Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
Pathogenesis Related (PR) Proteins in Plant Defense Mechanism
Saboki Ebrahim1, K.Usha1 and Bhupinder Singh2
1
Division of Fruits and Horticultural Technology, 2 Division of Nuclear Research Laboratory, Indian Agricultural
Research Institute, New Delhi, India
When resistant plants recognize cognate or matching elicitors, intracellular signal transduction pathways are activated that
ultimately result in the derepression of a battery of genes called defense response genes. These latter genes encode
producing various pathogenesis related (PR) toxic proteins such as chitinases, glucanases, lysozyme-active proteins, or cell
wall strengthening proteins such as hydroxyproline rich glycoproteins. Response proteins may also be enzymes in
biosynthetic pathways for lignification of cell walls or the production of phytoalexins, low molecular weight toxic
chemicals that antagonize the invader. In the following portion, biochemical response of plant defence mechanism related
to PR-protein including chitinase and glucanase, in addition to plant lignin content will be explained.
Keywords : Pathogenesis related (PR) protein, Chitinase, β-1,3-glucanase, Lignin.
Pathogenesis-Related (PR) Proteins
Higher plants have a broad range of mechanisms to protect themselves against various threats including physical,
chemical and biological stresses, such as wounding, exposures to salinity, drought, cold, heavy metals, air pollutants
and ultraviolet rays and pathogen attacks, like fungi, bacteria and viruses [1]. Plant reactions to these factors are very
complex, and involve the activation of set of genes, encoding different proteins. These stresses can induce biochemical
and physiological changes in plants, such as physical strengthening of the cell wall through lignification, suberization,
and callose deposition; by producing phenolic compounds, phytoalexins and pathogenesis-related (PR) proteins which
subsequently prevent various pathogen invasion [2]. Among these, production and accumulation of pathogenesis related
proteins in plants in response to invading pathogen and/or stress situation is very important. Phytoalexins are mainly
produced by healthy cells adjacent to localized damaged and necrotic cells, but PR proteins accumulate locally in the
infected and surrounding tissues, and also in remote uninfected tissues. Production of PR proteins in the uninfected
parts of plants can prevent the affected plants from further infection [3, 4]. PR protein in the plants was first discovered
and reported in tobacco plants infected by tobacco mosaic virus [5]. Later, these proteins were found in many plants.
Most PR proteins in the plant species are acid-soluble, low molecular weight, and protease-resistant proteins [6, 7]. PR
proteins depending on their isoelectric points may be acidic or basic proteins but they have similar functions. Most
acidic PR proteins are located in the intercellular spaces, whereas, basic PR proteins are predominantly located in the
vacuole [8, 9, 10]. The PR proteins have been classically divided initially into 5 families based on molecular mass,
isoelectric point, and localization and biological activity [11]. Currently PR-proteins were categorized into 17 families
according to their properties and functions (Table 1), including β-1,3-glucanases, chitinases, thaumatin-like proteins,
peroxidases, ribosome-inactivating proteins, defenses, thionins, nonspecific lipid transfer proteins, oxalate oxidase, and
oxalate-oxidase-like proteins [10]. Among these PR proteins chitinases and β-1,3-glucanases are two important
hydrolytic enzymes that are abundant in many plant species after infection by different type of pathogens. The amount
of them significantly increase and play main role of defense reaction against fungal pathogen by degrading cell wall,
because chitin and β-1,3-glucan is also a major structural component of the cell walls of many pathogenic fungi. β-1,3glucanases appear to be coordinately expressed along with chitinases after fungal infection. This co-induction of the two
hydrolytic enzymes has been described in many plant species, including pea, bean, tomato, tobacco, maize, soybean,
potato, and wheat [12, 13, 14, 15, 16, 17, 18, 19, 20].
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Table 1: Classification of pathogenesis related proteins [10].
Families
Type member
Properties
PR-1
Tobacco PR-1a
Antifungal
PR-2
Tobacco PR-2
β-1,3-glucanase
PR-3
Tobacco P, Q
Chitinase type I,II, IV,V,VI,VII
PR-4
Tobacco ‘R’
Chitinase type I,II
PR-5
Tobacco S
Thaumatin- like
PR-6
Tomato Inhibitor I
Proteinase- inhibitor
PR-7
Tomato P69
Endoproteinase
PR-8
Cucumber chitinase
Chitinase type III
PR-9
Tobacco ‘lignin forming peroxidase’
Peroxidase
PR-10
Parsley ‘PR1’
Ribonuclease like
PR-11
Tobacco ‘class V’ chitinase
Chitinase, type I
PR-12
Radish Rs- AFP3
Defensin
PR-13
Arabidopsis THI2.1
Thionin
PR-14
Barley LTP4
Lipid- transfer protein
PR-15
Barley OxOa (germin)
Oxalate oxidase
PR-16
Barley OxOLP
Oxalate oxidase-like
PR-17
Tobacco PRp27
Unknown
Synergistic Effect of Chitinase and β-1, 3-Glucanase in Transgenic Plants
Chitinase genes, alone or together with β-1,3-glucanase genes, have been transferred to a number of plant species and
expressed and those effects were studied. In most cases, the resulting transgenic plants exhibit enhanced levels of fungal
disease resistance or delayed symptom development as compared to the control plants [15, 21, 22]. But several studies
have showed that plants transformed with chitinase or β-1, 3-glucanase gene alone did not exhibit resistance to certain
pathogens or showed less resistance compared to plants which were transformed with both the β-1, 3-glucanase and
chitinase genes. Like β-1, 3-glucanase, chitinases inhibit only a limited number of fungal species therefore these two
enzyme have synergistic effect. Plant chitinases alone usually affect only the hyphal tip and are unable to effectively
degrade harder chitin structures of fungi. But whenever these two enzymes are combined, a synergic effect can usually
be observed. For example, tomato plants expressing tobacco class I β-1,3-glucanase and chitinase transgenes showed
increased tolerance to infection by Fusarium oxysporum f.sp. lycopersici [22]. Also in tobacco plants transformed with
a barley class II basic β-1, 3-glucanase along with a barley class II basic chitinase gene showed enhanced levels of
protection against Rhizoctonia solani as compared to plants transformed with a single gene [15].
Age-Related Pathogen Resistance
Many studies reported a direct relationship between plant age and pathogen resistance or susceptibility
[23,24,25,26,27,28,30,31,32,33,34,35]. In most plants, resistance to pathogen usually increases with increasing age; but
in few cases susceptibility of plants to pathogens increases with development of plant. This difference in resistance to
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pathogens depending on plant age is called age-related pathogen resistance. For example, the older leaves from bottoms
of the shoots of grapevines showed more β-1, 3-glucanase and consequently they exhibited a higher resistance to
Plasmopara viticola than younger leaves from upper shoots [31]. In addition, both young and mature leaves in older
tobacco and wheat plants showed higher resistance than the leaves in younger plants [25, 26]. Another study in tobacco
showed that tobacco plants naturally become resistant to blue mold caused by Peronospora tabacina as its age
increases. β-1,3-glucanase, chitinase and peroxidase activities increased in tobacco with age [35]. Enzyme activities
were observed higher in leaf tissue from the main stalk (resistant to blue mold) as compared to leaf tissue from
suckering stems (susceptible to blue mold) on the same plant [35].
Chitinase in Plant Defense
Chitinases (E.C. 3.2.1.14) are enzymes catalyzing the cleavage of a bond between C1 and C4 of two consecutive Nacetyl-D-glucosamine monomers of chitin. They are widely distributed in nature, occurring in bacteria, fungi, animals,
and plants. Chitin is a common component of fungal cell walls and of the exoskeleton of arthropods [36]. Plant
chitinases are usually endo-chitinases capable of degrading chitin, a major constituent of certain fungal cell walls as
well as inhibit fungal growth [37, 38]. Some class I chitinases are localized in the vacuole, whereas other chitinases,
including class III chitinases, are located outside the cell [39]. Extracellular chitinases may directly block the growth of
the hyphae invading intercellular spaces and possibly release fungal elicitors, which then induce additional chitinase
biosynthesis and further defense reactions in the host [40, 41,]. There are strong indications that chitinase, together with
β-1, 3 glucanase participate in the plant defense system against fungal pathogens. Chitinase are found in leaves of many
plants, whereas has no known function in growth and development of plant. Higher plants do not contain chitin and any
other known endogenous substrate for chitinase. However chitin and β-1, 3-glucanase are major components in the cell
wall of many fungi and there is possibility of plant chitinase and β-1, 3- glucanase target fungi cell wall components as
substrate and has anti fungal function [42.43].
Classification of Plant Chitinase
Chitinase are classified into two categories, endochitinases and exochitinases. Endochitinases cleave chitin randomly at
internal points within the polymer, producing soluble, low molecular weight multimers of N-acetylglucosamine, such as
chitotrose, chitotetraoseand and the dimer, di-acetylchitobios. Exochitinases are divided into two subcategories:
chitobioses, catalyzing release of di-acetylchitobioses from nondeoxydizing end of the chitin microfibril, and β-1, 4-Nacetylglucoseaminidase, cleaving oligomer products of endochitinases and chitobiosidases with the production of
monomer N-acetylglucosamine [29].
Based on their primary structures, plant chitinases have been classified into seven classes, class I through VII.
Different chitinase classes have no apparent correlation to being present in a particular plant species and plant organ or
tissue. However, certain chitinase isoforms are sometimes induced by a particular elicitor. For example, in potato, both
acidic and basic chitinase, can be active. Also only particular isoforms have antifungal activities and some isoforms
have shown another role like antifreeze activity [44, 45].
Class I chitinases have a cysteine-rich N-terminal chitin-binding domain (CBD) that is homologous to havein, a
chitin-binding lectin from the rubber tree. CBD is separated from the catalytic domain by a proline and glycine-rich
hinge or spacer region, variable both in size and composition. Class I chitinase, in tobacco due to deletion of CBD and
the spacer region singly or in combination reduces the hydrolytic activity by 50%, also antifungal function is reduced by
80% [46]. Class I is divided into subclasses Ia and Ib that include acidic and alkaline chitinases, respectively. Class I
chitinases occur only in plants, whereas chitinases of class II, also in fungi and bacteria. They are induced by the
pathogen attack and lack both N-terminal domains as the C-terminal signal peptide ensuring vacuolar localization. Class
I chitinase, in tobacco due to deletion of CBD and the spacer region singly or in combination reduces the hydrolytic
activity by 50%, also antifungal function is reduced by 80% [46]. Class II chitinases are similar to class I but they lack
the N-terminal CBD and the hinge region. Also, they have acidic properties.
Class III chitinases are unique in structure and have no relationship to any other class of plant chitinases. These
chitinases belong to the PR-8 family and family 18 of glycosyl-hydrolases. Class III chitinases generally have lysozyme
activity and appear to be more closely related to the bacterial chitinases. Class III chitinase enzyme have high amino
acid sequence homology to the bifunctional chitinase lysozyme from Hevea brasiliensis and include the chitinase from
Azukin bean and cucumber. This is unusual for a class III chitinase since they do not have a chitin-binding domain.
Class III chitinases show a wide range of isoelectric points, activity over a wide range of pH, and temperature stability
at 60-70oC. The B. hispida chitinase has a pH optimum of 2 and retains approximately 50% activity at pH 8 [47]. Some
class III chitinases, such as a yam enzyme, show two pH optima and heat stability at 80oC [48]. There is no posttranslational modification reported for class III enzymes analyzed to date.
Class IV, V, VI, and VII chitinases belong to the PR-3 family of pathogenesis related proteins. The structure of class
IV chitinases is similar to class I chitinases containing cysteine rich domain and a conserved main structure, except that
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they are shorter due to four deletions. Class IV chitinases represent a group of extracellular chitinases, Like class II
chitinases, they are found mainly in dicotyledon plants. Class V was not similar to any known plant chitinase but have
significant sequence similarity to bacterial exochitinases. Class VI chitinase are homologous, to sugar beet chitinase I
possessing only half of chitin binding domain and a long prolin rich sapacer.
I-VII classes of chitinase [49] have been reclassified into 4 families (Corresponding to 4 PR-protein families) namely
Chia, Chib, Chic and Chid and having total of nine classes [39].This classification is based on the presence or absence
of an N-terminal havein domain and on sequence similarity to an archetypal catalytic domain. Chia1 chitinase have an
N-terminal havein domain and a catalytic domain that is at least 50% identical to tobacco Chia1chitinase. The Chia2
chitinase lack an N-terminal havein domain, but contain catalytic domain that are at least 50% identical to the catalytic
domain of tobacco Chia1 chitinase. The Chia4 chitinase have N-terminal havein domain but shorter than Chia1
chitinase. They are at least 50% identical to Phaseolus vulgaris PR-4 chitinase. Chib1 chitinase sequence are different
than Chia1, Chia2 and Chia4 chitinases, but are at least 30% identical to tobacco Chib1 chitinases which also have
lysozyme activity. Chic1 chitinases have no similarity whit Chia1, 2, 4 or Chib1 but their amino acid sequences are at
least 50% identical to a group of tobacco chitinase that are similar to bacterial exochitinases. Chia5 chitinase has two
chitin binding domains and Chia6 chitinase has half chitin binding domain and are related to Chia1.Chia chitinase
belong to PR-3 protein family, Chib1 belong to PR-8 and Chic1 belong to PR-11 protein family. PR-4 has been found
to have chitinase activity, and makes the family Chid.
Chia1 enzymes in general have more specific activity than chia2. Compared to chia2, Chia1 has 3 fold more activity
in barley [50] and 6 to 15 fold more specific activity in tobacco [8]. Chia1 chitinase account for the majority of the
chitinolytic activity in plant. For example, two chia1 isoforms account for 68% of total activity, yet constitute only 25%
of the chitinolytic protein [8].
Purified chitinases show varying degrees of antifungal activities in vitro. In general, class I chitinases have the
highest antifungal activity, perhaps due to the presence of a chitin-binding domain [44]. All other chitinase classes have
lower to no antifungal activity as compared to class I chitinases.
Induction and Functions of Chitinases in the Plant
Chitinase have been known to be induced in the plant by fungal infection or other biotic and abiotic factors. Inhibitory
effect of plant chitinase about fungal growth was demonstrated by in vitro studies on the growth of fungi that contain
chitin as a component of their cell wall [12, 13, 38]. The induction of chitinases was initially shown in pea plants
infected with Fusarium solani [12]. Inhibitory effect of protein extracted from pea plant was infected by Fusarium
solani and showed to inhibit growth of 15 of the 18 fungal species tested in vitro.
Purified chitinase inhibited growth of only one fungal species whereas a combination of chitinase and another PRprotein, β-1, 3-glucanase, inhibited the growth of all fungi tested showing a synergism in activities [13]. The number of
studies verified these results in tobacco [51], grapes [52], chickpea [53], rice [54] and other plants. These studies
demonstrated that specific isoforms are induced in response to a particular pathogen and only certain isoforms are able
to inhibit specific fungi [44, 55]. For example, a class I chitinase from tobacco showed antifungal activity against
Fusarium solani [15].
Various studies have shown that chitinase expression against phyto-pathogen systems is higher and induction is
stronger in the resistant varieties in comparison to susceptible varieties in the sugar beet [56], wheat [57] and tomato
varieties [58]. Also another report showed no difference in the induction timing or amounts of PR-protein in resistant
and susceptible cultivars of cotton [59].
However, quick response in the resistant cultivars might affect the cell wall of germinating fungal spores, releasing
elicitors leading to the expression of PR-genes and disease resistance. It was shown for Alternaria solani that a basic
chitinase was only active on the germinating spores and not on the mature fungal cell wall for generation of elicitor
molecules to induce disease resistance [58].
Kragh [60] demonstrated chitinase activity in susceptible primary leaves of barley (Hordeum vulgare L.) fivefold and
threefold, respectively 7 days after inoculation with powdery mildew fungus (Erysiphe gramini f. sp. hordei). Metraux
and Boller [61] reported in cucumber, chitinase is induced up to 600-fold locally and up to 100-fold systemically in
response to infection by various pathogens. They showed systemic induction of chitinase in response to a localized
treatment and was correlated with the systemic induction of resistance against C. lagenarium. O'Garro and
Charlemange [62] studied response of the pepper leaves and flowers to infection with Xanthomonase campestris Pv.
Vesicaroria and showed chitinase and β-1, 3-glucanase were coordinately induced in infected leaf and flower tissue. A
grape class III chitinase was also shown to be induced in infected and non-infected leaves upon fungal infection [63].
The induction showed two maxima at 2 d and 6 d in the susceptible Vitis vinifera whereas the level was steeply induced
up to 4 d and declined to the basal level by day 7 in the resistant V. rupestris.
Induction of chitinase was elevated in transgenic plants. In bean (Phaseolus vulgaris) chitinase gene under the
control of CaMV 35S promoter was introduced into tobacco plants through Agrobacterium mediated transformation.
Level of chitinase activity was 20-40 folds as compared to control in the transgenic plants. Transgenic plants showed
increased resistance to infection by pathogenic fungi Rhizoctonia solani and delayed development of disease symptoms.
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The transgenic plants showed no resistance to the non-chitin containing fungus Pythium aphanidermatum [64]. In
another study, tomato chitinase gene was transferred to oilseed rape (Brassica napus) and was challenged with three
different fungal pathogens at two field locations [65]. Over a period of 52 days, the protection level against three fungi
was 23% to 79% with both delayed appearance of symptoms and reduced lesion numbers in transgenic plants. The
highest protection was against Cylindrosporium concentricum which contains chitin as a major component in its cell
wall and at the tip of growing hyphae. The protection was lower against two other fungi whose cell walls have different
distributions and amounts of chitin.
Transformation of chitinase genes were performed in grapevine, rice and peanut and enhanced disease resistance has
been achieved. In grapevine, rice chitinase gene enhanced resistance of these plants against powdery mildew caused by
Uncinula necator [66]. . Chitinase gene transformed into rice showed enhanced resistance to sheath blight caused by
Rhizoctonia solani [67]; a tobacco chitinase gene transformed into peanut increased resistance to leaf spot disease
caused by Cercospora arachidicola [68].
Non-plant chitinases have also been transferred to model plants which enhanced resistance in susceptible varieties. A
chitinase gene from the mycoparasitic fungus Trichoderma harzianum was transformed into tobacco and potato plants.
The transgenic plants increased resistance to various fungal pathogens including Alternaria alternata, A. solani, Botrytis
cinerea and Rhizoctonia solani [69]. Another fungal chitinase gene from Rhizopus oligosporus showed suppression of
disease symptoms in transgenic tobacco plants when challenged with pathogenic fungi [70].
Plant β-1, 3-Glucanases in Plant Defense
Plant β-1, 3-glucanases are pathogenesis-related (PR) proteins, which belong to the PR-2 family of pathogenesis-related
proteins and are believed to play an important role in plant defense responses to pathogen infection. In addition to plant,
β-1, 3-glucanases have been found in yeasts, actinomycetes, bacteria, fungi, insects, and fish [71, 72].β-1,3-glucanases
are able to catalyze the cleavage of the β-1,3-glucosidic bonds in β-1,3-glucan [73].β-1,3-glucan is a another major
structural component of the cell walls of many pathogenic fungi [74, 75]. For example, Phytophthora infestans is an
oomycete pathogen that causes late blight of potato and tomato. Oomycetes have a cell wall that is comprised of 8090% β-1,3-glucan. Syntheses of these enzymes can be induced by pathogens or other stimuli. β-1,3-glucan (called
callose in plants) is unlike chitinases, the substrate for β-1,3-glucanases is widespread in plants and therefore these
enzymes may have other physiological functions as well as in plant defense. For example, callose acts as a permeability
barrier in pollen mother cell walls [76] and muskmelon endosperm envelopes [77]. β-1,3-glucanases in the plant also
have function, such as cell division and cell elongation [78, 79, 80], fruit ripening [81], pollen germination and tube
growth [ 82, 83], fertilization [84], somatic embryogenesis [85], seed germination [86, 87] and flower formation [88,
89].Plant β-1,3-glucanases are pathogenesis-related proteins and have been suggested as an important component of
plant defense mechanisms against pathogens [90, 41, 91, 92]. It has been suspected that β-1,3-glucanases have direct
effect in defending against fungi by hydrolyzing fungal cell walls, which consequently causes the lysis of fungal cells.
In addition, β-1,3-glucanases was showed to have an indirect effect on plant defense by causing the formation of
oligosaccharide elicitors, which elicit the production of other PR proteins or low molecular weight antifungal
compounds, such as phytoalexins [93, 94, 95].
Classification of Plant β-1, 3-Glucanases
β-1, 3-glucanase genes have been reported in a number of plants, including tobacco [96], soybean [97], rubber tree [98],
banana [99], and rice [100]. There are different β-1, 3-glucanase genes in different plant species and a single plant
species may have various copies of β-1, 3-glucanase genes. For instance, more than 14 β-1,3-glucanase genes have been
reported in tobacco plants, [6], A variety of β-1,3-glucanase genes have been identified in a wide range of plant species.
Plant β-1,3-glucanases having size from 30-40 kDa, with both acidic and basic isoforms. Based on their amino acid
sequence, structural properties and cellular localizations, β-1, 3-glucanases were classified into two major classes I and
II and two minor classes [84, 6, 101,102,103, 104, 105]. Most of these hydrolytic enzymes identified so far are class I
and class II enzymes. Most class I β-1,3-glucanases are usually basic proteins which are localized in the cell vacuole,
while most class II, III, and IV β-1,3-glucanases are acidic proteins which are secreted into the extracellular space.
Class I β-1, 3-glucanases contain a N-terminal signal peptide and a short C-terminal signal sequence that is
glycosylated. The C-terminal signal sequence commonly referred to as the C-terminal extension, is believed to be the
signal sequence responsible for vacuolar localization of these enzymes [84]. Classes II, III and IV β-1, 3- glucanases are
usually acidic and lack this C-terminal signal and therefore are secreted to extracellular spaces. Some β-1, 3-glucanases
are solely developmentally regulated and do not show a stress-related induction. Two examples are tobacco styler β-1,3glucanase [106] and a tobacco anther β-1,3-glucanase [107]. Several β-1, 3-glucanases in class I like enzymes from
tobacco and tomato have inhibitory activity on the growth of certain pathogenic fungi in vitro. Combinations of class I
β-1, 3-glucanases and class I chitinases showed synergistic effect. In contrast, class II β-1,3-glucanases from tobacco
and tomato do not have the in vitro fungal growth inhibitory effect [44, 108,109]. Class I β-1, 3-glucanase accumulated
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only at the site of tobacco mosaic virus (TMV) infection in tobacco plants. In contrast class II and III β-1, 3-glucanases
accumulated both at the site of infection and systemically [110, 111]. Similar to chitinases, β-1, 3-glucanases can
degrade the fungal cell wall by disrupting hyphal tips, especially in combination with a chitinase [12, 13].Glucanases
can also play roles in plant defense by generating pathogen-derived elicitors. Like class II enzymes β-1, 3-glucanases,
digest fungal cell walls, leading to the release of oligosaccharide elicitors which stimulate the production of PR proteins
and other defense-related molecules [112].
Many studies have shown that the synthesis of β-1, 3-glucanases is stimulated by pathogen infections. For example,
The induction of the β-1,3-glucanase gene transcript on tobacco usually occurs within 24 h to 48 h and β-1,3-glucanase
amount increasing up to 21-fold by the bacterium Pseudomonas syringae pv syringae [113, 114]. In barley, two β-1,3glucanase isozymes, GI and GIII, were observed in healthy barley leaves, while three isozymes, GI, GII, and GIII, were
observed in infected leaves at different expression levels [115], the β-1,3-glucanase activity also can change during
plant development. This change corresponds to the age-related pathogen resistance in plants [31].
Induction of β-1,3-Glucanases in the Plant
β-1,3-glucanases usually expressed at low concentration in plants, but when plants are infected by fungal, bacterial, or
viral pathogens, β-1,3-glucanases enzyme concentration increases dramatically, . For example, Van Kan et al [103]
showed that mRNA for a tomato acidic β-1, 3-glucanase accumulated to a higher level in leaves infected by the fungal
pathogen Cladosporium fulvum. In tobacco by infection with the bacterium Pseudomonas syringae pv syringae level of
β-1,3-glucanase was transcriptionally induced up to 21-fold [113, 114]. These increasing levels showed locally or/ and
systemically [116, 117, 118, 119]. Induction of β-1, 3-glucanases by pathogens can vary in different clones of same
plant species. For example, when the production of a β-1, 3-glucanase upon infection with Corynespora cassiicola was
compared in different clones of Hevea brasiliensis, variability of the enzyme’s activity was observed significantly
among different clones during pathogenesis. Enzyme activity increased in the tolerant clone, while in the susceptible
clone enzyme decreased [120].
Several studies showed that the expression levels of these enzymes increased after infected with pathogens, such as
barley infected by powdery mildew [121], maize infected with Aspergillus flavus [122]. pepper infected with
Xanthomonas campestris pv. vesicatoria and Phytophthora capsici soybean infected with Pseudomonas syringae [123],
wheat infected with Fusarium graminearum [20], chickpea infected with Ascochyta rabiei (Pass.) Labr. [124] and
peach infected with Monilinia fructicola [125].
Induction of β-1, 3-glucanases and other PR proteins in the plant can also occur due to some components of
pathogens or degraded components of pathogens. These elicitors may be components of the cell surface of the pathogen
that are released by host enzymes, including fungal β-glucan, chitin, chitosan, glycoproteins and Nacetylchitooligosaccharides [126, 127, 128]. They may also be synthesized and released by the pathogen after it enters
the host in response to host signals.
Plant β-1, 3-glucanases are induced not only by pathogen infection, but also by other factors. For example, salicylic
acid induced accumulation of mRNAs of classes II and III β-1, 3-glucanases in wild-type tobacco plants [9,102].Similar
effect was reported for abscisic acid (ABA) in tobacco [129, 130, 131] and methyl jasmonate, ethylene and gibberellin
A3 in tomato seeds and leaves [132]. Stress factors like wounding, drought, exposure to heavy metals, air pollutant
ozone, and ultraviolet radiation can stimulate synthesis of β-1,3-glucanases in some plants [89, 125, 133, 134, 135].
These various factors often appear to interact, resulting in a dynamic response to biotic, as well as abiotic stimuli.
Lignin
Lignin is principally derived from phenylpropanoid hydroxycinnamyl alcohols and it has an important role in host
defense against pathogen invasion. Lignification is a mechanism for resistance in plants. Lignin deposition during
pathogen attack is well documented as a plant defense response [136, 137]. After pathogen invades the plant, during
plant defense response, lignin or lignin-like phenolic compound accumulation was shown to occur in a variety of plantmicrobe interactions during the plant defense responses [138]. Plants assemble cell wall apposition at the sites of
attempted penetration of biotrophic fungi such as powdery mildew [139]. Lignin has critical role in cell wall apposition
(CWA)-mediated defense against pathogen fungus penetration into plant. Lignin, a major component of cell walls of
vascular plants, was shown to accumulate in cell wall apposition and surrounding halo areas [139, 140]. Hence it is
considered as first defense barrier against successful penetration of invasive pathogens. Lignification renders the cell
wall more resistant to mechanical pressure applied during penetration by fungal appressoria and water resistant and thus
less accessible to cell wall-degrading enzymes. Lignification is necessary for the structural integrity of plant cell walls
and that is crucial for plant development [141], but the monomeric composition of lignin can vary depending on the
developmental process: thus, defense lignin accumulated by an elicitor treatment was shown to be significantly different
from lignin in vascular tissues. Lange et al. [142], suggestied that lignin biosynthesis is differentially regulated.
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Lignification may have several roles in defense as pointed out by Ride [143]. He suggested five ways that
lignification might hinder fungal growth through plant tissue. 1) Lignin may make walls more resistant to mechanical
penetration. It is generally held that lignin increases resistance of walls to compressive forces such as would be
expected at the growing point of a penetration peg. 2) Lignification of the wall at the point of attack may render it
resistant to dissolution by fungal enzymes. Induced lignification of wheat leaves makes them resistant to enzyme
degradation [144]. 3) Lignification of walls may restrict diffusion of enzymes and toxins from the fungus to host, and
water and nutrients from the host to fungus, in essence starving the fungus [145]. 4) Low molecular weight phenolic
precursors of lignin and free radicals produced during polymerization, may inactivate fungal membranes, enzymes,
toxins, and elicitors. Many phenolic compounds have antifungal activity. 5) The hyphal tip may become lignified and
lose plasticity necessary for growth [130]. Each of these roles can be tested and therefore used to evaluate lignification
as a resistance mechanism. Thus, if lignification is to play a primary role in restricting pathogen development, it must
occur early in the host-pathogen interaction and it must be localized near the invading pathogen [146].
Recently, Bhuiyan et al.,[147] by using an RNAi gene-silencing assay, they showed that monolignol biosynthesis
plays a critical role in cell wall apposition mediated defense against powdery mildew fungus penetration into diploid
wheat. Silencing monolignol genes led to super-susceptibility of wheat leaf tissues to an appropriate pathogen, Blumeria
graminis f. sp. tritici (Bgt), and compromised penetration resistance to a non-appropriate pathogen, B. graminis f. sp.
hordei. Autofluorescence of cell wall apposition regions was reduced significantly at the fungal penetration sites in
silenced cells. Their work indicates an important role for monolignol biosynthetic genes in effective cell wall apposition
formation against pathogen penetration. In this addendum, they show that silencing of monolignol genes also
compromised penetration resistant to Bgt in a resistant wheat line.
Conclusion
Higher plants protect themselves against fungal infection or other biotic and abiotic factors in different ways. Plants
defend themselves against such factors by physical strengthening of the cell wall through lignification, suberization, and
producing various pathogenesis-related (PR) proteins such as chitinases, β-1, 3-glucanases.
Pathogenesis-related proteins, including hydrolytic enzymes chitinases and β- 1, 3-glucanases, in plants in response
to invading pathogen are very important. Plant pathogenesis-related proteins are implicated in plant defense responses
against pathogen infection. Production of PR proteins in the remote uninfected parts of plants can lead to the occurrence
of systemic acquired resistance, protecting the affected plants from further infection.
It has been suspected that PR proteins have direct effect in defense mechanism against fungi by hydrolyzing fungal
cell walls, which consequently causes the lysis of fungal cells. In addition, PR proteins were showed to have an indirect
effect on plant defense by causing the formation of oligosaccharide elicitors, which elicit the production of other PR
proteins or low molecular weight antifungal compounds, such as phytoalexins.
Lignin is has an important role in plant defense against pathogen invasion. Lignification is a mechanism for
resistance in plants. After pathogen invades the plant, lignin or lignin-like phenolic compound accumulation was shown
to occur in a variety of plant-microbe interactions during the plant defense responses.
Endogenous enzymes chitinase, β-1, 3-glucanases and lignin content in plant leaves can be used as biochemical
markers for identifying plant varieties resistant to fungal infection or other biotic and abiotic factors. Also by
transferring pathogenesis-related (PR) proteins such as chitinases, β-1, 3-glucanases genes can induce resistance in
plants to various photogenes.
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