCs/RGAs). Reports on recent cloning of several

R-genes by various strategies such as transposon tagging, map-based

positional cloning has revealed the amino acid domains with extensive

44

sequence homology (Madsen et al. 2003). Designing simple PCR-based

strategies with degenerative primers for amplification and isolation of

similar sequences in other plant species has been possible due to the

presence of conserved domains in genes (Kanazin et al. 1996). The P-loop,

the Kinase-3a, and the hydrophobic GLPL motifs have been the most

commonly used (Cordero et al. 2002). The PCR amplification products are

separated by gel electrophoresis. The expected DNA fragments are excised

and cloned to a suitable vector (pGEM-T or pMD-18T) and transformed

into competent cells of Escherichia coli. The transformed colonies are

selected and the recombinant plasmid DNA is isolated using alkaline lysis

and subjected to double digestion to detect the presence of expected DNA

insert. The DNA inserts are sequenced and similarity search is performed

with BLAST program to identify putative resistance gene candidates. The

RGAs are validated by performing multiple sequence alignment with

known R-genes using the ClustalX software. Further analysis on phylogeny

and evolutionary aspects can also be done by various other bioinformatics

tools (Figure 2.6). Many resistance genes had been cloned and

characterized in crop plants that show resistance against particular diseases

through transposon and map- based approaches such as Hm-1 and Rp-ID in

maize (Johal and Briggs 1992), Xa21, Pi-ta, Pi-b and Pi-kh in rice (Song et

al. 1995; Wang et al. 1999; Bryan et al. 2000; Sharma et al. 2005 b) and

Cre3, VRN1 and Lr21 in wheat (Lagudah et al. 1997; Yan et al 2003;

45

Huang et al. 2003) However, it is unlikely to use transposon tagging as a

routine method to clone R-genes because it is very difficult inactivate a

functional R-gene by transposon insertion. Similarly it is also very difficult

for cloning R-genes via map-based cloning, because of highly repetitive

sequences. Alternatively, candidate gene approach may be very promising

in cloning R-genes due to conserved motifs shared by them. Seah et al.

(1998) used a similar approach to isolate five distinct NBS RGAs from

wheat and barley of which two were mapped to wheat chromosomes 2A

and 2B. Chen et al. (1998) did not clone any RGAs but used the conserved

motifs to design primers and identified polymorphisms associated with

resistant wheat, rice and barley lines. Recently, RNA fingerprinting and

data mining approach has been used by Dilbirligi and Gill (2003) to isolate

220 expressed R-gene candidates in wheat. Of these, 125 sequences

structurally resembled known R-genes. Maleki et al. (2003) used motifs in

nucleotide binding site-leucine-rich repeat (NBS-LRR) resistance genes

and two conserved motifs within R-gene kinases to design degenerate

primers, isolated 8 NBS-LRR, and 26 kinase analogs in common wheat.

There are now sufficient sequences available from rice, to reveal the

diversity and general nature of NBS–LRR- three genes and related genes in

cereal genomes due to the availability of complete rice genome sequence

(Bai et al. 2002). NBS–LRR-genes in rice are a large and diverse class with

more than 600 genes, at least three to four times the complement of

46

Figure 2.6: Degenerative primer based PCR amplification and subsequent

cloning for isolation of resistance gene candidates (RGCs). RGCs can be

confirmed by aligning them with known resistance protein sequences from

other plants.

47

Arabidopsis. Zhou et al.( 2004) most of them occur in small families

containing one or a few cross-hybridizing members .Isolation and

characterization of RGAs has been successfully carried out in many

commercial crops. Tan et al. (2003) utilized R-gene degenerate primers

designed from the NBS motifs of tobacco N protein, Arabidopsis RPS2

protein and Flax L6 protein to amplify and clone PCR products in the

250bp size range Gossypium arborium. This enabled cloning of 33 putative

cotton RGAs containing the highly conserved NBS R- protein motif.

Primers designed from motifs representing TIR-NBS-LRR and non-TIR-

NBS-LRR classes of R-proteins has been used by He et al. (2004) for

identification of 61 unique sequences containing high similarity to R-genes

in polyploid cotton (Gossypium hirsutum L.). Hinchliffe et al. (2005) used

the same approach and isolated 165 clones of which 57 has novel

nucleotide sequences in Gossypium hirsutum cv. Auburn 635 RNR. Rossi

et al. (2003) identified 55 RGAs within the sugarcane EST database with

homology to typical disease R-genes including Cf-2.1, Xa21, Hcr2, I2, Xa1,

RPR1, Pib, Hv1LRR, Gpa2, TMV, Rp1-D, Pto and Pti1. Many diverse R-

gene homologues in the sugarcane EST collections has been revealed by

homology searches with known cloned R- genes. Fifty-fiive RGAs were

identified within the sugarcane EST database with homology to typical

disease R-genes including Cf-2.1, Xa21, Hcr2, I2, Xa1, RPR1, Pib,

Hv1LRR, Gpa2, TMV, Rp1-D, Pto and Pti1 (Rossi et al. 2003). Similarly,

48

McIntyre et al. (2005a) isolated 54 RGAs from the Australian sugarcane

cultivar, Q117 using degenerate oligonucleotide primers. Degenerative

primer approach was used by Totad et al. (2005) to amplifiy and clone 13

RGAs from Sorghum bicolor cv. M35-1, out of these, nine SRGAs showed

significant sequence similarity with known R-genes and classified into two

clusters-cluster-I comprising only SRGAs and cluster- II comprised of

known R-gene sequences along with three SRGAs with the identity ranging

from 31-91%. These RGAs are being used in mapping and tagging of traits

related to disease resistance.

Horticultural crops such as tomato, potato, grapes and citrus are often used

as alternative food source after rice, wheat and maize. They grow in a wide

range of environmental conditions and are often susceptible to diverse

pathogens. Ohmori et al. (1998) used the genomic DNA of two tomato

lines carrying ToMV resistance genes, Tm1 and Tm2 and cloned three PCR

amplified fragments homologous to class I R-genes like tobacco N,

Arabidopsis RPS2 and Flax L6 and another two fragments homologous to

class II R-genes such as Cf-9 and Cf-2. Pan et al. (2000) isolated genomic

sequences conferring race specific resistance to pathogens using a variety

of primer pairs based on ubiquitous NBS motifs in tomato. They also

concluded that there exist a high degree of conservation of NBS

homologous between tomato and potato, which suggest rapid evolution of

R-gene homologous during diversification of plant families. Chen et al.

(2007) isolated 22 RGAs grouped into five distinct clusters in Ipomoea

batatas using the NBS degenerative primers. These RGAs are now being

used as markers for differentiating resistance and susceptible sweet potato

varieties. Recently, in citrus Deng et al. (2000) isolated ten classes of citrus

49

sequences similar to NBS-LRR class R-genes and identified ≈77 contigs

that contain 150 copies of NBS-LRR R-gene sequences. Di Gaspero and

Cipriani (2002) used the NBS based reverse genetics approach to isolate 12

grapes (Vitis amurensis) genomic sequences belonging to the NBS-LRR-

gene family one of which has been used for designing candidate markers

for disease resistance genes. Pulses and legumes complement cereals in

both production and consumption. Nine classes of resistance gene analogs

have been isolated by Kanazin et al. (1996) in soybean and he mapped

them on six linkage groups. Cloning and sequencing of PCR amplified

product from cicer has been done by Huettel et al. (2002 and he isolated

thirteen different RGAs and classified them into 9 distinct classes.

Sequence similarity grouped them as NBS-LRR type with N-terminal

region being TIR or coiled-coil (CC) sequence. Alfalfa (Medicago) is

another important legume often used as a model for-genetic study. It is very

difficult to determine the associations of molecular markers with disease

resistance in alfalfa due to the auto-tetraploid genetic structure. Cordero et

al. (2002) solved rhis problem by using the degenerative primer approach

to isolate eighteen families of NBS containing R-genes exhibiting a

polyphyletic origin.

2.8.9. Resistance sequences in cultivated and wild spice crops

Spices crops are valued world over as culinary herbs, home remedy and

medicinal crops. Continuous domestication of preferred spice genotypes

coupled with their exclusive vegetative propagation have eroded the

genetic base of these crops thus making them susceptible to all major

diseases such as soft rot, leaf blotch and bacterial wilt. Only means is

through resistance gene characterisation. of protecting these plants and

retaining their productivity. Nair et al. (2007) used the degenerative PCR

method and identified 42 Zingiber resistance gene candidates (RGCs) in

the wild species Zingiber zerumbet, which were classified into five classes

50

of the non-TIR-NBS-LRR-gene family. Although the genetic variation for

disease resistance is low in the cultivated Curcuma. The degenerative PCR

approach was used by Joshi et al (2010) for identification of 17 DNA

sequences out of which only 5 DNA sequence information (CRGA1,

CRGA2, CRGA3, CRGA4, and CRGA5) gave high level of accuracy to

NBS sequences. However not a single sequence was found to be

expressive. Again it complicates the situation to develop or to identify

expressive resistance sequences towards race specific or non-specific

pathogens. Mining expressive resistance sequences in Curcuma using

Zingiber RGCs may not be useful as NBS-LRR sequences also exhibit a

phenomenon called „restricted taxonomic functionality‟ (Tai et al. 1999,

Joshi et al. 2013). That means the class of NBS-LRR family unevenly

distributed among different plant taxa as a result sequence retrieved from a

genus may not work in another genus even if they belongs to same family.

This problem could be solved if wild relatives of Curcuma would be used

to identify expressive R-genes. Utilization of wild turmeric for isolation

and characterization of RGCs can be an alternative as they can evolve

resistance specificities more efficiently than cultigens as seen in many

other plants (Ebert and Hamilton 1996). It is well established that wild

populations harbour significantly higher-genetic diversity than does the

cultivated one (Provan et al.1997; Oka 1988; Sun et al. 2001). Sun et al.

2001 studied the genetic diversity of O. rufipogon and O. sativa from more

than ten Asian countries, and found that the genetic diversity of O.

rufipogon was much higher than that of cultivated rice and that a great

number of genes that occurred in O. rufipogon could not be found in

cultivated rice. To-date, although many studies have been conducted on

cultivated Curcuma, the resistance gene isolation and characterisation of

wild Curcuma and other spice crop are less well known (Joshi et al. 2013).

2.9. Population genetic structuring and evaluation of resistance source

51

Population genetics is the study of allele frequency distribution and change

under the influence of the four main evolutionary processes: natural

selection, genetic drift, mutation and gene flow. It also includes the factors

of recombination, population subdivision and population structure and

attempts to explain such phenomena as adaptation and speciation.

Population genetics was a vital ingredient in the emergence of the modern

evolutionary synthesis. The goal of population genetics is to describe and

quantify genetic variation in populations and to use this variation to make

inferences about evolutionary processes affecting populations (Hartl and

Clark 1997, Hedrick 1985). Evolutionary forces such as mutation,

migration, genetic drift, selection and recombination change gene

frequencies in populations and shape their genetic structure. Population

geneticists focus on genetic variation and evolutionary processes below the

species level (microevolution) although the distinction between population

genetics and systematics (macroevolution) is not always clear. Population

genetic structuring hold a great promise to improve disease management

(Burdon, 1993). The ability of a population to adapt to its environment

through natural selection is determined by theextent of genetic variability

of that population. Reed and Frankham (2003) reported that the possible

combinations of genes that can confer fitness and vigor in response to

critical changes in the environmental conditions are considerably reduced

with reduction in genetic variability. According to isolation by distance

hypothesis the genetic differentiation between the populations increased

with geographical distance. Holsinger and Gottlieb (1991) reported that it is

essential to determine genetic diversity and structure of natural plant

populations in order to assess conservation strategies. Knowledge of the

extent and distribution of genetic variation may become a very helpful tool

not only for conservation purposes, but also to design rational ways of

economic exploitation. Molecular markers in combination with spatial

52

statistical tools have contributed immensely to the understanding of the

distribution of genetic diversity and differentiation, gene flow, and

population size implications for a wide range of plant species (Cruzan

2001, Wang and Ge 2006, Raji et al. 2009, Yuan et al. 2012). Molecular

markers have been particularly useful for studies involving intra- and inter-

species genotypic variation (Robinson et al. 1999) in relatively small

populations due to their neutral nature. Molecular random markers are able

to provide a more precise understanding of genetic diversity through the

identification of genomic segments that differentiate individuals or

populations without the need for-genetic information about the genome

(Young et al.1996). Weising et al.(1995) reported that different methods of

DNA fingerprinting have proved to be useful, with a wide range of

applications in plant population studies, such as the detection of genetic

variation within and between populations, the characterisation of clones,

the analysis of breeding systems, and the analysis of ecogeographical

variation. PCR based RAPD (random amplified polymorphic DNA)

(Williams et al. 1990) is one of the most commonly used method which has

been employed in many plant population structure studies in recent years

(reviews in Bartish et al. 1999, Bussell 1999, Nybom and Bartish 2000,

Nybom 2004, Romeiras et al. 2007). Inter simple sequence repeats (ISSR)

has also been effectively used as a molecular marker to study genetic

diversity (Chen et al. 2007). Recently, microsatellites or simple sequence

repeats (SSRs) have become the markers of choice for a wide spectrum of

genetic, population and evolutionary studies in many plants (Zhao and

Kochert 1992; Wu and Tanksley 1993; Panaud et al. 1996). The rapid and

inexpensive development of SSRs from expressed sequence tag (EST)

databases has also been shown to be a feasible option for obtaining high-

quality nuclear markers (Gupta et al. 2003; Bhat et al. 2005). Moreover,

the National Center for Biotechnology Information (NCBI) EST database

53

(dbEST; Boguski et al. 1993) contains an ever-increasing number of these

„single-pass‟ cDNA sequences, meaning that the resources necessary for

the efficient development of large numbers of so-called EST-SSRs already

exist for a wide variety of taxa. In general, EST-SSRs have been found to

be significantly more transferable across taxonomic boundaries than are

traditional „anonymous‟ SSRs (Chagne et al. 2004; Liewlaksaneeyanawin

et al. 2004; Gutierrez et al. 2005; Pashley et al. 2006), and reports of EST-

SSR transferability have become increasingly common. EST-SSRs also

have clear potential for use in basic evolutionary applications, such as

population genetic analyses (Ellis et al. 2006).

However, Amplified Fragment Length Polymorphism (AFLP) is robust,

and proficient at revealing population diversity and estimating genetic

distance between samples and populations. Furthermore, AFLP has the

potential to screen a large number of genetic loci in a single experiment and

does not require prior information about the genome of the species under

investigation. A number of conservation genetic studies have been

conducted using AFLP markers to evaluate genetic diversity and

differentiation in endangered plant species (e.g., AFLP analysis was used to

describe patterns of genetic variation and population structure in seven

extant populations of Isoetes sinensis (Kang et al. 2005), wild populations

of Sinopodophyllum hexandrum (Xiao et al.2006), and apricot (Prunus

armeniaca L; Yuan et al. 2007).Population genetic structuring has been

carried out in many plant species to study inter as well as intra species

genotyping , evolutionary relationship, economic exploitation etc . but as

per recent review no population structuring has been carried out in

Curcuma longa with respect to disease resistance. Molecular markers such

as RAPD, ISSR, SSR, AFLP etc. in combination with spatial statistical

tools have contributed immensely for development of strategy towards

disease management. These highlights important attribute of these markers

54

which can lead to develop SCAR markers to discriminate plant species

towards disease resistance at intra as well as inter- specific level.

2.10. Development of SCAR/STS marker and identification of

resistance trait

This technique is used where the random marker termini are sequenced and

longer primers are designed (22–24 nucleotide bases long) for specific

amplification of a particular locus. These are also similar to STS markers

(Olson et al.1989) in construction and application. The presence or absence

of the band indicates variation in sequence. These markers are better

reproducible than RAPD, ISSR, AFLP etc. SCARs have exhibit several

advantages in mapping studies, map-based cloning as they can be used to

screen pooled genomic libraries by PCR, physical mapping, locus

specificity, etc. SCARs are also used in comparative mapping or homology

studies among related species, thus making it an extremely adaptable

concept in the near future. There are many advantages of SCAR markers

for their specificity, low cost, ease and fast use. SCAR markers have been

successfully employed in plant and animal species identification (Parent

and Page 1998, Mariniello et al. 2002, Yau et al. 2002, Bautista et al.

2003). SCAR markers have been developed usually from RAPD

fingerprints (Parasnis et al. 2000, Koveza et al. 2001, Arnedo-Andrés et al.

2002, Bautista et al. 2003) or from AFLP fingerprints (Negi et al. 2000, Xu

and Korban 2002, Schmidt et al. 2003). Besides, ISSR fingerprints

(Zietkiewicz et al. 1994, Reddy et al. 2002) have also been used, (Bornet et

55

al. 2005, Wang et al. 2008) to develop SCAR markers, because they are

known to be very reproducible, abundant, polymorphic and because of the

specific targeted regions of the genomes (Zietkiewicz et al.1994, Bornet

and Branchard 2001, Bornet et al. 2002). ISSR markers can be further

converted into the more specific sequence-characterized amplified region

(SCAR) markers (Albani et al. 2003). Combination of ISSR and SCAR

strategies for strain differentiation in Lentinula edodes, an important edible

mushroom has been reported by Qin et al. (2006). SCAR markers have

been successfully used in research and breeding programs to characterize

or identify resistance genes in many plants such as tomato (Simons G, et

al.1998, Arens et al. 2010), durum wheat (Cao et al. 2001), melon

(Brotman et al. 2005), and sorghum (Singh et al. 2006).This technique has

also help in a breeding program for selection of male fertility (Ashutosh et

al. 2007) and male sterility genes (He et al. 2009; Lee et al. 2010). The

usefulness of SCAR markers in identifying the resistance to biotic stresses,

such as Gall Midge in rice (Sardesai et al. 2002), covered smut in barley

(Ardiel et al. 2002), topovirus in tomato (Dhyaneshwar et al. 2006), rust in

Asparagus (Li et al. 2007), rust in common bean (de Souza et al. 2007),

club-rot disease in cabbage (Hayashida et al. 2008), root-knot nematode in

pepper (Wang et al. 2009), bacterial wilt in egg plant (Cao et al. 2009) etc.,

have been well exploited in breeding disease tolerant genotypes. In

sugarcane also, a SCAR marker linked to smut resistance gene has been

56

identified and utilized in the breeding program (Que et al. 2008). However,

until now, the applicability and authenticity of SCAR markers have been

found to be limited in the case of biotic stresses or for the identification of a

particular trait corresponding to a single gene.

2.10.1. Importance of the SCAR/STS study in Curcuma longa L.

Turmeric (Curcuma longa L.), belonging to the family Zingiberaceae, is an

important cash crop in tropical and sub-tropical countries. The dried

underground stem (rhizome) of turmeric is valued world over not only as

condiment and spice but also as the raw material for preparation of various

drugs. Soft-rot disease caused by different species of the oomycete Pythium

severely affects turmeric cultivation in all turmeric-producing regions of

the world (Lawrence 1984). In view of the fact that, no effective fungicides

or biological control methods are available till date for controlling Pythium

species (Folman et al. 2003) an effective strategy towards early detection

of Pythium infection can help in protection of existing crops from the

severity of infection. Molecular DNA markers linked to resistance genes

can be utilized to screen for resistant/tolerant lines in the genetic

improvement programme of turmeric. PCR based molecular markers are

the most appropriate assays for molecular breeding applications because

they are relatively simple to handle and can be easily automated. SCAR

markers designed from RAPD and ISSR analysis has been used to detect

resistance specificity against various pathogens as evidenced from a variety

57

of plants like wheat, banana, sorghum etc (Paran and Michelmore 1993;

Mutengwa et al. 2005).

Further, detecting rhizome rot resistance genes in turmeric by their linkage

to DNA markers makes it possible to screen for many different resistance

mechanisms simultaneously, without a need for inoculation with the

Pythium fungus. This will not only result in broad spectrum realization of

resistance in turmeric against Pythium but also will lead to improved

production and export of this highly demanded spice crop.