JXB Advance Access originally published online on February 9, 2009
Journal of Experimental Botany 2009 60(2):351-352; doi:10.1093/jxb/erp001
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eXtra Botany |
Cell wall appositions: the first line of defence
Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871-Frederiksberg C, Denmark
* E-mail: dbc{at}life.ku.dk
Most interactions between plants and fungi or Oomycetes do not result in a successful infection but are thwarted by the host (Thordal-Christensen, 2003; Lipka et al., 2008). In many cases, the obvious visible first line of defence is the papilla, a cell wall apposition (CWA) laid down by the host at the site of attempted penetration by the fungus or Oomycete on the inside of the host cell wall. CWA are observed in many plant species but are best studied in the cereal–powdery mildew interactions where the CWA comprises the inner papilla surrounded by an outer halo. Histological and chemical analyses demonstrate the complexity of the papillae, which comprise callose, protein, and various phenolic compounds and inorganic compounds, especially opal silica and, at least transiently, reactive oxygen species (see Zeyen et al., 2002, for a review). It is clear that papillae play a significant role, since penetration is often arrested by papillae, but as to which components of papillae are important for successful defence against a particular species pathogen is unclear. In other words, the demonstration of the complexity of papillae is not a demonstration of their role in disease resistance and presents the question as to why they are so complex. What is the role of the individual components of the papilla? There are two approaches which use the tools of forward and reverse genetics to determine the role of individual components of papillae which will answer this question, at least in part.
The regulation of the formation of papillae has been studied genetically in both barley and Arabidopsis. Mutants have been obtained in barley exhibiting resistance, and indeed the mlo mutant confers race-non-specific resistance in both species (Consonni et al., 2006). Other Arabidopsis mutations contribute to the assembly of papillae (Collins et al., 2003, 2007). The physiological role of the Arabidopsis gene products is uncertain in some cases (Lipka et al., 2008). Thus the forward genetics approach has favoured the identification of regulatory, like mlo, and mechanistic components, like Pen1, but has not yielded mutations in the phenolic biosynthetic pathways associated with papilla formation.
The nature of the first line of defence in cereals is still something of a mystery despite many years of study. We know much but there is still much to learn. Wei and colleagues have previously developed Triticum monococcum L. (Einkorn, a diploid wheat) as an excellent model system to use as an alternative to barley. In this issue, the study by Bhuiyan et al. (2009) uses a forward genetic approach to study the role of papilla components in Einkorn wheat infected with the powdery mildew fungus Blumeria graminis. The result is compelling evidence that lignification of papillae plays an important role in defence against penetration by this fungus.
Lignification is, in essence, the generation of wood. The process can be considered to comprise two phases, firstly, the biosynthesis of monolignols, the building bricks, and, secondly, the assembly and polymerization of these bricks in the papilla. This paper represents an in-depth study of the first phase, which is illustrated in Fig. 1 of Bhuiyan et al. (2009). The approach taken has been to screen an expression sequence tag (EST) library prepared from the epidermis of T. monococcum infected with B. graminis f. sp. tritici (Bgt) (Liu et al., 2005; Bhuiyan et al., 2007) which is predicted to be enriched for first line defences against Blumeria graminis (Wei et al., 1998; Collinge et al., 2002). This yielded 13 cDNAs representing eight genes encoding enzymes involved in monolignol biosynthesis, according to their bioinformatical analysis. That six of these transcripts exhibit nearly synchronized accumulation patterns, as revealed by Northern blotting of entire leaves and epidermal expression, as revealed by RT PCR, is an excellent indication of their role in papilla formation.
Proof of the importance of monolignol biosynthesis comes from the reverse genetics of transient gene silencing of four of the biosynthetic genes and an additive effect was obtained by combining three of the genes pair-wise. Thus, silencing gave a statistically significant increase in the efficiency of penetration. In the control, roughly 40% of penetration attempts were successful by the host powdery mildew fungus Bgt and increased 4-fold in the non-host powdery mildew fungus, Bg. f.sp. hordei, which infects barley. The double silenced constructs gave over 70% successful penetration rates. The genes included TmPAL, which encodes the first enzyme in the phenylpropanoid pathway, and can be predicted to have other roles in defence in grasses. For example, flavonoid biosynthesis has been seen to be induced in barley in response to B. graminis (Christensen et al., 1998a, b). The combination of TmPAL with genes encoding the two final biosynthetic enzymes of monolignols suggests strongly that, during penetration in this system, the only relevant product of the phenylpropanoid pathway is monolignols and therefore polyphenolics, and that these polyphenolics are essential components of effective papillae. The study continues by studying the chemical composition of the papillae formed in silenced cells. Silencing using RNAi by particle bombardment does not abolish gene expression, but reduces it in the affected cells and this is seen here by the demonstration that the papillae produced in the ‘silenced cells’ contain reduced amounts of the fluorescent phenolic components compared with ‘unsilenced’ cells. Finally, a pharmacological approach was used: treatment with well-documented inhibitors of two of the enzymes (PAL and CAOMT) also led to increased rates of penetration of papillae in the treated tissue.
The study is not perfect. For example, the RT-PCR is semi-quantitative rather than quantitative and expression data are provided for a subset of the genes and for only two time-points for the tissue expression. Nevertheless, this study provides convincing evidence that lignification plays an important role in making effective barriers against penetration by Blumeria graminis in a cereal. Remaining questions for future study lie in determining whether the prevention of production of other components would have a similar effect, for example, callose or silica. This paper does not address the effects of over-expression of individual components where over-expression of a rate-limiting component might lead to enhanced resistance, as has been seen for other defence genes expressed in tissues producing papillae (Schweizer et al., 1999; Jensen et al., 2007). However, in defence of this study in its present form, the genes that have exhibited an effect encode regulators or components which are predicted to have a direct antimicrobial effect.
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