داروخانه گیاه پزشکی تابنده

Pathogenesis-Related Proteins for the Plant Protection

 

V. Borad, S. Sriram*

Department of Biochemistry and Biotechnology,

Institute of Science,

Nirma University of Science and Technology,

Ahmadabad (Gujarat); India

 

Abstract : Fungi are far more complex organisms than viruses or bacteria and can developed

numerous diseases in plants that cause loss of big portion of the crop every year. Plants have

developed various mechanisms to defend themselves against these fungi which include the

production of low molecular weight secondary metabolites, proteins and peptides having

antifungal activity. In this review, brief information like biochemistry, source, regulation of gene

expression, mode of action of defense mechanism of various pathogenesis-related proteins is

given. Proteins include pathogenesis-related protein 1, â-glucanases, chitinases, chitin binding

protein, thaumatine like protein, glycine-histidine rich proteins, ribosome inactivating protein,

and some newly discovered antifungal proteins.

Key words : Pathogenesis-related Proteins, â-Glucanase, Chitinases, Thaumatine like protein,

Glycine-histidine rich proteins and Ribosome inactivating protein.

 

 

granulomatous disease, severe combined

Introduction

immunodeficiency, chronic mucocutaneous

Fungi are an extremely diverse group of

organisms, with about 250,000 species widely

candidacies,
hyper-IgE
syndrome,

myeloperoxidase deficiency, leukocyte

distributed in essentially every ecosystem. They

can use almost any surface e.g., bathroom tile,

skin, or leaves for their growth. They are

proficient at colonizing and using plants,

humans, and animals as substrates.

During the past two decades, invasive

fungal infections have emerged as a major

threat to immunocompromised hosts. Fungal

infections are a frequent cause of death among

immunocompromised patients, and the

increasing number of immunosuppressed

patients has spurred development of new

antifungals (Shoham and Levitz, 2005). Patients

with primary immunodeficiencies exhibit

immune deficits that confer increased

susceptibility to fungal infections. Numerous

fungi, have been invariably implicated in

causing disease in patients with chronic

adhesion deficiency, defects in the interferon-

ã/interleukin-12 axis, DiGeorge syndrome, X-

linked hyper-IgM syndrome, Wiskott-Aldrich

syndrome and common variable

immunodeficiency (Antachopoulos et al.,

2007). Unfortunately there are few species

of fungi that infect the human and animals.

But among the all microbes fungi are the most

causative agent of disease in plant.

When a pathogen attacks a plant, it either

successfully infects the plant or plant prevents

the infection. Plants do not have circulating or

phagocytic cells. Instead their cells have a

thick, complex wall that acts as a barrier to

invasion. Plants display an innate pathogen-

specific resistance by producing responses like

oxidative burst of cell, change of cell wall

composition that prevent infection and de-novo

 

* Corresponding author : Dr. Sriram Seshadri, Department of Biochemistry & Biotechnology, Institute

of Science, Nirma University of Science and Technology, Ahmadabad, Gujarat; Ph.: +91-2717-241901

Extn. 618/627 (O); Fax: : +91-2717-241916; E-mail: sriramsjpr@gmail.com, sriramsjpr@rediffmail.com

 

189

 

 

 

 

 

 

Borad V. and Sriram S. (2008) Asian J. Exp. Sci., 22(3), 189-196

 

 

synthesis of compounds like phytoalexin and

pathogenesis-related proteins. All this

responses can be triggered by exposing the

plant to virulent, avirulent, and nonpathogenic

microbes, or artificially with low molecular

weight and sometimes volatile molecules like

such as salicylic acid, jasmonate (Delaney et

al., 1994; Xu et al., 1994; Wu and Bradford,

2003), 2,6-dichloro-isonicotinic acid or

benzo(1,2,3) thiadiazole-7-carbothioic acid S-

methyl ester(BTH) (Vallad and Goodman,

2004). These types of resistance are called as

Systemic Acquired Resistance (SAR) or

Induced Systemic Resistance (IAR). Among

all induced responses, production of

“Pathogenesis Related (PR) proteins” is most

important because they can lead to the

increased resistance of the whole plant against

a pathogenic attack (Adrienne and Barbara,

2006). Large numbers of small, basic, cysteine-

rich antimicrobial proteins are produced by

many organisms throughout all kingdoms. They

display a great variety in their primary

structure, in species specificity, and in the

mechanism of action (Leiter et al., 2005).

There are more than 13 different pathogenesis-

related proteins are known to us.

Antifungal PR proteins are of great

biotechnological interest because of their

potential use as food and seed preservative

agents and for engineering plants for resistance

to phytopathogenic fungi (Dempsey et al.,

1998). Various studies have revealed that

transgenic plants over expressing genes of the

PR-1, PR-2, PR-3, and PR-5 families mediate

host plant resistance to phytopathogenic fungi.

Co-expression of multiple antifungal protein

genes in transgenic plants seems to be more

effective than expression of single genes

(Bormann et al., 1999).

Pathogenesis-related (PR) protein 1

The first PR- 1 protein was discovered in

1970. Since then, a number of PR-1 proteins

have been identified in Arabidopsis, Hordeum

vulgare (barley), Nicotiana tabacum

 

190

(tobacco), Oryza sativa (rice), Piper longum

(pepper), Solanum lycopersicum (tomato),

Triticum sp. (wheat) and Zea mays (maize)

(Liu and Xue, 2006). These PR-1 having 14 to

17 kD molecular weight and mostly of basic

nature. Non-expressors of Pathogenesis-

Related Genes1 (NPR1) regulate systemic

acquired resistance via regulation pathogenesis-

related 1 (PR-1) in Arabidopsis thaliana. The

interaction of nucleus-localized NPR1 with

TGA transcription factors, after reduction of

cysteine residues of NPR 1 by salicylic acid

(SA) results in the activation of defense genes

of PR-1. In the absence of TAG 2 and/or SA

expression of PR-1 not occur in Arabidopsis

thaliana (Després et al., 2000; Rochon et al.,

2006). PR-1 proteins have antifungal activity

at the micromolar level against a number of

plant pathogenic fungi, including Uromyces

fabae, Phytophthora infestans, and Erysiphe

graminis (Niderman et al., 1995). The exact

mode of action of the antifungal activities of

these proteins are yet to be identified but a

PR-1-like protein, helothermine, from the

Mexican banded lizard have been found to be

interacting with the membrane-channel

proteins of target cells, inhibiting the release

of Ca 2+ (Monzingo et al, 1996).

â-Glucanase (PR2):

Plant ß-1,3-glucanases (ß-1,3-Gs)

comprises of large and highly complex gene

families involved in pathogen defense as well

as a wide range of normal developmental

processes. ß-1,3-Gs have molecular mass in

the range from 33 to 44 kDa (Hong and Meng,

2004; Saikia et al., 2005). These enzymes have

wide range of isoelectric pH. Most of the basic

â-1,3-Gs are localized in vacuoles of the plant

cells while the acidic ß-1,3-Gs are secreted

outside the plant cell. Wounding, hormonal

signals like methyl jasmonate and ethylene (Wu

and Bradford, 2003), pathogen attack like

fungous Colletotrichum lagenarium (Ji and

Ku, 2002) and some fungal elicitors releases

from pathogen cell wall (Boller, 1995) can also

induced â-1,3-Gs in the various parts of plant

 

 

 

 

 

 

Pathogenesis-Related Proteins for the Plant Protection

 

 

(Wu and Bradford, 2003; Saikia et al., 2005).

The enzyme â-1,3-Gs was found to be strongly

induced by ultraviolet (UV-B; 280–320 nm)

radiation in primary leaves of French bean

(Phaseolus vulgaris), so that UV-induced

DNA damage is a primary step for the induction

of â-1,3-Gs.(Kucera et al., 2003). â-1,3-

glucanases and chitinases are down regulated

by combination of auxin and cytokinin while

Abscisic acid (ABA) at a concentration of 10

µM markedly inhibited the induction of â-1,3-

glucanases but not of chitinases (Rezzonico,

1998; Wu et al., 2001). These enzymes are

found in wide variety of plants like Arachis

hypogaea (peanut), Cicer arietinum

(chickpea), Nicotiana tabacum (tobacco), etc.

and having resistivity against various fungi like

Aspergillus parasiticus, A. flavs, Blumeria

graminis, Colletotrichum lagenarium,

Fusarium culmorum, Fusarium oxysporum,

fusarium udum, Macrophomina phaseolina

and Treptomyces sioyaensis (Rezzonico, 1998;

Wu and Bradford, 2003; Hong and Meng,

2004; Wróbel-Kwiatkowska et al., 2004, Liang

et al., 2005; Roy-Barman et al., 2006). â-1,3-

glucanases are involves in hydrolytic cleavage

of the 1,3-â-D-glucosidic linkages in â-1,3-

glucans, a major componant of fungi cell wall

(Simmons, 1994; Høj and Fincher, 1995). So

that cell lysis and cell death occur as a result

of hydrolysis of glucans present in the cell wall

of fungi.

Chitinases (PR3)

Most of Chitinase having molecular mass

in the range of 15 kDa and 43 kDa. Chitinase

can be isolated from Cicer arietinum

(chickpea) (Saikia et al., 2005), Cucumis

sativus (cucumber), Hordeum vulgare

(barley) (Kirubakaran and Sakthivel, 2006),

Nicotiana tabacum (tobacco) (Pu et al.,

1996), Phaseolus vulgaris (black turtle bean)

(Chu and Ng, 2005), Solanum lycopersicum

(tomato) (Wu and Bradford, 2003) and Vitis

vinifera (grapes) (Sluyter et al., 2005).

Chitinases can be divided into two categories:

Exochitinases, demonstrating activity only for

 

191

the non-reducing end of the chitin chain; and

Endochitinases, which hydrolyse internal â-1,4-

glycoside bonds. Many plant endochitinases,

especially those with a high isoelectric point,

exhibit an additional lysozyme or lysozyme like

activity (Collinge et al., 1993; Brunner et al.,

1998; Schultze et al., 1998; Subroto et al.,

1999). Chitinase and â-1,3-Glucanase are

differentially regulated by Wounding, Methyl

Jasmonate, Ethylene, and Gibberellin.

Wounding and methyl jasmonate induces gene

chi 9 for Chitinases expression in the tomato

seeds (Wu and Bradford, 2003). In some study,

it is also found that Chitinase gene are also

expressed in response to stress like cold up to

-2 to -5ºC (Yeh et al., 2000). These Chitinases

have significant antifungal activities against

plant pathogenic fungi like Alternaria sp. For

grain discoloration of rice, Bipolaris oryzae

for brown spot of rice, Botrytis cinerea for

blight of Tobacco, Curvularia lunata for leaf

spot of clover, Fusarium oxysporum, F. udum,

Mycosphaerella arachidicola, Pestalotia

theae for leaf spot of tea and Rhizoctonia

solani for sheath blight of rice (Chu and Ng

2005; Saikia et al., 2005; Kirubakaran and

Sakthivel, 2006). The main substrate of

Chitinases is chitin - a natural homopolymer of

â-1,4- inked N-acetylglucosamine residues

(Kasprzewska, 2003). The mode of action of

PR-3 proteins is relatively simple i.e. Chitinases

cleaves the cell wall chitin polymers in situ,

resulting in a weakened cell wall and rendering

fungal cells osmotically sensitive (Jach et al.,

1995).

Chitin Binding Protein (CBP, PR4):

All chitin binding proteins do not possess

antifungal activities. CBP can be isolate from

plant Beta vulgaris (suger beat), Hydrangea

macrophylla (hortensia), Nicotiana tabacum

(tobacco), Piper longum (pepper), Solanum

lycopersicum (tomato) and Solanum

tuberosum (potato) and bacteria like

Streptomyces tendae (Nielsen et al., 1997;

Bormann et al., 1999; Lee et al., 2001, Yang

and Gong, 2002,). Moleculer weight of the

 

 

 

 

 

 

Borad V. and Sriram S. (2008) Asian J. Exp. Sci., 22(3), 189-196

 

 

CBP was found to be in the range of 9 kDa to

30 kDa and having basic isoelectric pH

(Nielsen et al., 1997; Bormann et al., 1999;

Yang and Gong, 2002,) Expression of the

CACBP1 chitin-binding protein isolated from

cDNA library of pepper (Capsicum annuum

L.) (CACBP1) gene was rapidly induced in

the incompatible interactions upon pathogen

infection, ethephon, methyl jasmonate or

wounding (experimental model plant pepper).

The CACBP1 gene was organ-specifically

regulated in plants. High level of expression

occurs in phloem of vascular bundles in leaves

of pepper (Lee et al., 2001; Wan et al., 2008).

CBP shows strong inhibitory effect against

fungi Aspergillus species, Cercospora

beticola, Xanthomonas campestris and many

more and several crop fungal pathogen

(Nielsen et al., 1997; Bormann et al., 1999;

Lee et al., 2001; Yang and Gong, 2002).

Enzymeticaly CBP has not any function but it

binds to insoluble chitin and enhances hydrolysis

of chitin by other enzyme like Chitinase

(Houston et al., 2005; Vaaje-Kolstad et al.,

2005).

Thaumatin-Like Protein (TLP, PR5):

Thaumatin-like proteins comprise of

polypeptides classes that share homology with

thaumatin, sweet protein from

Thaumatococcus danielli (Bennett) Benth

(Cornelissen et al., 1986). Thaumatin-like

proteins can be isolated from Hordeum

vulgare (barley), Actinidia deliciosa

(kiwifruit), Zea mays (maize), Pseudotsuga

menziesii (douglas-firs), Nicotiana tabacum

(tobacco), Solanum lycopersicum (tomato)

and Triticum sp. (wheat) (Wurms et al., 1999;

Fecht-Christoffers et al., 2003; Anand et al.,

2004; Zamani et al., 2004). Most of the TLPs

have a molecular weight in the range of 18

kDa to 25 kDa and have a pH in the range

from 4.5 to 5.5 (Fecht-Christoffers et al., 2003;

Zamani et al., 2004). Constitutive levels of

Thumatin-Like Protein is typically absent in

healthy plants, with the proteins being induced

exclusively in response to wounding or to

 

192

pathogen attack like Uncinula necator,

Phomopsis viticola (Monteiro et al., 2003).

Although the specific function of many PR5 in

plants is unknown, they are involved in the

Acquired Systemic Resistance and in response

to biotic stress, causing the inhibition of hyphal

growth and reduction of spore germination,

probably by a membrane permeabilization

mechanism and/or by interaction with pathogen

receptors (Thompson et al., 2007). Linusitin is

a 25-kDa Thaumatin-Llike Protein isolated

from flax seeds. Linustin shows antifungal

activity against Alternaria alternata by the

mechanism of membrane permeabilization.

Concentration of protein and lipid and

composition of cell wall of fungi play a major

role in these mechanisms (Anzlovar et al.,

1998). In one study by Menu-Bouaouiche et

al., (2003), Thaumatin-like proteins were

isolated from cherry, apple and banana shows

antifungal activity against Verticillium albo-

atrum and having endo- â1,3-glucanase

activity.

Glycine-Histidine Rich Protein

Many insects like holotrichin and flesh fly

synthesized some Glycine-Histidine Rich

Antifungal Proteins. The mode of action of this

protein is not understood completely.

Phenoloxidase Interacting Protein (POIP)

isolated from Tenebrio molitor (Tenecin)

interacts with phenoloxidase (Yoo et al., 2001)

and inhibits some fungi like Candida albicans

and Saccharomyces cerevisiae (Kim et al.,

2001) and bacteria like Bacillus subtilis,

Proteus vulgaris and Streptococcus aureus

(Kim et al., 2001).

Ribosome Inactivating Protein (RIP,

PR10)

RIP has an inherent antifungal activity. It

has been isolated from Arachis hypogaea L.

(peanut), Mirabilis expansa (mauka)

(Vivanco et al., 1999), Nicotiana tabacum

(tobacco) (Kim et al., 2001), Pisum sativum

(pea) (Ye et al., 2000), Solanum surattense

(nightshade) and Volvariella volvacea

 

 

 

 

 

 

 

.