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Keywords:

  • disease resistance;
  • hypersensitive response;
  • peroxidase;
  • Physcomitrella patens;
  • plant pathogen resistance;
  • R genes

The Vermin only teaze and pinch

Their Foes superior by an Inch.

So Nat’ralists observe, a Flea

Hath smaller Fleas that on him prey,

And these have smaller Fleas to bite ‘em,

And so proceed ad infinitum

– Swift. On Poetry: a Rhapsody (1733)

Jonathan Swift's satiric verses warning aspiring poets of the adverse opinions of critics have much to recommend them as advice to biologists, struggling to persuade reviewers of the value of their work or committees of the ‘impact’ of their research grants. But these lines, in particular, serve to remind us how all organisms (not just poets and biologists) engage in a struggle with pathogens, pests and parasites, and must needs deploy a variety of defensive strategies with which to repel them. In this issue of New Phytologist (pp. 432–443), Mikko Lehtonen and his colleagues at the University of Helsinki describe how the war between plant and pathogen is waged by bryophytes, using the unique tools afforded by the model moss, Physcomitrella patens.

‘Using the highly efficient gene targeting technology available in Physcomitrella to create knockout mutant lines they have demonstrated that these peroxidase genes form an essential part of the moss defence mechanism.’

It is estimated that 20–40% of crop yield, worldwide, is lost to pathogen attack – principally plant pathogenic fungi – every year, and that this proportion would be even greater were it not for the intervention of crop-protection strategies. Clearly, plant pathogens pose a threat to agricultural monocultures. However, a glance out of the window reminds us that, left to their own devices, the plants which dominate our natural environment ward off these depredations very effectively. Without a doubt, close observation of plants reveals incidences of pathogenesis, but it also reveals the frequent and characteristic fingerprints of effective defence against pathogens, usually in the form of the small and sporadic lesions that mark the activity of the hypersensitive response to infections.

Flowering plants deploy highly effective countermeasures against microbial infection that rely on their ability to recognize a potential pathogen and neutralize it through the rapid release of a range of fungistatic substances, including reactive oxygen species (ROS), phytoalexins and specific antifungal enzymes such as chitinases. These are allied with programmed death of the infected cell and the modification of the surrounding cell walls to restrict the further passage of infection. The speed with which this response is mounted is the key to its effectiveness, and has been characterized, genetically, as a specific interaction between resistance (R) genes within the host plant and pathogen-encoded avirulence (avr) loci. This ‘gene-for-gene’ response is explained through a molecular recognition between an R-gene product – essentially a plant receptor protein – and a fungal avr-specified component to trigger an intracellular signal transduction pathway that triggers these events. Both R and avr loci are highly polymorphic, a consequence of the evolutionary ‘arms race’ that develops as host and pathogen compete to survive (Dangl & Jones, 2001).

Among the most prominent features of the plant response to infection is the ‘oxidative burst’: a rapid release of ROS – in particular hydrogen peroxide (Bolwell et al., 2002). Hydrogen peroxide is multifunctional. It can serve directly as an antimicrobial agent, through its antiseptic properties, as well as participating in mechanical defence through the generation of papillae (Thordal-Christensen et al., 1997), the modification of cell walls (cross-linking and lignification through the incorporation of phenolic compounds) to restrict fungal spread, the synthesis of phytoalexins and as a potent intracellular and intercellular signalling compound to activate further responses (Torres et al., 2006; Almagro et al., 2009).

By contrast, comparatively little is known about the pathogens of the ‘lower’ plants, although pathogenic effects of fungal infection have been reported for a number of mosses (comprehensively reviewed by Davey & Currah, 2006). However, until recently, little was known of the mechanisms that the bryophytes deploy to resist infection. Because the dominant gametophyte stage of the moss life cycle lacks a protective cuticle, mosses should be particularly susceptible to infection. Add to this organs that are typically one cell thick, and the consequences of a successful infection are not difficult to envisage. However, it is clear that the mosses are a very successful taxon. They have flourished for the past 450 million years, and are the dominant form of vegetation in some ecosystems (e.g. polar regions). Comprising c. 10 000 species, the mosses are second only to the angiosperms in species diversity. It is not surprising therefore that this group of plants exhibits a robust response to infection. The wide range of molecular genetic tools, developed by the Physcomitrella research community, now provide researchers with the resources necessary to undertake a comprehensive molecular analysis of moss pathogen responses, and through comparative analysis, to place the existing information regarding the resistance of flowering plants within an evolutionary framework.

Lehtonen et al. (2009) have isolated naturally occurring fungal bryo-pathogens of a moss species in the field (Racomitrium japonicum) and have shown that these are equally pathogenic to Physcomitrella. Having identified secretion of peroxidase as a rapid response to elicitation by the fungal cell-wall-derived compound chitosan, they have identified the genes encoding this enzyme as a pair of duplicated Class III peroxidase genes clustering in a moss-specific phylogenetic clade. Using the highly efficient gene-targeting technology available in Physcomitrella to create knockout mutant lines, they have demonstrated that these peroxidase genes – that either use or generate hydrogen peroxide – form an essential part of the moss defence mechanism. The questions that arise from this are as follows.

• How does Physcomitrella – and by analogy other mosses – recognize the presence of the fungal elicitor?

• To what extent do the defence responses of mosses resemble those of higher plants?

• What can we learn from comparative studies about the evolution of the plant pathogen response?

In recent years, considerable progress has been made in determining the molecular features of plant–pathogen resistance mechanisms: an enterprise facilitated greatly through the use of the model angiosperm, Arabidopsis thaliana, and the extensive molecular genetic resources available for this species. Genomic analysis reveals that over 200 R genes are required for resistance, not only to fungal pathogens, but also to infection with bacteria and viruses, as well as to predation by invertebrate pests such as nematodes and insects. These genes display common features and are often clustered within the genome, suggesting that copy number expansion and diversification has been an important feature of their evolution (Sterck et al., 2007). In particular, the presence of leucine-rich repeat (LRR) sequences as a common feature of R-gene products indicates the basis for specific recognition of avr-dependent elicitors because these domains commonly participate in protein–protein interactions and in specific ligand-binding (Kobe & Deisenhofer, 1995), and it is these regions of the R-gene products that display significantly high levels of polymorphism, indicative of the highly active selection operating on these loci (Dangl & Jones, 2001; Bakker et al., 2006). This combination of gene duplication and polymorphic variation provides plants with an adaptively flexible means of repelling pathogens.

It is now recognized that plant resistance to pathogens is multilayered: an initial response to contact with pathogen-specific elicitors (or ‘pathogen-associated molecular patterns’: PAMPs), backed up by a second phase of recognition of pathogen-derived ‘effectors’ with which the pathogen seeks to suppress the response to an initial recognition event (Jones & Dangl, 2006; Boller & Felix, 2009). Notwithstanding this added complexity, the recognition of each of these pathogenic signals is based on R–avr interactions, and can lead to the activation of hypersensitive cell death and the release of antifungal microbial compounds.

The R-gene-mediated pathogen response is clearly essential for the health of flowering plants, but how much is known about the evolution of this mechanism? Some features of the response are clearly ancient. R-gene products themselves share considerable similarity with proteins involved in the mammalian programmed cell death pathway (Dangl & Jones, 2001), suggesting that apoptosis is an ancient feature of multicellular organisms that has been recruited by evolution to provide a solution to similar problems in different organisms. Most R genes are distinctively modular, the most abundant class comprising sequences encoding nucleotide-binding (NB) domains, and a domain homologous with the Drosophila Toll and mammalian interleukin-2 receptors (TIR) that is thought to have an intracellular signalling role. To what extent are these activities widespread among the Plant Kingdom? The development of Physcomitrella for comparative functional genomic studies promises to shed light on this question.

How then do mosses perceive and respond to pathogenic infection? Probably by using some of the same mechanisms that exist in flowering plants: previous studies have demonstrated that Physcomitrella will respond to infection with known angiosperm pathogens, such as Erwinia and Botrytis, in a manner analogous to angiosperms (Andersson et al., 2005; Ponce de Léon et al., 2007) and pathogen receptor type LRR-containing genes have been isolated from Physcomitrella (Akita & Valkonen, 2002) and also identified in other basal lineages, including liverworts and charophycean algae (Sasaki et al., 2007). With the publication of reference genomes for the mosses (Physcomitrella) and lycopods (Selaginella), we can now examine the extent of the gene families encoding LRR-domain genes.

A rapid (and by no means exhaustive) search of these two genomes reveals over 150 (Physcomitrella) and 110 (Selaginella) LRR-containing genes, respectively. However, compared with Arabidopsis, only a handful have the characteristic motifs of the abundant flowering-plant TIR–NB–LRR family: in Physcomitrella, I found only 6 gene models containing TIR motifs, whilst 28 have NB domains; in Selaginella the corresponding numbers are 2 and 16, respectively. Therefore, it seems that whilst the expansion of LRR repeat genes is widespread among land plants, the evolutionary acquisition of the signalling domains may be a feature of the angiosperm radiation. This may underpin taxon-specific differences that have been recorded in the recognition of specific PAMPs (Qutob et al., 2006). It will be both practical and fruitful to apply the powerful reverse genetics tools afforded by Physcomitrella to investigate the functions of those receptors that do exhibit a high degree of similarity with their angiosperm counterparts. Nevertheless, responses elicited by microbial infection in Physcomitrella are similar in many ways to those in flowering plants, with the release of active oxygen species, mobilization of salicylic acid signalling (Andersson et al., 2005) and cell death (Ponce de Léon et al., 2007).

It is tempting to speculate that necessary for the successful transition of green plants from an aquatic environment to colonize terrestrial habitats c. 470 million years ago was the evolution of an effective set of defences against airborne pathogens, and that the expansion and diversification of pathogen receptor recognition domains (the LRR domains) date from this time. This is clearly a question that can be addressed through comparative genomics, although at present no genome sequences exist for a taxon representative of the last common ancestor of the land plants.

It is generally believed that the land plants are derived from the Charophycean algae (Karol et al., 2001), and whilst it is to be hoped that the genome sequence of either Chara or Coleochaete will be determined in the not-too-distant future, at present these taxa are currently only the subject of expressed sequence tag (EST) programmes, represented by fewer than 500 ESTs available in the NCBI Trace Archive. New sequencing technologies will undoubtedly play a part in rectifying this missing link in the genome databases, and it will clearly be of interest to determine the representation of the LRR gene set in either of these organisms. Until such times, we can only inspect the genome of the unicellular chlorophyte, Chlamydo-monas reinhardtii, and that of its colonial relative Volvox carteri. Each of these organisms contains only a very restricted set of LRR genes (< 20), but these algae diverged from the green plant lineage approx. 1 billion years ago, and additionally are single-celled organisms. It is not unreasonable to suggest that a defence mechanism which operates primarily through the programmed death of host cells might not be the most evolutionary effective strategy for such organisms. Whatever the future holds, it is clear that there is much to be learnt about the acquisition of pathogen defence through the study of basal taxa.

References

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