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Introduction

During pathogen attack, plants employ a complex of physical and chemical defence strategies to combat attempted invasions. Pathogen penetration attempts are often met with targeted cell wall fortification, and the formation of these localized cell wall appositions (CWAs) or papillae has long been recognized as a common plant response to fungal penetration attempts (de Bary 1863). Because the occurrence of CWAs often correlates to increased penetration failure, CWA formation is considered to be an important resistance mechanism. CWAs form in both susceptible and resistant hosts, as well as in nonhosts, and therefore constitute a basal or ancient form of resistance, halting the early stages of fungal ingress into host cells. Since commercial release 25 years ago, recessive mutations in the Mlo gene of barley have provided durable broad spectrum resistance to penetration by most powdery mildew races, based largely on strong oversized CWAs (reviewed by Lyngkjaer et al. 2000). Although CWA formation has been recognized for over a century, the phenomenon continues to challenge researchers and many questions remain unanswered. The aim of the present review is to provide a brief synopsis of the state of CWA research, and to highlight future directions in understanding CWA genesis and function.

Cytoplasmic and cytoskeletal reorganization

One of the earliest recognizable changes in an epidermal cell responding to attempted pathogen penetration is cytoskeletal rearrangement. Penetration attempts cause microtubules and microfilaments in host cells to become radially arranged around the site of attack (Gross et al. 1993; Kobayashi et al. 1992). Treatment of barley, onion, Arabidopsis or tobacco with cytochalasin, an actin polymerisation inhibitor, partially compromises nonhost resistance at the penetration stage for fungal pathogens (Kobayashi and Hakuno 2003; Kobayashi et al. 1997; McLusky et al. 1999; Yun et al. 2003). Concurrent to cytoskeletal rearrangement, cytoplasmic streaming towards the pathogen contact site accelerates, and small volumes of cytoplasm termed cytoplasmic aggregates, which hold rough endoplasmic reticulum and vesicles presumably containing precursors necessary for CWA formation, begin to accumulate beneath the pathogen-plant contact site (Freytag et al. 1994; Gross et al. 1993; Kobayashi et al. 1992). As the cell becomes poised for pathogen attack, the cell nucleus migrates towards the pathogen contact site and the cell begins to produce a CWA (Skalamera and Heath 1998).

Vesicle traffic

Recently, the ArabidopsisPEN1 (PENETRATION1) and barley ROR2 (REQUIRED FOR MLO RESISTANCE2) genes we re isolated and characterised as homologous synaptome-associated protein receptors (SNAREs) (Collins et al. 2003). Arabidopsis or barley plants with mutations in their respective SNARE genes show increased penetration by barley powdery mildew. In addition to ROR2, a SNAP-25 homologue was also shown to be required for full resistance in barley, and ROR2 and SNAP-25 interacted in yeast two hybrid experiments. While the functions of PEN1, ROR2, and SNAP-25 are still not completely known, Collins et al. (2003) suggested that the proteins may be involved in the facilitation of vesicle exocytosis or the homotypic fusion of vesicles as they carry cargo towards the CWA.

Cell wall modification and reactive oxygen species

Although the structural rearrangements preceding CWA formation are now becoming clearer, the identity of the payload carried by CWA-destined vesicles remains somewhat mysterious. The structure and composition of CWAs show remarkably heterogeneity, but they commonly contain callose, pectic substances, phenolic derivatives, suberin, proteins, and some metal ions (reviewed by Smart 1991). The synthesis, deposition, and assembly of these materials are apparently accompanied by the localized release of reactive oxygen species (ROS), predominantly H2O2 (Thordal-Christensen et al. 1997; Fig. 1). Studies in our lab have shown that ROS production beneath the germinating conidium can be detected as early as 3 h after inoculation, before an appressorium is formed by the fungus (Wei et al., unpublished results). The oxidative burst, a rapid and transient ROS production in response to pathogen attack, has been prescribed several roles including direct toxic effects on the pathogen, the oxidative cross-linking of wall components, and local and systemic induced resistance signalling (reviewed by Hücklehoven and Kogel 2003). H2O2 has also been found in vesicles in transit to CWAs (Collins et al. 2003; Hücklehoven et al. 1999), suggesting that ROS may play a role upstream of CWA deposition, or that CWA materials are oxidatively cross-linked before arrival at front line. The origin of ROS in CWAs remains largely unknown, despite compelling evidence for specific ROS generating systems in several different pathosystems. For example, while pharmaceutical and genetic approaches indicate that H2O2 is generated from O2- produced by the NADPH oxidase complex in Arabidopsis (Torres et al. 2002) and tobacco (Yoshioka et al. 2003), ROS generation in barley (Hückelhoven and Kogel 1998) and wheat (Wei et al., unpublished) show low sensitivity to diphenyleneiodonium, an inhibitor of the NAD(P)H oxidase, suggesting an different unidentified ROS generating system in cereals attacked by pathogens.

Figure 1

Penetration attempts of Blumeria graminis f. sp. tritici on wheat leaves are met with cell wall appositions and reactive oxygen species production.

Penetration attempts of Blumeria graminis f. sp. tritici on wheat leaves are met with cell wall appositions and reactive oxygen species production.

Inoculated leaves were subjected to 3,3-diaminobenzidine (DAB) uptake treatment overnight (Thordal-Christensen et al. 1997) and examined under a light microscope. The dark DAB staining indicates the presence of H2O2. c, condium; agt, apressorial germ tube; CWA, cell wall apposition; pp, penetration peg.

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Current research program on CWAs of powdery mildew attacked wheat

Recent work in our lab has focused on the Triticum monococcum L. epidermal transcriptome during attack by Blumeria graminis f. sp. tritici. The diploid wheat epidermis-powdery mildew system has proven to be an excellent platform for the study of cell wall mediated defence. On the plant side, we used T. monococcum because it has a simple wheat ‘AA genome’ and well defined resistance responses; we have lines that exhibit complete CWA resistance, late single cell hypersensitive response, and full susceptibility. One current project is to develop isogenic lines that differ only in CWA-mediated resistance and susceptibility. We used the epidermis specifically because it is the first line of defense against environmental insults, and because it is a relatively inert tissue that, when challenged, focuses the majority of its transcriptional effort on defence. On the pathogen side, we chose B. graminis f. sp. tritici because it only infects the epidermis, and because it has highly synchronous and well defined penetration, infection, and reproduction processes. We have sequenced 3000 expressed sequence tags (ESTs) from a powdery mildew infected T. monococcum epidermis cDNA library, and have found very few ‘housekeeping genes’ and over 1000 stress/defence related genes. Much of the transcriptional effort of the infected epidermis is devoted to ROS generation and scavenging. For example, 1.5% of all of the ESTs sequenced so far encode type III peroxidases (Liu et al., unpublished). We have found genes involved in many known aspects of CWA formation including cytoskeletal organisation and vesicle traffic (e.g. clathrin, COPI and COPII structural and regulatory elements), cell wall modification (e.g. enzymes involved in synthesis of cell wall polysaccharides and phenolic derivatives, and protein and lipid alterations), and generation and scavenging of ROS (e.g. NADPH oxidase, germin-like proteins, xanthine oxidases, ascorbate/glutathione cycle enzymes, superoxide dismutases). These ESTs provide seemingly endless angles from which to tackle the CWA phenomenon, and a major challenge now is to identify key components that regulate major steps leading up to CWA formation.

Challenges and future directions

Although CWAs are presumed to stop fungal penetration by reinforcing the cell wall at the point of attack, a recent paradox has emerged suggesting that there is more to CWAs than was first supposed. Two independent reports have shown that Arabidopsis plants with mutated callose synthase genes are in fact more resistant to attack by some pathogens (Jacobs et al. 2003; Nishimura et al. 2003). Nishimura et al. (2003) found that while callose synthase mutants were resistant to powdery mildew, double mutants with a blocked salicylic acid (SA) pathway had restored susceptibility, suggesting that callose could be involved in repressing other defence pathways, and an absence of callose allows for an enhanced SA response. Therefore, successful pathogenic fungi might have adapted to the plant surveillance system of cell wall integrity to establish their parasitism.

Recent studies have suggested that bacterial flagella are recognized by plants and induce cell wall-mediated defences, which can then be suppressed by secreted bacterial effectors (DebRoy et al. 2004; Hauck et al. 2003). The discovery of a common fungal signature that is able to induce CWA formation in the absence of a pathogen could be central to understanding early events in the CWA formation signal. Another long-standing question has been the origin of the ROS produced during plant responses to pathogen attack. Apparent differences between monocot and dicot systems (e.g. Hückelhoven and Kogel 1998 vs. Torres et al. 2002) have compounded this problem, and in monocot systems no clear ROS generating system has been defined. Significantly, recent work in our lab suggests that in cereals the oxidative burst may be generated primarily by redox active metals rather than by enzymatic means (Wei et al., unpublished). Although major strides have been made in understanding aspects of vesicle trafficking (Collins et al. 2003), the further identification of components involved in CWA-destined vesicle formation, fusion and exocytosis will surely provide important insights into the CWA formation process as a whole. Finally, although this review has focused primarily on basic research concerning CWA-mediated resistance, the barley mlo example illustrates the importance of this ancient form of resistance in combating ‘real world’ agricultural problems. The identification and eventual deployment of CWA regulators in other crops could provide durable broad spectrum resistance for years to come.