The effective papilla hypothesis

‘Chowdhury et al. used cell wall probes and antibodies for a beautiful visualization and quantification of cell wall polymers in barley papillae under attack from B. graminis f. sp. hordei.’ Cell wall papillae have been observed, and their role in penetration resistance discussed, for decades (Zeyen et al., 2002; see also the introduction of Chowdhury et al.). In the interaction of cereals with grass powdery mildew fungi, susceptible host plants restrict fungal penetration to a degree that varies greatly depending on the degree of background resistance (synonymous with basal resistance, quantitative resistance, partial resistance, horizontal resistance). Background resistance can lead to fungal penetration failure in the majority of attempts. This is observed as a cell-autonomous phenomenon, meaning that similar neighboring epidermal cells show differing success in stopping fungal penetration from an appressorium and subsequent establishment of a haustorium (the fungal feeding structure) in the intact host cell. Penetration success is vitally important for the fungus because, as a biotroph, it needs access to host resources, and haustoria may additionally serve as an interface for delivering virulence effectors into the host cell cytoplasm. This explains why nonsuccessful fungi are unable to develop epicuticular hyphae for fungal spreading on the leaf surface. Instead, B. graminis develops a second, or even a third, appressorial lobe when penetration fails (Fig. 1). This shows that the fungus initially survives penetration failure, suggesting that nonfungitoxic principles of host defense are effective in grass penetration resistance to powdery mildew. However, evidence that structural papilla components are crucial for penetration resistance is lacking. The term ‘an effective papilla’ is not precisely defined – I use it here to describe a cell wall apposition that is formed during a local response to an attempted fungal penetration and that cannot be penetrated by the fungus because of structural cell wall components, which confer resistance to fungal hydrolytic or mechanical force. The concept of an effective papilla is difficult to prove experimentally and it has been challenged by genetic studies of powdery mildew resistance in Arabidopsis thaliana, which showed that indole glucosinolates are crucial for penetration resistance to nonadapted B. graminis f. sp. hordei, whereas papillary callose only marginally contributes to penetration resistance in this experimental system (Jacobs et al., 2003; Bednarek et al., 2009). Loss of function of the callose synthase GSL5/PMR4, which is responsible for the biosynthesis of the majority of callose found in papillae, even leads to deregulation of salicylic acid-mediated defense responses and loss of susceptibility to adapted powdery mildew pathogens (Jacobs et al., 2003; Nishimura et al., 2003). On the other hand, overexpression of GSL5/PMR4 leads to enhanced papillary callose deposition and complete penetration resistance to powdery mildew, showing that cell wall carbohydrates can contribute to penetration resistance (Ellinger et al., 2013). Attempts to unravel the function of papillae in penetration resistance may profit from knowing the chemical composition of NPP and PP in a wildtype situation. Few apoplastic defense compounds have been found to actually mark NPP but not PP. Cell wall autofluorescence deriving from phenolic substances is immobilized and thus becomes insensitive to saponification over time during attack from B. graminis f. sp. hordei. Immobilization of autofluorescence manifests earlier in penetration-resistant mlo (MILDEW RESISTANCE LOCUS O) mutants compared with susceptible wildtype MLO (von Röpenack et al., 1998). One can further visualize peroxidase-dependent hydrogen peroxide (H2O2) accumulation by 3,3-diaminobenzidine (DAB) staining and protein immobilization, presumably as the result of oxidative crosslinking in papillae (Thordal Christensen et al., 1997). H2O2 formation in papillae appears to be further supported by Fe3+ accumulation in the apoplast (Liu et al., 2007). H2O2 is much more frequently detected in association with NPP than with PP in both background and mlo-mediated resistance and could thus be important for crosslinking cell wall components (Hückelhoven et al., 1999, 2000). In this respect, it was suggested that CatB, a catalase secreted by B. graminis f. sp. hordei at sites of penetration, is involved in scavenging H2O2 and might thus interfere with penetration resistance (Zhang et al., 2004). In analogy, suppression of the oxidative burst in corn is gained by Pep1, a key virulence effector from the corn smut fungus Ustilago maydis. Pep1 inhibits the apoplastic oxidative burst by directly interacting with, and inhibition of, type III peroxidases (Hemetsberger et al., 2012). However, there is little direct evidence that the actual carbohydrate composition of papillae distinguishes NPP from PP. Chowdhury et al. used cell wall probes and antibodies for a beautiful visualization and quantification of cell wall polymers in barley papillae under attack from B. graminis f. sp. hordei. This revealed a layered composition of papilla with autofluorescent phenolic substances, callose, arabinoxylans, and cellulose. Callose, arabinoxylans, and cellulose are enriched in NPP compared with PP. These substances might be partially interlinked and further crosslinked via phenolic bridges. The papilla has a layered structure, with phenols in its core, callose and arabinoxylans through the body of the papilla, and an outer encapsulation by arabinoxylan and cellulose. This might give the papilla a resistant structure, which cannot be dissolved or mechanically broken by the fungus. The outer cellulose layer might provide further structural strength (Fig. 2). These findings now allow many questions on the cell biology and genetics of NPP formation to be better addressed, as follows. How are NPP carbohydrates synthesized and transported to NPP? Do fungal virulence effectors specifically interfere with biosynthesis or transport of these carbohydrates or do fungal xylanases specifically adapt to host xylans? In this context, it would be important to compare carbohydrate patterns in papilla formed in response to adapted and nonadapted powdery mildew fungi. Reverse genetic approaches may allow for identification of key elements on both sites of this apoplastic interaction: for example, host glycosyl transferases, fungal hydrolases, host inhibitors of fungal hydrolases, and fungal virulence effectors. In general, genetic evidence is lacking for the effective papilla hypothesis. It will be important to see whether ROR2 (REQUIRED FOR mlo-SPECIFIED RESISTANCE 2) and mutant ror2 genotypes differentially accumulate carbohydrates in papillae as they do for H2O2 (Hückelhoven et al., 2000). ROR2 is the barley ortholog of PEN1/SYP121, a plasma membrane resident syntaxin, which is postulated to act in vesicle-mediated secretion and is required for penetration resistance of Arabidopsis to B. graminis (Collins et al., 2003). Further mechanisms of endomembrane transport, tethering, and fusion might be involved in delivering carbohydrates to NPP. Cellulose is most probably deposited via plasma membrane-localized cellulose synthases, which are dynamically positioned along microtubules. Microtubule organization differs greatly between cells with NPP and those with PP (Hoefle et al., 2011). In particular, nest-like focal accumulation of microtubules around NPP could function in positioning of cellulose synthases for papilla compaction (Hoefle et al., 2011). The actual function of a papilla might be complex. Therefore, it will also be important to resolve the timing of the carbohydrate apposition in papillae. Carbohydrates could be deposited as a secondary late response of the resistant cell to seal the cell wall and possibly to down-regulate cell wall damage-induced defense responses. By contrast, early deposition of carbohydrates would make it more likely that they operate in penetration resistance. Additionally, it is challenging to distinguish cell wall strengthening against penetration by fungal hyphae from cell wall insulation against penetration by fungal virulence effectors, which partially act in the host cytoplasm (Fig. 2). Recessive mlo-mediated resistance is considered as a form of extreme background resistance and shares some genetic components in grasses and dicots (Collins et al., 2003). It would therefore be interesting to visualize papilla carbohydrates in mlo-mediated penetration resistance in both monocots and dicots. Notably, GSL5/PMR4-dependent callose biosynthesis (and thus papillary callose) is not only expendable for defense against nonadapted powdery mildews (discussed earlier), but is also dispensable for mlo-mediated resistance in Arabidopsis (Consonni et al., 2010). Finally, the evolution of papilla formation and the exact composition of NPP vs PP in dicots are not understood. These questions reveal the stimulating nature of Chowdhury et al.'s study and the great challenge of understanding papilla function in plant–microbe interactions. Experimental work in the laboratory of R.H. is supported by the German Research Foundation (SFB924; HU886/8), the German Federal Ministry of Education and Research (0315955E) and the Helmholtz Association (Portfolio topic: Sustainable Bioeconomy).