fungal interaction in plants

7/31/2019 fungal interaction in plants

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6

Recognition of Fungi and Activationof Defense Responses in Plants

B.N.Chakraborty

One of the most difficult and intriguing aspects in the study of biology is an understanding

of the significant events of the interaction between a plant and a micro-organism at the cellular

and subcellular level. However, advances made in the formulation of concepts and techniques

of modern, quantitative cell biology in recent years have paved the way for a basic understanding

molecular events in developing resistance during plant host-pathogen interactions. In spite of

this, what still eludes our grasp is the answer to the high degree of specificity of the host-

pathogen combinations observed in nature. In fact, if one considers the multitude of micro-

organisms to which plants are being continuously exposed in nature, the significance of

specificity becomes more apparent. Until and unless the mechanisms whereby a host and

pathogen recognize the potential for establishing a compatible or incompatible relationship

are identified, and the devices whereby specificity for that relationship is established, it cannot

be claimed that a full understanding of host-pathogen interactions has been achieved.

Plants are constantly being challenged by aspiring pathogens, but disease is rare. Why?

Broadly, there are three reasons for pathogen failure. Either (1) the plant is unable to support

the niche requirements of a potential pathogen and is thus a nonhost; or (2) the plant possesses

preformed structural barriers or toxic compounds that confine successful infections to specialized

pathogen species; or (3) upon recognition of the attacking pathogen, defense mechanisms areelaborated and the invasion remains localized. All three types of interaction are said to be

incompatible, but only the latter resistance mechanism depends on induced responses. Successful

pathogen invasion and disease (compatibility) ensue if the preformed plant defenses are

inappropriate, the plant does not detect the pathogen, or the activated defense responses are

ineffective.


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Recognition of Fungi and Activation of Defense Responses in Plants 71

The success or failure of infection is determined by dynamic competition and the finaloutcome is determined by the sum of favourable and unfavuorable conditions for both the

pathogen and host cells. There is evidence that tolerance of the parasite by the host increases

with increasing antigenic similarity, whereas, resistance of the host is characterized by an

increasing disparity of antigenic determinants (DeVay and Adler, 1976; Chakraborty, 1988). In

this context, the concept of common antigens has acquired a special meaning due to its

coincidence in compatible host-parasite relationships and because it may give some clue to the

high degree of specificity.

Fungal interactions with plants that result in plant diseases cause one of the most serious

problems in the production of food and fiber. Other types of fungi (Mycorrhizae) that establish

mutually beneficial symbiotic relationships with plants enhance plant nutrition. Characteristics

of these interactions clearly suggest that the fungal spores and plants begin to communicate

with each other as soon as they get close to each other.

1. RECOGNITION

Plants have mechanisms to keep a homeostatic balance with potential pathogen or non-

pathogens in the immediate environment. A basic requirement for the proper functioning of

even a simpler cellular system is communication between its components. In this sense,

communication means transmitting information to and receiving information from the

environment, then transforming it to signals or instructions which the cell can understand and

respond to. This exchange of information can be mediated by soluble agents (transmitters) or

by direct cell-cell interaction. It is assumed that compatibility and incompatibility are initiated

by surface-surface contact.

The importance of the plasma membrane in the maintenance and regulation of a cellsphysiological state, its interaction with other cells and its antigenic individuality have been well

established. In addition, by serological methods it has been established that antigen is located,

at least in part, on or near the cell surface. Antigens may involve information transfer and/or

the maintenance of membrane integrity during the cell-to-cell interaction of host and parasite.

When two cells touch, the interfacial energy in the area of contact becomes very low. This can

have a large effect on both the protrusion of membrane proteins in that area and on their local

aggregation resulting in protein transfer following cell-cell contact. The mutual recognition of

different cell types could result from the transfer or interaction of membrane proteins by these

mechanisms. Membrane proteins aggregate in response to a variety of triggering substances

including antigens and mitogens.

The relative unspecificity of fungi and hosts in all kinds of mycorrhiza comes as a surprise

to those who might be led to expect that organisms which form permanent mutualistic

associations would be more likely to be highly specific than pathogenic biotrophs whose

association is temporary, ending perhaps with the premature death of the host. Indeed it might

further be expected that ectomycorrhizal fungi, symbiotic with long-lived perennials, might be

more specific than those of annual herbs, but that is not so either. The explanation lies in the

selective advantage of the associated state and a consequent selection against variation towards


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72 Molecular Approaches for Plant Fungal Disease Management

incompatibility. In addition to the nutritional advantages mentioned, there is for the fungus afurther selective advantage arising out of non-specificity. There will always be, in any soil or in

any ecosystem, suitable habitat, i.e. suitable host roots, for a non-specific fungal symbiont.

Mycorrhizal dependency may be defined as the degree to which a plant is dependent on the

mycorrhizal condition to produce maximum growth or yield at a given level of fertility of soil.

A great variation of dependency in Citrus seedlings using Glomus fasciculatum in artificial soil

cultures of different fertility has been demonstrated. Mycorrhizal dependency is essentially a

crop or host-oriented concept where interest centres on the growth or development of the

host rather than of the fungus. Mycorrhizal effectiveness considers the effectiveness of the

mycorrhizas as organ active in some physiological processes e.g. nutrient absorption, but it is

often measured in similar terms, of dry weight increment per unit time compared to an uninfected

control, as mycorrhizal dependency.

Very little experimental work has been performed on mutual recognition of mycorrhizalfungi and their hosts. Their specificity is not close; a single fungal isolate may form vesicular-

arbuscular mycorrhiza with a wide range of species of host of all the phyla of land plants in

laboratory experiments. Specificity seems to be closer in the competition of natural vegetation

than in pure culture. The extent to which this impression is real is questionable. At all events,

any mycorrhizal host is usually compatible with a wide range of fungi and each mycorrhizal

fungus with a wide range of hosts ( Roy et al., 2002; Chakraborty and Sunar, 2009). Moreover

at a single time one individual plant may associate with several species of fungi. Mycorrhizal

fungi have extensive compatibility with potential hosts which is perhaps only limited by the

inhibitory properties in the host, which itself can be universal or selective in response to the

fungi.

Roots of every plant are colonized by root-surface populations of microorganisms which

also includes the mycorrhizal fungi (Fig. 6.1 D-I & J-M). In the first stage of mycorrhizal

infection these fungi colonize the surface in competition with other members of the population.

The members of the root surface populations rely for growth particularly on the presence of

essential nutrients including carbohydrates, organic and amino acids and a multiplicity of other

compounds including vitamins and growth factors in the root exudates. The mycorrhizal fungi

must all possess the ability to penetrate in the middle lamella region of the cortical tissues and

they must have common property that allows them to do this, and that property differentiates

them from the other denizens of the root surface which do not penetrate.

The available evidence suggests that penetration occurs in a region of the root where the

cells of the epidermis and cortex are reaching or have reached their full size and are in the

process of laying down their cell walls. The electron microscopic observations of many

mycorrhizas show evidence of an interfacial matrix, a contact zone or an involving layerderived from the wall of the host (and perhaps that of the fungus also) separating the hypha

from plasmalemma of the host , both in intercellular and intracellular infections. This indicates

an action of the fungus upon the process of condensation of polymers to form the cell walls of

the host. Such an action could arise as proposed in the case of pathogens, by the proteins

(enzymes) of the fungal walls complexing with the condensing enzymes forming the cell walls

of the host and inhibiting them. It could also arise if the fungus produced on or from its


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surface an inhibitor of the host enzymes. The appearance of the interfacial matrix in

endomycorrhizas gives the impression of the host plasmalemma continuing to secrete vesicles

and to form carbohydrate fibres, but being unable to organize them as a coherent wall. Theability to form a wall is recovered when the fungus senesces, degenerates and becomes

encapsulated within the cell. It is a corollary of the views that variations of the constitutions of

the enzymes which bring about wall-building of the host, or of the proteins of the fungal

surface, will provide a primary explanation of the limitation of ectomycorrhizal fungi to particular

groups of host, ericoid mycorrhiza fungi to others, and vesicular-arbuscular mycorrhiza fungi,

to a wide range of hosts.

Fig. 6.1:(A&B) Conidia of Colletotrichum gloeosporiodes and (C) Erysiphe graminis showing appressoria formation duringgermination. (D-G; J&K) Glomus sp. (H-I& L-M) Gigaspora sp. (A, C, J-M) Scanning electron microscopical

observations. (N&O) Root colonization with AM fungi


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This hypothesis clearly needs examination, especially into the nature of the enzymes orsurface proteins or glycoproteins of the walls or developing walls of both host and fungus. It

is implicit in it that wall-synthesizing enzymes of the host are affected in their activity by

substances on the fungal wall. In addition, a special examination is needed of the exact location

where infection occurs and the condition there of the host cells and their walls. The hypothesis,

however, has the great merit of explaining how it is that fungi which do not secrete enzymes

hydrolyzing carbon polymers such as pectins or cellulose penetrate the cortex and into the

cells.

2. CELL SURFACE INTERACTION

Physical features of the plant surface and chemicals from the plant, both have been

considered to promote germination of the fungal spores and the early differentiation processes,

such as the formation of structures that are needed for penetration into the host. In fungi that

penetrate into the host through stomata, sensing of the physical structure of stomata was

reported to cause formation of appressorium. Thus, germinating spores of Colletotrichum

gloeosporioides, a foliar pathogen of tea, are induced to form appressoria by the cuticular

wax, while the rust fungi form appressorium when the tip of the fungal germ tube contacts the

stomata ( Fig. 6.1. A-C; Fig. 6.2. B). In a plant root-fungal culture, the fungal germ tubes take

contact with the root, branching and growing on the root surface. Appressoria shaped as flat

and swollen bodies, 20-40 mm long, are formed adhering to the host surface as a result of the

contact. It is not known whether topographical signals induce the formation of appressoria in

AM fungi as has been shown for rust fungi. However, they are usually observed as lens shaped

structures lying between adjacent epidermal cells. Leek (Allium porrum) roots colonized by

Glomus versiforme revealed that two infection hyphae are usually formed by the appressoria.

In this plant, they penetrate the underlying epidermal layer passing between two adjacentepidermal cells, developing either into short pegs or large hyphae. The contact of hypodermal

cells and inside them, or in the underlying cortical cell, they loop to form coils. Appressoria

appear as multinucleate structures, slightly vacuolated and rich in lipid. Hyphae which penetrate

intracellularly are encased by a thick deposition of material continuous with the cell wall, and

by the invaginated host plasma membrane. This type of surface interaction is maintained along

the whole infective process. Alternative patterns of fungal penetration across the outer root

layer have also been observed, generally related to the type of host plant. Instead of penetrating

through tight junctions between two epidermal cells, the fungus can pass across the tangential

epidermal cell wall or through root hairs. Roots of woody plants the fungus runs under the

sloughing cells, penetrating the first intact cortical cell layer. All these different patterns of

penetration seem to be influenced by the features of the host cell surface. When the fungus

penetrates the epidermal cells, a localized thickening of the plant wall in the epidermal and inthe underlying cortical cell has been observed in pea roots. The nature of this thickening has

not further investigated, but it is morphologically comparable to the papillae described during

plant-pathogen interactions, although the size in mycorrhizal plants is very reduced.

The characteristics of the plant cell surface may also influence the successive steps in

infection process, namely the passage from the epidermal to the underlying cortical cells,


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which sometimes develop a well-defined layer called the hypodermis. Dimorphic hypodermishave been described and are characterized by deposits of suberin on the radial cell walls.

Suberification is a secondary modification of the cell wall that starts in the zone of differentiating

cells. It is physiologically important because it can deeply influence the apoplastic radial transfer

Fig.6.2: (A- K) Indirect immunofluorescence of Healthy tea leaf tissues (A,C&D) Blister blight infected tea leaf tissues (F-K),

Germinated spores of Exobasidium vexans (B), sclerotia of Corticium invisum. Autofluorescence of cuticle

(A), Major cross reactive antigens in healthy leaf tissue (susceptible variety) probed with IgG of C. invisum (C&D),

Infected leaf tissue probed with IgG of E. vexans and labelled with FITC(F-I); Transmission electron micrographs of

immunogold labelled blister blight infected leaf tissue probed with IgG of E. vexans (J&K).


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of solutes out of or into the root. However, during colonization of roots by AMF, the role of thedimorphic hypodermis is to channel fungal growth and development inside the root. The

invading fungus tends to penetrate and colonize those cells where there is little or no suberin

deposited in the wall. Hypodermal cells where suberin is clearly detectable are avoided by the

fungus and appear dead in contrast to the active and living colonized cells. When the fungus

finds itself surrounded by suberin-lined cells, it stops further colonization. Similar behaviour

has been observed in many other herbaceous woodland plants, where suberization of the

hypodermis may occur before or after the passage of AMF. Thus, morphologic, cytochemical

and genetic analysis point to the importance of the root cell surface in the early events of the

formation of AM symbiosis (Fig.6.1 N&O), being primary physical site of contact between

the plant and the fungus.

3. SIGNAL MOLECULES

3.1. Antigenic Response

There is no simple definition for antigens but a good working concept is to consider them

as substances which are capable of inducing antibody formation as evidenced by various

reactions including specific immunity to infectious agents. A variety of macromolecules can

behave as antigens-virtually all proteins, many polysaccharides, lipoproteins, nucleoproteins,

numerous synthetic polypeptides and many small molecules if they are suitably linked to proteins

or to synthetic polypeptides. The actual significance of the term antigen lies in its capacity to

stimulate the formation of the corresponding antibodies, i.e. immunogenicity, and its ability to

react specifically with these antibodies. However, this has been modified by common usage so

that it now also includes those portions of antigen molecules which have the ability to only

react with antibody but are unable to just stimulate antibody formation. Substances known ashaptens are not immunogenic but do react specifically with the appropriate antibodies.

Immunogenecity is not an inherent property of a macromolecule, as for example, its molecular

weight or absorption spectrum; rather, it is dependent on the system and the condition employed.

Although plants do not produce antibodies, as animals do, against invading pathogens

such as fungi, bacteria or viruses, still some kind of immunological response may be operating

in plants. The striking similarities of cell surface characteristics in plants, animals and micro-

organisms with regard to structural components, antigenic determinants and various functional

characteristics have been critically emphasized. The complexity of the interactions that affect

the selection of parasites and allow their establishment and survival among host cells is manifest

in the frequency and variability of cell surface antigens. Some intriguing research work suggests

that antigenic similarity between host and pathogens may be a prerequisite for compatible

reactions or, in other words, successful establishment of the pathogen in its host depends

upon some kind of molecular similarity between the two partners.

It is clear from the experimental evidences considering various host-pathogen combinations

that when a given host variety does not have an antigen that is present in a particular pathogen

race, the variety is resistant to that race, indicating that susceptibility and resistance are due to

presence or absence of specific pathogen antigen in the plant varieties. Conversely, antigens of


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different races of a pathogen may differ, but susceptibility or resistance of a variety dependson the presence or absence of the specific host antigen in the pathogen race. The active role of

common antigens is rather speculative, but can be visualized in certain instances as a substance

which provides both a stabilizing influence between interacting cells of different organisms

and a basis for continued compatibility (Alba and DeVay, 1985; Chakraborty et al., 2009).

Recognition is thought of as a process initiated by binding of pathogen signal compounds and

host -cell receptors, where receptor includes all kinds of molecules that recognize a specific

configuration of signal compounds. Molecular recognition and specificity determined at the

host-pathogen interface are much more complex than have been presumed. The involvement

of cross-reactive antigens (CRA) in host-parasite compatibility is strongly supported by

immunolocalization in tea leaf tissues (Fig.6.2 C-E). Immunogold localization of CRA has also

been successfully demonstrated for the first time in tea leaf tissues using IgG ofExobasidium

vexans, causal agent of blister blight disease (Chakraborty and Sharma, 2007). With the adventof immuno-cytochemistry subcellular level studies have gained enormous importance in

substantiating preliminary findings with novel and sophisticated techniques. An attempt was

made to detect pathogen (E. vexans) in tea leaf tissue during development of blister blight

disease following indirect immunofluorescence (Fig.6.2 F-I). Encouraging results were also

obtained following immunogold cytochemical labelling of ultra thin tea leaf sections (Fig. 2

J&K).

The outer layers of the root demonstrate a strong heterogeneity in cell wall composition.

Abundant UV autofluoresent (Fig. 2 A), cell wall bound phenols associated with the layered

and complex texture of the cellulosic wall are visible. These features are comparable to those

found in the cell wall of many epidermal tissues of shoots and leaves. Other species, such as

pea, possess epidermal cell walls which, on the contrary, are weakly autofluoresnt and which

have an amorphous texture. Pectin and cellulose have been detected by using monoclonalantibodies against polygalacturonic acid, and a purified cellobiohydrolase binding to -1-4-glucans. Many factors make it difficult to quantify antigens based on the density of gold

particles after gold immunolabeling. However, a dual labeling approach suggests that, in leek

roots, cellulose is very abundant in the epidermal cell walls, while pectin antigens are rare on

the same walls. In pea, walls in the root hairs and epidermal cells are in contrary, strongly

labelled by the probe for pectin. These are consistent with chemical analysis of the pectin

content between dicots and monocots, which indicate that monocots are poorer in pectin

material. It is known that pectins represent the more flexible wall component, which probably

influences and facilitates the penetration of microorganisms. One can thus hypothesize that in

AMF the preferential site for the fungal penetration is mainly controlled by the physicochemical

features of the epidermal cell walls. More specific mechanisms, however, cannot be ruled out.

3.2. Metabolomic Approach

Metabolomics is a developing post-genomic technology which focuses on small metabolites

from an organelle, cell, tissue, organ or organism. Metabolite profiling is a metabolomic approach

which employs nuclear magnetic resonance (NMR) or mass spectroscopy (MS) and can

allow the metabolites to be ultimately identified. Metabolite fingerprinting makes no attempt to


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identify the metabolites but provides a specific chemical signature. These could be used inbreeding programmes and also for early detection of plant pathogens using serological and

molecular methods as documented by Chakraborty and Chakraborty (2003) and Gawande

et al. (2006).

Plant resistance to disease may be thought to be mediated primarily through changes in

metabolites. Defense gene expression is to be a great extent regulated through the production

of metabolites which act as signals. Equally, several of the anti-microbial compounds that are

produced are chemical toxins. It is also the case that pathogens often generate metabolites

which suppress plant defences and cause disease symptoms. Plant pathogen interactions

therefore, represent complex metabolite exchanges which are ideal scenarios for investigation

using metabolomic approaches.

Metabolomic approaches were used to elucidate some key metabolite changes occurring

during interactions ofMagnaporthe grisea the cause of rice blast disease with an alternate

host,Brachypodium distachyon (Routledge et al., 2004; Allwood et al., 2006 ),Exobasidium

vexans blister blight pathogen (Chakraborty and Mur, 2009) as well asHelopeltis theavora

pest interaction with tea plants (Mur et al., 2009). Fourier-transform infrared (FT-IR)

spectroscopy provided a high-throughput metabolic fingerprints of such interactions Principal

component-discriminant function analysis (PC-DFA) allowed the differentiation between

developing disease symptoms and host resistance. Examination of PC-DFA loading plots indicated

that fatty acids were one chemical group that discriminated between resistant (ABR5) and

susceptible (ABR1) response ofB. distachyon by M. grisea . Seven metabolites were

subsequently identified as phopsholipids (PLs) by electrospray ionization mass spectrometer

(ESI-MS) as well as by MS-MS which suggests considerable and differential PL processing of

membrane lipids during interaction which may be associated with the elaboration/suppression

of defense mechanisms or developing disease symptoms. A metabolite fingerprinting was

obtained fromExobasidium vexans challenged tea plants from various plantations using FT-

IR. Four sample types were assessed, healthy (H) and infection stages 1 (S1, purple spots), 2

(S2, showing hyperplasia and blisters) and 3 (S3, necrotic). The derived spectra were analysed

using multivariate analyses, mainly Discriminant Function Analyses (DFA). DFA allowed the

discrimination of fingerprints of H from infected samples particularly at the S3 stage

(Fig. 6.3). There was also some evidence of discrimination according to some plantations sites.

4. DO SIGNAL MOLECULES EXIST IN MYCORRHIZAL SYMBIOSIS?

The dual in vitro system for culturing AM fungi has clearly demonstrated that the presence

of the root is required for fungal growth. Soluble factors from the plant can significantly

stimulate the growth of the fungus before physical contact with the root surface. If there areany signal molecules involved, the first questions concern their chemical nature and their range

of specificity, considering the fact that 80% of land plants harbour Mycorrhizal symbiosis. In

this context, the importance of plant phenolic compounds in the early stages of communication

between plants and pathogenic or symbiotic bacteria have been demonstrated. Wounded plant

cells produce phenolic molecules that activate virgenes inAgrobacterium, while roots exude


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flavanoids that act as activators ofnodgenes inRhizobium. These regulatory genes are necessary

in both cases for infection of the host. When some flavanoids viz. Naringenin, hesperitin and

apigenin were added to AM fungi at a concentration of 0.15 to 1.5 M, hyphal growth ofG.margarita was increased 2 to 10 fold over that obtained on water agar and there was also

increase in the rate of spore germination. Flvanoids released from seeds of alfalfa during

imbibition also increased mycelia branching in AM fungi. Plant exuded flavanoids may enhance

independent growth of AM fungi. However, it is not clear whether they act as true signalmolecules or simply as a nutritional factor. A wider range of flavanoids should be tested to find

out whether related molecules may have a different effect or some degree of host specificity.

5. WHAT DOES THE FUNGUS COMMUNICATE TO THE PLANT?

Numerous signals are thought to be exchanged between the host-fungal partners during

Fig. 6.3: Contribution of particular wavenumbers to the DFA models. DF1 (A) appears to indicate a greater contribution fromaromatic and polysaccharides metabolites as a common response in infection at all stages (S1, S2, S3). (A) General

(DF1) and (B) stage specific responses to infection (DF2) appear to involve changes in fatty acids and amide groups.

(C&D) Infection stage specific Discriminant Function Analysis.


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both compatible and incompatible interactions. In plant-pathogen interactions, many of thesignals produced during early stages of infection induce a hypersensitive reaction (HR) in the

plant. Penetration into the root and intercellular growth of the AM fungi involves a complex

sequence of biochemical and cytological events and intracellular modifications (Bonfante, 2001),

which imply that the fungus must clearly be recognized by the host plant. Evidence suggests

that, as in plant-pathogen interactions, the induction/suppression of mechanisms associated

with plant defence play a key role in AM fungal colonization and compatibility with its host.

The extensive fungal colonization of mycorrhizal rootsraises some questions as to how

the fungi sustain a long-term infection without evoking or becoming susceptible to a host

defense response. Furthermore, how is the viability of host tissue maintained upon colonization?

The cell walls of both microbes and plants are considered to be one of the major structures

from which signaling molecules are generated during interaction. During the infection process,

an important role in producing these signal molecules may be played by the hydrolytic enzymesproduced by the fungus which degrade cell wall components. To penetrate the plant tissue,

invading microorganisms loosen the wall through the localized secretion of hydrolytic enzymes.

At this stage, oligomeric fragments are released from the wall of the host plant which could

regulate physiological events such as elicitation of the plant defense response.

6. DEFENSE RESPONSES OF HIGHER PLANTS

Plants are compelled to withstand stresses of all kinds, be it biotic, abiotic or anthropogenic

as a consequence of their immobility (Chakraborty 1996, 2007). Higher plants protect themselves

from various stresses, such as pathogen attacks, wounding, application of chemicals including

phyto-hormones and heavy metals, air pollutants like ozone, ultraviolet rays, and harsh growing

conditions. This reaction is known as the defense response of higher plants, and a series ofproteins actively synthesized with this reaction is called defense-related proteins. Such constraints

lead to production of a wide array of defense compounds, which are either induced or preformed.

One of the ways in which plants respond to biotic and/or abiotic stress factors are the

accumulation of various novel proteins collectively referred to as pathogenesis-related proteins

or PR-proteins (Datta and Muthukrishnan, 1999; Chakraborty 2005). Such protective plant

proteins specifically induced in pathological or related situations have been intensively studied

from the agricultural interest. On the other hand, many of the reserve proteins accumulated in

seeds and fruits take the constitutive defense function against microbial pathogens and

invertebrate pests in addition to their storage function. These inducible or constitutive defense

mechanisms of higher plants are relatively conserved in the course of evolution. Accordingly,

most plants produce or accumulate similar proteins under certain situations irrespective of

their morphological differences.These proteins were first observed in tobacco, Nicotianatabacum, cultivars reacting hypersensitively to tobacco mosaic virus (Van Loon and Van

Kammen, 1970; Gianinazzi et al., 1970). The occurrence of these proteins was not pathogen

specific but determined by the type of reaction of the host plant, indicating that these proteins

were of host origin. Since the proteins were induced under specific pathological conditions,

they were named pathogenesis-related proteins (Ornstein, 1964). PRps were found in small


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amounts in senescing leaves of flowering plants and in relatively larger quantities when necrosiswas more severe. This led to the assumption that these polypeptides were stable proteolytic

breakdown products of larger leaf proteins. By definition, these proteins abbreviated as PRps,

were described as proteins coded for by the host plant but induced specifically only in

pathological or related situations. PRps are most often of low molecular weight, selectively

extractable in lowpH, highly resistant to proteolytic degradation/ or endogenous proteases and

localized predominantly in the intercellular space. PRps have been found in several plant species

belonging to various families (Table 6.1).

Proteins that are induced exclusively during disease development in compatible host-

pathogen combinations have hardly been considered as PRps. PRps have been grouped into

families on the basis of similarities in molecular weights, amino acid composition and serological

properties confirmed by nucleotide sequencing of corresponding cDNAs (Van Loon, 1975).

The classification has set a convenient standard for other plant species, in which these familynumbers now similarly designate PRps with properties homologous to tobacco PRps. Initially

the PRps were grouped in to five main classes consisting of the 10 major acidic PRps of

tobacco characterized both by biochemical and molecular biological techniques and designated

as PR-1 to -5 (Gianinazzi et al., 1977; Van Loon 1985).

A unifying nomenclature was proposed based on their grouping into eleven families,

classified for tobacco and tomato, sharing amino acid sequences, serological relationships

and/ or enzymatic or biological activity. The criteria for inclusion of new families of PRps

were (i) protein(s) must be induced by a pathogen in tissues that do not normally express the

protein(s), and (ii) induced expression must have shown to occur in at least two different

plant-pathogen combinations, or expression in a single plant-pathogen combination must have

been confirmed independently in different laboratories (Van Loon and Van Strien, 1999). Each

PR family is numbered and the individual family members are assigned lower case letters in the

order in which they are described. In accordance with the recommendations of the Commission

for Plant Gene Nomenclature, PR-genes are designated asypr, followed by the same suffix as

of the family. Later on three more peptides, which were capable of inducing defense responses

of plants, were identified. These three families (PR-12, -13 and -14) comprise the pathogen

induced plant defensins (PR-12) (Fraser, 1981) thionins (PR-13) (Antoniw et al., 1981) and

lipid transfer proteins or LTPs (PR-14) (Garcia-Olmedo et al., 1995). So far, 17 families of

PRps have been recognized. However, the properties of all these proteins have not yet been

elucidated.

Besides proteins newly defined mRNAs (cDNAs) are often considered as additional members

of the existing families were shown to be induced by pathogens or specific elicitors. However,

because PRps are generally defined by their occurrence as protein bands on gels, and classifiedwithin each family once the protein has been characterized, cDNA or genomic sequences

without information on the corresponding protein cannot be fitted in to the adopted nomenclature.

Thus for naming it is necessary to gather information at both the nucleic acid and the protein

level when dealing with a stress-related sequence falling within the definition of PRps. Conversely,

homologies at the cDNA or genomic level may be encountered without information on the

expression or characteristics of the encoded protein. Such sequences obviously belong to the


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PR-type families, but yet cannot, be considered to correspond to pathogen-induced PRps andnamed accordingly. In a more than a few situations, it is difficult to distinguish PRps from related

proteins/ mRNAs that are present in some organs or appear during specific developmental stages.

Homologous proteins/ mRNAs in healthy tissues in which no induction by pathogen infection has

yet been demonstrated, are to be termed PR-like proteins (PRLs) and their genes yprl.

Localization of the major, acidic PRps in the intercellular space of the leaf seems to guarantee

contact with invading fungi or bacteria before these are able to penetrate. However, few of the

inducible acidic PRps associated with SAR have been shown to possess significant anti-

pathogenic activity. It could be that PRps make cells less conducive, but any such evidence is

lacking. Elucidation of the biochemical properties of the major, pathogen-inducible PRps of

tobacco and subsequent cloning of their cDNAs and/ or genes revealed that proteins with

substantial similarity to the classical PRps, which are mostly acidic and extracellular proteins,

the homologous counterparts are mostly basic and localized intracellularly in the vacuole. Asfar as it has been possible to deduce, they possess the same type of enzymatic activities, but

their substrate specificity and specific activity may be rather different.

Table 6.1: Plant species in which pathogenesis-related proteins have been identified

Family Plant species

Amaranthaceae Gomphrena globosa

Chenopodiaceae Chenopodium amaranticolor, C. quinoa, Beta vulgarisCompositae Gynura aurantiaca, Helianthus annuum

Cruciferae Arabidopsis thaliana, Brassica nigra, B. juncea, B. napus, B. rapa, Raphanus sativusCucurbitaceae Cucumis sativus , C. melo, Cucurbita maxima, C. Pepo

Gramineae Hordeum vulgare, Zea mays, Avena sativa, Oryza sativa,

Malvaceae Triticum aestivumPapilionaceae Gossypium hirsutum, Medicago sativa, Phaseolus vulgaris, P. lunatus, Cicer arietinum,

Vigna unguiculata, V. radiata, Arachis hypogea, Lablab purpureus, Pisum sativum,Glycine max

Rutaceae Citrus sinensis

Solanaceae Capsicum annuum, Petunia, Solanum demissum, S. nigrum,S. tuberosum, S. dulcamara,Nicotiana debneyi, N. glutinosa, N. langsdorfii, N. plumbaginifolia, N. rustica, N. sylvestris,

N. tomentosiformis, N. tabacum, Lycopersicon esculentumUmbelliferae Petroselinum crispum, Apium graveolens

Vitaceae Vitis viniferaPinaceae Picea abiess

PRps are, as such, a collective set of novel proteins which a plant produces in reaction to

a pathogen mainly in incompatible interactions and thus impedes further pathogen progress.The related situations in which PRps were found to be induced, seem to prove the point:

application of chemicals that mimic the effect of pathogen infection or induced some aspects

of the host response, as well as wound responses that give rise to proteins that are also

induced during infections, can induce both PRps and acquired resistance. The occurrence of

homologous PRps as small multigene families in various plant species belonging to different


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families, their tissue-specific during development and consistent localization in the apoplast aswell as in the vacuolar compartment and their differential induction by endogenous and exogenous

signaling compounds suggest that PRps may have important functions extending beyond their

apparently limited role in plant defense ( Chakraborty and Sharma, 2008).

During the hypersensitive reaction cellular damage and death is a major stress to the plant,

as exemplified by high increases in abscisic acid and ethylene. It is possible, therefore, that

PRps are stress proteins directed to alleviate harmful effects of cellular degradation products

on thus far untouched neighboring cells. Both acidic and basic PRps may be induced by high

concentrations of ethylene or physiological necrosis, or wounding. Such induction in the

absence of pathogenic attack might be taken to indicate protection of cellular structures, either

physically to stabilize sensitive membranes or macromolecules, or chemically to keep potentially

harmful saprophytic microorganisms on tissue surfaces or in intercellular spaces in check. In

virtually any natural stress condition e.g., heat, cold, drought, osmotic stress, water logging,anaerobiosis, metal toxicity, etc., plants are known to react by the synthesis of novel, and

sometimes partly overlapping, sets of proteins.

The various conditions under which PRps occur are reminiscent of those under which

heat-shock proteins (HSP) are induced. These proteins are ubiquitous in living organisms and

associated with the acquisition of thermotolerance to otherwise lethal temperatures, but a

causal connection is not evident. Interestingly, the promoters of all three tobacco PR-1 genes

that are expressed, as well as of a different type of PR in parsley, contain a heat shock

regulatory element, but the proteins are not induced to detectable levels by heat shock.

Nevertheless, PRps might have an analogous function, though quite different, chaperonin-like

function: unlike PRps, HSP are intercellular proteins that do not accumulate during heat shock.

However, the specific occurrence of individual PRps in some floral organs, but not in others,

points to other, more specific roles.

The relative ineffectiveness of PRps in determining resistance to pathogens does not preclude

an involvement in defense. As first proposed by Mauch and Staehelin (1989) acidic, extracellular

PRps might be involved in recognition processes, releasing defense-activating signal molecules

from the walls of invading pathogens. This would hold particularly for chitinases and glucanases

that could liberate elicitor-type carbohydrate molecules from fungal and bacterial cell walls.

Thus, a ,1,3 glucanase induced in soybean seedlings by infection or chemical stress releaseselicitor-active fragments from cell wall preparations of the fungus Phytophthora megasperma

f.sp. glycinea.Such elicitors could help stimulate defense responses in adjacent cells and thus

accelerate and enhance these reactions, as well as induce acquired resistance to further infection

(Ham et al., 1991). A role of PRps as specific internal signal generating enzymes would be

consistent both with their occurrence in specific organs and with their induction during thedevelopment and in response to stressful pathogen infections. The major chitinase of bean

leaves first described by Boller et al. (1983) to be induced by ethylene and located in the

vacuole, appears to be also induced in abscission zones at the stem petiole-junction together

with a PR-1-like protein, two isoforms of-1,3-glucanase, other chitinases, and a thaumatin-like protein (Staehelin et al., 1994).


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Fig. 6.5. Induction of chitinase (PR-3) in cell suspension of tea varieties in response to biotic and abiotic elicitors

[ EV Exobasidium vexans; HX Hexaconazole ; CR Catharanthus roseus]

The response of biotic and abiotic elicitors treated suspension-cultured tea cells alwaysindicated more significant chitinase activity in comparison to ,1,3 glucanase activity (Fig. 6.4& 6.5). The PR-3 (Chitinase) and PR-2 (,1,3 glucanase) were immunolocalized in the leaftissues (Fig.6.6 A&B) and in the cultured tea cells (Fig.6.6 C-E) as evident by bright apple

green fluorescence following labelling with FITC (Sharma and Chakraborty, 2004; 2005).

Accumulation of PR-3 (Chitinase) was also confirmed by ultrastructural immunocytochemical

studies in cellular compartments (Fig.6.6 F&G).

Fig. 6.4. Induction of -1,3-glucanase (PR-2) in cell suspension of tea varieties in response to biotic and abiotic elicitors[ EV Exobasidium vexans; HX Hexaconazole ;CR Catharanthus roseus]


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Fig. 6.6:(A-E) Indirect immunofluorescence and (F&G) Transmision electron micrographs of immunogold labelled salicylic acid

(SA) treated tea leaf tissue and cell cultures probed with PAb-chitinase [ A,B, E-G] and PAb b-1,3-glucanase [D].

The versatile multicomponent defense system of plants is adequate to provide them pro-

tection against most of their potential pathogens (Chakraborty et al., 2005 a, b, c) only a few

of them can overcome this defense and cause disease. Just before or concomitant with the

appearance of a hypersensitive reaction (HR) the synthesis of PR-proteins is increased. Inaddition to the localized HR, many plants respond to pathogen infection by activating defences

in uninfected parts of the plant (systemic acquired resistance, SAR). As a result, the entire

plant is more resistant to secondary infection. SAR is long lasting and confers broad-based

resistance to a variety of pathogens. The synthesis of antimicrobial products, including phy-

toalexins and PR-proteins, correlates well with the development of both HR and SAR. (Fig.6.7).


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Fig.6.7: Signal transduction pathway illustrating cell surface interactions of plant and pathogen and induction of defense

response

7. DEFENSE RESPONSES IN ARBUSCULAR MYCORRHIZAL SYM-BOSIS

The endosymbioses formed between plants and micro-organisms play an important role in

agriculture natural ecosystems. The most widespread mutualistic endosymbiotic interactions

are formed between plant roots and AMF. The successful establishment of this mutualistic

association constitutes a strategy to improve the nutritional status of both partners. The fungi

receive fixed carbon compounds from the host plant, while the plant benefits from the associationby the increased nutrient uptake of phosphorus, enhanced tolerance to abiotic stress, and

resistance to pests and pathogens (Smith and Read 2008; Bhargava et al., 2008). Generally,

AMF show little or no specificity and the factors that determine whether mycorrhiza are

formed or not appear to depend on the genotype of the host plant (Koide and Schreiner 1992).

Evidence for this is provided by the existence of non-host plant species (Giovannetti and

Sbrana 1998) and Myc mutant plants unable to form AM symbiosis (Gollotte et al., 1993).

7.1. During the Early Stages of Development of Arbuscular Symbiosis

Recognition of a potential invader is a prerequisite for the initiation of an effective defence

response by the plant which is achieved through the recognition of specific signal molecules

known as elicitors. Exogenous elicitors are secreted from the microbe or whereas endogenous

elicitors formed as a result of physical and/or chemical cleavage of the plant cell wall. Afterperception of an elicitor, a number of biochemical changes contribute to the early response in

host cells. These processes include changes in the ion permeability of the plasma membrane,

the activation of plasma membrane-bound enzymes, the activation of kinases, phosphatases,

phospholipases, and the production of signal molecules, including active oxygen species. The

result of these processes is the transcriptional activation of defence-related genes (Somssich


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and Hahlbrock, 1998). Some events such as signal perception, signal transduction and defencegene activation, similar to those found in plant-pathogen interactions have also been found in

the plant interaction with AMF. AM fungi secrete similar chitin elicitors, which could induce a

defence response (Salzer and Boller, 2000). For instance, the elicitor derived from an extract

of extraradical mycelium ofGlomus intraradices was able to induce phytoalexins (glyceollin)

synthesis in soybean cotyledons (Lambais, 2000). Chalcone synthase, the first enzyme in the

metabolism of a flavonoid compound inMedicago truncatula was induced by G. intraradices

(Bonanomi et al., 2001). Furthermore, a more hypersensitive-like response, commonly observed

when the plant is confronted with a pathogen, could be observed in compatible AM associations.

An oxidative burst could be detected at sites where hyphal tips ofG. intraradices attempted to

penetrate a cortical root cell ofM. truncatula (Salzer et al., 1999). Moreover, necrosis and cell

death have been observed at sites ofGigaspora margarita infection ofMedicago sativa roots

(Douds et al., 1998). The most evident effects were observed in incompatible associationsbetween AM fungi and non-host plants or Myc mutant plants. In the case of non-host plants,

the molecular bases for incompatibility with AM fungi remain unclear, but in some associations

like resistance reaction seems to involve a hypersensitive defence response, characterized by

the deposition of callose, PR-1 protein, and phenolics has been observed when pea Myc

mutant plants were challenged with an AMF (Gollotte et al., 1993).

Transient increases of catalase and peroxidase activity were also observed in mycorrhizal

plants, during appressoria formation and fungal penetration into the tobacco, onion and bean

roots (Blilou et al., 2000a, Spanu and Bonfante-Fasolo, 1998; Lambais, 2000) which coincided

with the accumulation of salicyclic acid (SA), a signal molecule involved in the signal transduction

pathway activated in plant-pathogen reactions (Chakraborty and Chakraborty, 2008). A transient

accumulation of SA during the early stages of infection has also been observed in the interaction

between rice and Glomus mosseae (Blilou et al., 2000b) which was correlated to an increase inthe expression of genes encoding lipid transfer protection (LTP) and phenylalanine ammonia-

lyase (PAL). Most of the defense-related genes which express during the early stages of AM

fungal penetration are also activated by pathogen infection, treatment with elicitors, or by SA.

In this context, the induction of defence gene expression could be considered to be a result of

fungal elicitor recognition and signal transduction pathway activation. The weak and transient

character of the plant defense response could be a consequence of the low capacity of the

fungus to trigger such a response and/to induce a plant mechanism which suppresses an

already activated defence response at several levels to allow for fungal growth within the plant

tissue.

7.2. Defense Responses in Plant Cells Containing Arbuscules

In the early stages of root colonization by AMF, the plant defense response is characterizedby a weak and transient activation, but at later stages, after arbuscule formation, this activation

appears to become stronger, although only in those cells which contain fungal structures. For

instance, the use of specific probes in studies ofin situ expression revealed that some mRNAs

of genes associated with the plant defense response specifically accumulated in plant cells

containing arbuscules. Likewise, members of different classes of plant defense genes, including


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genes encoding hydroyproline-rich glycoproteins (HRGP), enzymes involved in metabolism ofphenlypropanoid, reactive oxygen species and plant hydrolase have been detected in plant cells

containing arbuscules. An increase in the quantity of mRNA encoding HRGP was observed in

the root cells that contained arbuscules (Blee and Anderson, 2000).

Genes encoding enzymes that catalyse core reactions of the metabolism of phenlypropanoid

have been expressed in cells with arbuscules. In situ localization of phenlyalanine ammonia

lyase (PAL) and chalcone synthase (CHS) transcripts were observed in cells containing

arbuscules. However, expression of other genes encoding enzymes such as chalcone isomerase

(CHI) or isoflavone reductase was not significantly affected in mycorrhizal roots (Harrison

and Dixon, 1994). Genes involved in the catabolism of reactive oxygen species, such as

catalase and peroxidase have also been localized in bean and wheat root cells containing arbuscules

(Blee and Anderson, 2000) which play a role in the catabolism of hydrogen peroxidase and/or

in cross-linking reactions between proteins and polysaccharides in the interface between thearbuscule and the plant cell plasma membrane. Corroborating the biochemical data, differential

gene expression of acidic and basic forms of chitinase and -1, 3-glucanase has been observedduring mycorrhiza formations in different plant-fungal combinations (Lambais and Mehdy,

1998; Salzer et al., 2000, Chakraborty et al., 2007). Accumulation of basic forms of chitinase

and b -1.3-glucanase transcripts have been observed in the intercellular region between cortical

cells containing arbuscules (Blee and Anderson, 1996; Lambais and Mehdy, 1998), suggesting

that these enzymes might be involved in the control of intraradical fungal growth. Interestingly,

the accumulation of-1,3-glucanase mRNA in cells containing arbuscules was modulated byphosphorus (P) concentration. The level of mRNA accumulation increased as concentration

of phosphorus decreased. In comparison, the amount of basic chitinase transcripts did not

change with mycorrhization or P concentration (Lambais and Mehdy, 1998). The enzymatic

activity and chitinases and -1, 3-glucanases could form part of the defence response by theplant to the invading fungus. A specific induction of a class III chitinase gene family in mature

M. truncatula mycorrhizae is evident, which may be involved in suppression of plant defence

reactions in the later stages of the AM development (Salzer et al., 2000).

Results from the split root experimental system developed by Cordier et al., (1998) clearly

demonstrated for the first time that arbuscular mycorrhiza formation by Glomus mosseae

induces not only localized but also systemic resistance against Phytophthora parasitica in

tomato roots. The localized resistance of mycorhizal tissues is seen in the rare occurrence of

P. parasitica hyphae and their inability to invade arbuscules containing host cells. The ISR is

characterized by large reductions in root damage and in P. parasitica development within

nonmycorrhizal root systems, in comparison to roots of nonmycorrhizal plants. This

bioprotection is directly linked to root colonization by the Arbuscular Mycorrhizal fungus since

neither a microbial filtrate from G. mosseae inoculum nor low Mycorrhizal levels are sufficientto induce it. Cytomolecular investigations provide clear evidence that both the localized and

systemic protective effects induced by Arbuscular mycorrhiza involve the accumulation of

plant defense related molecules in association with the elicitation of wall reactions in the host

roots.


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8. EXPRESSION OF GENES INVOLVED IN THE DEFENSE REACTIONCultured plant cells are suitable experimental material to study the response of plant cells in

some plant diseases because the signal molecules distribute simultaneously to each cell. Isolated

bean cell culture following treatment with elicitor prepared from Colletotrichum lindemuthianum

very rapidly accumulated mRNA for the key enzymes such as phenylalanine ammonia lyase

(PAL), chalcone synthase (CHS) and chalcone isomerase (CHI) for isoflavonoid phtoalexin

biosynthesis reaching to a maximum level 3h after treatment and then decreased to the original

level.The mRNA encoding cinnamyl-alcohol dehydrogenase, an enzyme specific to the synthesis

of lignin monomers, accumulates in cultured bean cells very rapidly following treatment with

fungal elicitior. Similarly, the transcription of genes encoding chitinase, which is responsible

for the defense reaction by the degrading the cell wall of pathogenic fungi, is activated very

rapidly following treatment with fungal elicitor. The rapid activation of these gene expressions

in plant cells after elicitor treatment or fungal attack indicates that in plant cells very rapid stepsmay be involved in the signal transduction system from the recognition of microorganisms to

the transcriptional activation of these genes (Daniel and Purkayastha, 1995)

Hydroxyproline-rich glycoproteins (HRGPs) are usually found in low amounts in the cell

wall of higher plants. However, larger accumulations were found in melon seedlings infected

with Colletotrichum lagenarium and many host-parasite combinations. It is believed that HRGPs

play a role in resistance of plants to pathogens by acting as a structural barrier and as an

agglutinin. A remarkable increase in HRGPs occurs not only during infection but also following

wounding and elicitor treatment. The activation of these genes is slower than compared with

the PAL or CHS genes, which suggests the involvement of a secondary signal substances

which originates indirectly from host cells.

The pattern of activation, structure and genomic organization of a gene (prpl) encoding apathogenesis-related protein (PR1) in potato cultivar carrying resistance gene R1 elicited by

infection ofPhytophthora infestans have been demonstrated. The coding sequence of theprpl

and the deduced amino acid sequence are strikingly similar to that of a 26-kDa heat shock

protein from soybean.

Genes encoding enzymes concerned with phenylpropanoid biosynthesis are activated

very rapidly after the elicitor treatment, but are restored to the original level within several

hours. The mechanism of this rapid decline of the accumulation of the product of PAL, may

play a role as a regulation signal in gene expression of the phenylpropanoid biosynthetic

pathway, because trans-cinnamic acid inhibits the transcription of PAL and CHS genes in

cultured bean cells.

9. REGULATION OF DEFENSE MECHANISMS IN AM SYMBIOSIS

Multiple signals and differential induction of gene expression mediate the complex interaction

between AMF and plant cells (Fig. 6.8). The differential activation of defence genes reflect

that some mechanism exists that regulated the suppression of the defence response to allow

compatible AM fungi to form a mutualistic symbiotic interaction with the plant. One possible

mechanism to attenuate the plant defence response in the AM symbiosis might be the degradation


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Fig.6.8. Hypothetical model representing the regulatory mechanisms involved in the plant defense response during AM

symbiosis ( Garcia-Garrido et.al., 2002)

of exogenous elicitor molecules produced by the AM fungi and/or the prevention of endogenous

elicitor release from the plant cell wall. Theoretically, hydrolytic enzymes regulate both processes

as plant hydrolases could hydrolyse fungal elicitor components while fungal enzymes could

hydrolyse plant cell wall components. During AM development the expression of constitutive

and mycorrhiza-specific chitinases, and -1, 3-glucanse isoforms (potential hydrolases offungal elicitors) are differently regulated, thereby pointing towards a central role for these

enzymes in AM formation (Salzer et al., 2000). Furthermore, the induction of a plant defenseresponse by an elicitor released from an extract of extraradical mycelium of G. intraradices

has been observed (Lambais, 2000). A similar mechanism for elicitor degradation and plant

defense attenuation in AM chitinase enzymes by the host plant was demonstrated (Salzer et al.,

2000; Salzer and Boller, 2000). It was proposed that constitutively expressed plant chitinase, in

the early stages, and the mycorrhiza- specific isoforms in the later stages, were the enzymes

responsible for elicitor degradation.


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The production of endogenous elicitors derived from the degradation of the cell wall doesnot seem to be an effective mechanism for the induction of the plant defence response. Evidence

supporting the hypothesis is that mycorrhizal fungi produce very little plant cell wall degrading

enzymes (Garcia-Garrido et al., 1992). Furthermore, the cell wall degrading enzymes produced

in mycorrhizal roots show similar electrophoretic and biochemical characteristics as plant

enzymes produced in non-mycorrhizal roots (Garcia-Garrido et al., 2000) which suggest that

the role of fungal enzymes is only selectively and specifically to break plant cell wall components

for fungal penetration, but not to participate in an unspecific and uncoordinated plant cell wall

degradation process, which could produce endogenous elicitors

Nevertheless, the intracellular colonization by AM mycorrhizal fungi creates a new interface

compartment composed of membranes from both partners separated by apoplastic material

containing molecules common to the plant primary wall, such as cellulose, pectin, xyloglucane,

and HRGP which are not assembled into a fully structured wall ( Bonfante, 2001). The lyticactivity of plant and/or fungal enzymes over these molecules could then putatively generate

oligo-fragments which could act as elicitors for a localized defensive response at the arbuscular

level.

Another possible mechanism to attenuate the plant defense responsible could be by blocking

components of the signal transduction pathway that activate this response. Among these

components, salicyclic acid and reactive oxygen species (ROS) have been implicated as second

messengers in AM associations. Although levels of H2O

2and other ROS have not been measured

in AM roots, indirect evidence exists that suggests that the levels of H2O

2in mycorrhizal

associations are increased (Salzer et al., 1999). Alterations in the pattern of anti-oxidative

enzymes, such as catalase and peroxidase in mycorrhizal roots may indicate that oxidative

compounds are produced during the colonization process (Lambais, 2000). The increase in

catalase and peroxidase activity could be due to their function as antioxidants for any active

oxygen molecules generated during the initial stages of fungal penetration. Since H2O

2and

other reactive oxygen species are involved in signal transduction cascades in plant-pathogen

interactions, it is possible that degradation of H2O

2by catalase in AM could be a possible

mechanism for avoiding the activation of defence response genes. Transient increases in catalase

and peroxidase activities in AM tobacco roots occur at the same time as the transient enhancement

of free SA (Blilou et al., 2000a). In this way there may be a synergistic relationship between

SA and H2O

2in some plant defence responses. The role and significance of SA in AM interactions

remains unclear. Nevertheless, the finding that transient increases of SA are detected in tobacco

and rice AM roots and that the exogenous application of SA to rice roots did not affect appressoria

formation , but did cause a transitory delay of mycorrhization of root (Blilou et al., 2000b)

suggest that the regulation of defence response in plants against AM fungi may be through the

SA pathway.

Alterations in the flux of nutrients and in the levels of plant hormones could generate cell-

signaling pathways that regulate the plant defence response during AM development. Evidence

for this is provided by the fact that phosphate levels in the plant are negatively, and carbohydrate

levels are positively correlated with root colonization by AMF. The exact mechanism and

molecular basis that could mediate the inhibition of AM colonization by P remain unknown.


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One possibility is that defense gene expression is mediated by a signaling mechanism thatsenses the level of P in the root, resulting in an upregulation of these genes in plants with high

levels of P. A sensor system based on the flux of carbohydrates regulates defence gene expression

in plant cells containing arbuscules. It has been postulated that an increase in the flux of

sucrose, glucose, and fructose and the activation of genes associated with the catabolism of

sucrose in the cells containing arbuscules constitute a mechanism responsible for the activation

of defense genes (Blee and Anderson, 2000). However, the mechanism that mediates defence

regulation by nutritional factors remains unknown.

It is intriguing that plant hormone levels in mycorrhizae are altered, as the ethylene level in

AM tobacco roots is low (Vierhlig et al., 1994) and cytokinin levels are significantly increased

in alfalfa and tobacco AM roots (Van Rhijn et al., 1997; Ginzberg et al., 1998). The significance

and role of plant hormones in AM symbiosis have been reviewed (Beyrle, 1995), but the exact

role of plant hormones in the regulation of gene expression during the plant AMF colonizationis not clear. Nevertheless, the effective role of plant hormones during AM is not clear and it is

difficult to determine if changes in hormone levels in AM symbiosis are directly implicated in

the process of mycorrhization. An investigation of the signalling mechanisms underlying the

activation and cross-talk processes that participate in the regulation of the AM symbiosis may

contribute to our understanding of the formation and functioning of this symbiosis, that these

mechanisms could have on the induction of the plant resistance against pathogens observed in

mycorrhizal plants.

Profiling of global Avr/R gene-triggered gene-expression responses points to a certain

degree of constitutive activity of R-gene-pathways. In the absence of infection , mutants

disrupted in distinct R pathways display reduced (or elevated) expression of defined gene sets.

This result supports the notion that R activation is tightly controlled. Ectopic R expression can

activate defense pathways in the absence of pathogen. After pathogen recognition, repression

of these pathways is completely removed and they operate with maximal capacity, fully activating

(or repressing) their target genes. The outcomes are the production of potentially toxic secondary

metabolites, programmed cell death and the creation of a locally inhospitable environment for

the pathogen. R- protein activation may be negatively regulated by intramolecular mechanisms,

although how this is achieved and how they are activated will require more examples and more

detailed biochemistry and structural biology. The role of accessory and partners proteins in R

activation is just at its beginning, and will require both clever forward and reverse genetics

approaches combined with proteome based solutions. Signaling pathways leading from activated

R proteins are being chipped away, and emerging concepts suggest that the resistant state is

achieved by breaching quantitative activation thresholds, possibly driven by a central SA-

driven positive feedback loop. In depth understanding of how the plant immune system functions

may one day lead us to the development of controlled, broad-spectrum resistant crops withoutthe deleterious fitness costs.

References

Alba, A.P.C. and DeVay, J.E. 1985. Detection of cross-reactive antigens between Phytophthora infestans(Mont.) de Bary

and Solanumspecies by indirect enzyme-linked immunosorbent assay. Phytopath Z.,112:97-104.


24/26


Allwood, J.W., Ellis, D.I., Heald, J.K., Goodacre, R. and Mur, L.A.J. 2006. Metabolomic approaches reveal that phosphatidicand phosphatidyl glycerol phopsholipids are major discriminatory non-polar metabolites in responses by Brachypodium

distachyonto challenge by Magnaporthe grisea. The Plant Journal: 1-18.

Antoniw, J.F., Kuch, J.S.H., Walkley, D.G.A. and White, R.F.1981. The presence of pathogenesis-related proteins in callusof Xanthi-nc tobacco. Phytopathologische Zeitschrift, 101:179-184.

Beyrle, H. 1995. The role of phytohormones in the function and biology of mycorrhizas. p365-390 In : Mycorrhiza structure,function, molecular biology and biotechnology. Varma, A. and Hock, B. (ed.). Springer-Verlag, Berlin.

Bhargava, S., Sharma, M.P., Pandey, R. and Gour, H.N. 2008. Suppression of plant parasites including nematodes by AM

fungi induced resistance in plants. Rev.Plant Pathol.,4:421-466.Blee, K.A. and Anderson, A.J. 1996. Defense-related transcript accumulation in Phaseolus vulgarisL. colonized by the

arbuscular mycorrhizal fungus Glomus intraradices, Schenk & Smith. Plant Physiol., 110:675-688.Blee, K.A., and Anderson, A.J. 2000. Defense responses in plants to arbuscular mycorrhizal fungi. p27-44 In: Current

advances in mycorrhizae research, Podila., G. K. and Douds., D.D. (ed.).The American Phytopathological Society,Minnesota, USA.

Blilou, I., Bueno, P., Ocampo, J.A. and Garcia-Garrido, J.M. 2000a. Induction of catalase and ascorbate peroxidase activities

in tobacco roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae. Mycol.Res.,104:722-725.Blilou, I., Ocampo, J.A. and Garcia-Garrido, M. 2000b. Induction of Ltp(Lipid transfer protein) and Pal(phenylalanine ammonia

lyase) gene expression in rice roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae. J. Exp. Bot.,51:1969-1977.

Boller, T., Gehri, A., Mauch, F., and Vgeli, U.1983. Chitinase in bean leaves: induction by ethylene, purification, properties,

and possible function. Planta, 157:22-31.Bonanomi, A., Oetiker, J.H., Guggenheim, R., Boller, T.,Weimken, A. and Vogeli-Lange, R. 2001. Arbuscular mycorrhizas in

mini mycorrhizotrons: first contact of Medicago truncatula roots with Glomus intrardicesinduces chalcone synthase.New Phytol., 150: 573-582.

Bonfante, P. 2001. At the interface between mycorrhizal fungi and plants: the structural organization of cell wall, plasma

membrane and cytoskeleton p45-61. In: The Mycota IX. .Hesser, K. and Hock, B. (ed.). Spinger-Verlag, Berlin,Heidelberg.

Chakraborty, B.N. 1988. In : Experimental and Conceptual Plant Pathology. Singh, R.S., Singh, U.S., Hess., W.M. andWeber., D.J. (ed.). Oxford & IBH Publishing, New Delhi. Antigenic Disparity p. 477-484.

Chakraborty, B. N. 1996. Biochemical defense strategies of plant against pathogens. p. 235-241 In: Contemporary Thoughtsin Plant Sciences. Pal., P. K. (ed.). Academic Staff College, Burdwan University, Burdwan.

Chakraborty. B.N. 2005.Antimicrobial Proteins in Plant Defence. P. 470-483. In: New Perspectives in the Frontiers of

Chemical Research. Chakravorti. S.S. (ed.). Royal Society of Chemistry (Eastern India Section), Kolkata.Chakraborty, B.N. 2007. Trade-off in plant defense against fungal pathogens and strategies for disease management p. 54. In:

Exploring the Vistas in Life Science. De., B. and Bera., s. (ed.). UGC-ASC and Department of Botany, University ofCalcutta.

Chakraborty, B.N. and Chakraborty, U. 2003.Immunodetection of Plant Pathogenic Fungi. p. 23-42. In: Frontiers of Fungal

Diversity in India. Rao, G.P. Manoharachari, C., Bhat, D.J., Rajak, R.C. and.Lakhanpal T.N., (ed.). InternationalBook Distributing Co. Lucknow.

Chakraborty, B.N. Chakraborty, U., Barman, B.C., Bhutia, L. and Ghosh, P.K. 2007. Induction of systemic resistance in teaplants against root rot pahogens upon field application of VAM, phoshphate solubilizing fungus and bacterium, p. 61-

71. In: Rhizosphere Biotechnology/Microbes in releation to plant health: Retrospects & Prospects. Roy, A.K.,

Chakraborty, B.N. Mukadam, D.S. and Rashmi (ed.). Scientific Publishers, Jodhpur.

Chakraborty, B.N. and Sharma, M. and Das Biswas, R. 2005a. Defense Responses in tea plants triggered by Exobasidiumvexans. p 226-232. In: Stress Biology, Chakraborty, U. and chakraborty, B.N. (ed.). Narosa Publishing House, NewDelhi.

Chakraborty, B.N. Sharma, M. Das Biswas, R. and Sharma, M. 2005b. Induction of resistance in tea plants against Curvulariapallescens by foliar application of leaf extracts. Journal of Hill Research,18(2):69-78.

Chakraboraty, B.N., Sharma, M. and Das Biswas, R. 2005c. Association of defense enzymes with resistance in tea plants

triggered by Exobasidium vexansMassee. Indian Phytopathology,58(3): 298-304.


25/26


Chakraborty, B.N. and Mur, L.A.J. 2009. Defining key metabolite changes in pathogenic interactions with tea. J. Mycol.Pl. Pathol,39:561-562.

Chakraborty, B.N. and Sharma, M. 2007 Serological detection and immunogold localization of cross-reactive antigens shared

by Camellia sinensisand Exobasidium. Journal of Applied Microbiology,103:1669-1680.Chakraborty, B.N. and Sharma, M. 2008. Pathogenesis related proteins in plant defence. Annual review of pathology,

4:105-138.Chakraborty, B.N. and Sunar, K. 2009. Arbuscular mycorrhizal fungal association in rhizosphere of Hevea brasiliensisNBU

Journal of Plant Sciences,3:67-70.

Chakraborty, U.and Chakraborty, B.N. 2008. Involvement of salicylic acid in plant defense against stresses. p. 233-246In : Abiotic Stresses in Plants. Khan., N.A. and Singh., S. (ed.). I.K.International Publishers, New Delhi.

Cordier, C., Pozo, M.J., Barea, J.M., Gianinazzi, S. and Gianinazzi-Pearson, V. 1998. Cell defense responses associatedwith localized and systemic resistance to Phytophthoraparasitica induced in tomato by an arbuscular mycorrhizal

fungus. Mol. Plant-Microbe Inter.,11:1017-1028.Daniel., M. and Purkayastha., R.P. 1995. Handbook of Phytoalexin Metabolism and Action. Marcel Dekker Inc., New York, 380 p.

Datta, S.K. and Muthukrishnan, S. 1999. Pathogenesis-related proteins in plants. CRC Press, LLC, 291p.

DeVay, J.E. and Adler, H.E.1976. Antigens common to hosts and parasites. Annu. Rev. Microbiol., 30:147-168.Douds, D.D., Galvez, L., Becard, G. and Kapulnik, Y. 1998. Regulation of arbuscular mycorrhizal development by plant host

and fungus species in alfalfa. New Phytol., 138: 27-35.Fraser,R.S.S.1981. Evidence for the occurrence of the pathogenesis-related proteins in leaves of healthy tobacco plants

during flowering. Physiological Plant Pathology,19:69-76.

Garcia-Garrido, J.M., Garcia-Romera, I. and Ocampo, J. A. 1992. Cellulase production by the vesicular-arbuscular mycorrhizalfungus G.mosseae. New Phytol., 121:221-226.

Garcia-Garrido, J.M., Tribak, M., Rejon-Palomares, A., Ocampo, J.A. and Garcia-Romera, I. 2000. Hydrolytic enzymes andability of arbuscular mycorrhizal fungi to colonize roots. J. Exp. Bot., 51:1443-1448.

Garcia-Olmedo, F., Molina, A., Segura, A. and Moreno, M.1995. The defensive role of non-specific lipid transfer proteins in

plants. Trends in Microbiology,3:72-74.Gawande, S.J., Chimote, V.P., Shukla, A. and Gadewar, A.V. 2006. Serological and molecular diagnosis of plant pathogens:

A plant protection approach. p.563-588 In: Plant Protection in New Millennium Gadewar., A.V. and Singh., B.P.(ed.)., Satish Serial Publishing House, New Delhi.

Gianinazzi, S., Martin, C. and Vall, J.C.1970. Hypersensibilit aux virus, temprature et protins solubles chez le NicotianaXanthi nc. Apparition de nouvelles macromolcules lors de la rpression de la synthse virale. C R Academy of

Science Paris,270D:2883-2886.

Gianinazzi, S., Pratt., H. M., Shewry, P. R. and Miflin, B. J.1977. Partial purification and preliminary characterization ofsoluble leaf proteins specific to virus infected tobacco plants. Journal of General Virology, 34:345-351.

Ginzberg, I., David, R., Shaul, O., Elad, Y., Wininger, S., Ben-Dor, B., Badani, H., Fang,Y., van Rhijn, P., Li Y., Hirsch, A.M. and Kapulnik, Y. 1998. Glomus intraradicescolonization regulates gene expression in tobacco plants. Symbiosis,

24:145-157.

Giovannetti, M. and Sbrana, C. 1998. Meeting a non-host: the behaviour of AM fungi. Mycorrhiza,8:123-130.Gollotte, A., Gianinazzi, P.V., Giovannetti, M., Sbrana, C. , Avio, L. and Gianinazzi, Z. 1993. Cellular localization and

cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a locus a mutant Pisum sativumL.Planta,191:112-122.

Ham, K.S., Kauffmann, S., Albersheim, P. and Darvill, A. G.1991. Host-pathogen interactions XXXIX. A soybean pathogenesis-

related protein with b-1,3-glucanse activity releases phytoalexin elicitor-active heat-stable fragments from fungal cell

walls. Molecular Plant-Microbe Interactions, 4:545-552.Harrison, M. and Dixon, R. 1994. Spatial patterns of expression of flavonoid/isoflavonoid pathway genes during interactions

between roots of Medicago truncatula and the mycorrhizal fungus Glomus versiforme.The Plant J., 6:9-20.

Koide, R.T. and Schreiner, R.P. 1992. Regulation of the vesicular arbuscular mycorrhizal symbiosis. Ann.Rev.Plant Physiol.PlantMol. Biol.,43:557-581.

Lambais, M.R. 2000. Regulation of plant defence-related genes in arbuscular mycorrhizae. p 45-59. In : Current advances in

mycorrhizae research, Podila., G.K. and Douds., D.D. (ed.). The American Phytopathological Society, Minnesota, USA.


26/26


Lambais, M.R. and Mehdy, M.C. 1998. Spatial distribution of chitinases and b-1,3 glucanase transcripts in bean arbuscularmycorrhizal roots under low and high soil phosphate conditions. New Phytol., 140:33-42.

Mauch, F. and Staehelin, L.A. 1989. Functional implications of the subecllular localization of ethylene-induced chitinase and b-

1,3-glucanase in bean leaves. The Plant Cell, 1:447-457.Mur, L.A.J., Lloyd, A. Heald, J. and Chakraborty, B.N. 2009. Metabolomics of Tea (Camellia sinensis) interactions with pest

and pathogens, J. Mycol. Pl. Pathol.,39:542-543.Ornstein, L. 1964. Disc electrophoresis I. Background and theory. Annals of the New York Academy of Sciences,

121:321-349.

Routledge, A.P., Shelley, G., Smith, J.V., Talbot, N.J., Draper, J. and Mur, L.A.J. 2004. Magnaporthe grisea interactions withthe model grass Brachypodiumdistachyonclosely resemble those with rice (Oryza sativa). Mol. Plant Pathol.,

5:253-265.Roy, A.K., Kumar, R., Chakraborty, B.N. and Chakraborty, U. 2002. VA Mycorrhiza in relation to growth of different tea

varieties. Mycorrhiza News,14:9-11.Salzer, P. and Boller, T. 2000. Elicitor- induced reactions in mycorrhizae and their suppression. p1-10. In : Current advances in

mycorrhizae research. Podila., G. K. and Douds., D. D., (ed.). The American Phytopathological Society, Minnesota, USA

Salzer, P., Bonanomi, A., Beyer, K., Vogeli-Lange, R., Aeschbacher, R.A., Lang, J., Wiemken, A., Kim, D., Cook, D.R. andBoller, T. 2000. Differential expression of eight chitinase genes in Medicago truncatula roots during mycorrhizal

formation, nodulation and pathogen infection. Mol. Plant-Microbe Int.,13:763-777.Salzer, P.,Corbiere, H. and Boller, T. 1999. Hydrogen peroxide accumulation in Medicago truncatula roots colonized by the

arbuscular mycorrhizal-forming fungus Glomus mosseae. Planta,208:319-325.

Sharma, M. and Chakraborty, B.N. 2004. Biochemical and immunological characterization of defense related proteins of teaplants triggered by Exobasidiumvexans. Journal of Mycology and Plant Pathology, 34:742-760.

Sharma, M. and Chakraborty, B.N. 2005. Hexaconazole and calixin mediated defense strategies of tea plants againstExobasidium vexansMassee. Journal of Mycology and Plant Pathology,35:417-431.

Smith, S.E. and Read D.J. 2008. Mycorrhizal symbiosis, 3rd edn. Academic Press Ltd, London, U.K.

Somssich, I.E. and Hahlbrock, K. 1998. Pathogen defence in plants: a paradigm of biological complexity. Trends Plant Sci.,3:86-90.

Spanu, P. and Bonfante-Fasolo, P.1998. Cell wall-bound peroxidase activity in roots of mycorrhizal Allium porrum: regulationand localization. Planta,177:447-455.

Staehelin, C. Granado, J., Mller, J. Wiemken, A., Mellor, R.B., Felix, G., Regenass, M., Broughton, W.J. and Boller, T.1994. Perception of Rhizobiumnodulation factors by tomato cells and inacitivation by root chitinases. Proceedings of

National Academy of Sciences U.S.A., 91:2196-2200.

Van Loon, L.C. 1975. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacumvar. Samsunand Samsun NN IV. Similarity of qualitative changes of specific proteins after infection with different viruses and

their relationship to acquired resistance, Virology, 67:566-575.Van Loon, L. C. 1985. Pathogenesis-related proteins. Plant Molecular Biology,4:111-116.

Van Loon, L.C. and Van Kammen, A.1970. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana

tabacumvar Samsum and Samsun NN II. Changes in protein constitution after infection with tobacco mosaic virus.Virology,40:199-211.

Van Loon, L.C. and Van Strien, E. A. 1999. The families of pathogenesis-related proteins, their activities, and comparativeanalysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 55:85-97.

Van Rhijn, P., Fang,Y., Galili, S., Shaul, O., Atzmon, N., Wininger, S., Eshed,Y., Lum, M., Li,Y., To,V., Kapulnik,Y. and

Hirch, A.M. 1997. Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways

used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved. PNAS, USA,94:5467-5472.

Vierhlig, H., Alt, M., Mohr, U., Boller, T. and Wiemken, A. 1994. Ethylene biosynthesis and activities of chitinase and b-1,3

glucanase in the roots of host and non-host plants of vesicular-arbuscular mycorrhizal fungi after inoculation withGlomus mosseae. J. Plant Physiol., 143:337-343.

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