Plant cell walls are vital features of all plants with numerous essential functions, such as to 1) provide definite shapes in different cell types to form tissues and organs, 2) allow intercellular communication to take place, 3) offer structural support and turgidity, as well as 4) defence against plant pathogens (Keegstra, 2010). At the simplest level, plant cell walls offer a physical barrier to aid in limiting pathogen attachment, invasion and infection. The plant cell wall is a multi-layered system that actively modifies and reinforces components precisely at local and distinct sites when it is in contact with potential pathogenic microbes. It consists of principal components such as high molecular weight polysaccharides that are cross-linked into an intensive network by ionic and covalent bonds (Carpita and McCann, 2002). Besides this, plant cell walls are dynamic reservoirs of a wide range of chemical defence compounds, such as antimicrobial proteins and plant secondary metabolites that can be activated rapidly to inhibit the growth of pathogens.
2.3.2.1 Nature of the plant cell wall
Plant cell walls are generally divided into two classes: the primary cell walls that are capable of growth to provide structural support and secondary cell walls that thicken beneath the primary walls after the cell stops growing. The most abundant and well characterised cell wall components are celluloses. They are complex polysaccharides and crystalline microfibrils that comprise ~ 30 to 36 chains of β-1, 4-linked glucose, which interact with one another via hydrogen bonds (Somerville, 2006) to form insoluble and inelastic crystalline compounds. Celluloses function as the fundamental building blocks of cell walls by giving strength and flexibility.
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Apart from celluloses, plant cell walls also consist of branched polysaccharides that can be categorised into two groups: hemicellulosic polysaccharides (xylans, glucomannans, xyloglucans and mixed-linkage glucans) (Scheller and Ulvskov, 2010) and pectic polysaccharides (homogalacturonan and rhamnogalacturonan I & II) (Harholt et al., 2010). Hemicellulose fibers are known as the cross-linking glycans that function to provide strength by cross-linking with cellulose microfibrils through hydrogen bonds. In contrast, pectic polysaccharides form hydrated gel-like compounds that aid in adhering neighboring cells to increase the firmness and regulating the water content of cell walls (Freeman and Beattieiowa, 2008). Plant cell walls also consist of various components, including suberin, cutin and waxes that offer protection towards plant pathogens. These components are fatty acid elements that are usually found to be deposited on the outer protective tissues of plants, such as bark and on the primary or secondary cell walls. Moreover, plant cell walls also contain lignin, a complex polymer comprised of phenolic compounds that contribute to cell rigidity. Lignin fills up the empty spaces in between the cellulose, pectin and hemicellulose components in cell walls (Freeman and Beattieiowa, 2008). Generally, the contents of plant cell walls are complex and significantly different from individual cell types and plant species (Vorwerk et al., 2004).
2.3.2.2 Changes to the plant cell wall during pathogenesis
From the physical perspective, plant cell walls synthesise and deposit callose between the cell walls and membranes next to the invading pathogen rapidly as an immediate response towards pathogen attack. Deposition of callose is associated with papillae, which are polysaccharide polymers that limit the entrance of plant pathogens to the underlying protoplast at the point of infection (Freeman and Beattieiowa, 2008). Furthermore, papillae also act as a reservoir for the accumulation of antimicrobial secondary metabolites (Bednarek et al., 2009).
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Hence, higher rates of papilla formation are often associated with higher resistance in plants towards fungal penetration, while lower rates of papilla formation will lead to higher susceptibility to pathogens (Collins et al., 2003).
The presence of a waxy cuticle on plant cell walls also forms an initial barrier to pathogen invasion. Dickinson (1960) proposed that the cuticle layer repells water films on the surface of cell walls and subsequently prevents the deposition of pathogen inoculums. A well-developed cuticle, epidermal membrane and lignified cell wall is usually thick, rigid and highly impermeable to pathogens (except for white-rot fungi), so they act as a defensive barrier in plants (Serrano et al., 2014). Previous studies have reported that overexpression of a transcription factor; S1SHINE3 that is mainly expressed in the epidermal layer results in a higher content of cutin monomers in tomato (Buxdorf et al., 2014). This leads to an increase in tomato resistance to B. cinerea and Xanthomonas campestris attack. Nevertheless, there are also reports on failure of linking pathogen infectivity with the thickness of plant cell walls (Martin, 1964). This could be due to the nature of the plant cell walls that are highly hydrated and have gel-like structures which subsequently act as the mediators of hydrolytic enzymes produced by plant pathogens. According to Baron-Epel et al. (1988), proteins and polysaccharides below the size of 17 kDa and 50 kDa are able to diffuse through standard plant cell walls in a matter of minutes and less than an hour respectively. Hence, hydrolytic enzymes and other by-products produced by pathogens, which range between 30 to 40 kDa normally can diffuse from cell walls to the internal plasma membrane.
In addition to its possible role as a physical barrier, the plant cell wall also produces a wide range of proteins and glycoproteins, including enzymes and structural proteins (Rose and Lee, 2010). These compounds thicken and strengthen cell walls upon contact with potential pathogens and act as signaling molecules to transfer infection signals within plant cells. For instance, arabinogalactan is a structural protein complex located at both plant cell walls and
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plasma membranes; it is believed that this complex plays a role in pathogen recognition and signaling events in Arabidopsis (Ellis et al., 2010). Besides proteins and enzymes, plant cell walls also secrete various components that have antagonistic effects to the fungal pathogen, such as benzoxazolinone, phenolic compounds, alkaloids and coumarin derivatives (Martin, 1964). Detection of potential pathogens by plant cell walls can trigger the production of oxidative burst catalysing enzymes, which in turn increases the level of highly reactive oxygen molecules to cause damage to the outer cells of invading pathogens (Freeman and Beattieiowa, 2008). These molecules also assist in strengthening plant cell walls by catalysing cross-linkage processes between the polymers and conveying infection signals to the neighboring cells.
2.3.2.3 Cell wall appositions
Living plant cell walls respond to fungal penetration rapidly at the site of infection through the deposition of cell wall materials directly onto the inner layer of cellulose, through a process known as cell wall apposition (Israel et al., 1980). Papillae play a significant role in this process, together with the deposition of several cell wall-associated structures as illustrated in Figure 2.6 (Micali et al., 2011; Underwood, 2012).
Ultra-structural observations and immuno-cytochemical characterisations of haustorial encasements and collars have confirmed that they are the extensions of the papillae (Zeyen et al., 2002). They contain similar cell wall and membrane lipid materials and multi-vesicular bodies as compared to papillae (Meyer et al., 2009). Haustorial encasements play a role as a defensive structure, instead of being responsible for the accommodation of fungal haustorium. This is because they are absent around the haustoria of fungal pathogens during a compatible interactions, signifying their formation is activated by the incompatible interactions with host plants (Meyer et al., 2009).
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Figure 2.6 Cell wall-associated structures during cell wall apposition. (A) Deposition of cell wall-associated structures to halt a fungal penetration. (B) Formation of a haustorial neck-band or collar around the neck of fungal haustorium upon successful penetration. (C) Partially surrounded fungal haustorium by a haustorial encasement. (D) A fully encased haustorium. CW, cell wall; PM, plasma membrane; C, conidiospore; PGT, primary germ tube; AGT, appressorial germ tube; PP, penetration peg; H, haustorium; EHM, extra-haustorial membrane; NB, haustorial neck-band; P, papilla; E, haustorial encasement (Underwood, 2012).
Histological and chemical analyses have shown that the structures of papillae are rather complex and often associated with different classes of compounds including callose, phenolic compounds such as lignin and phenolic polyamines, peroxidases, ROS, inorganic compounds, cell wall polymers such as pectin and xyloglucans, and cell wall structural proteins such as arabinogalactan and hydroxyproline-rich glycoproteins (Zeyen et al., 2002; Collinge, 2009). Bhuiyan et al. (2009b) revealed the role of lignin associations with papillae in Einkorn wheat infected with Blumeria graminis using a forward genetic approach. It was suggested that lignification of papillae increased plant defences against the penetration of B. graminis as the presence of lignin helped to strengthen the papillae structure.
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