Tree immunity: growing old without antibodies

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Opinion

Tree immunity: growing old without antibodies Peri A. Tobias and David I. Guest Department of Plant and Food Sciences, Faculty of Agriculture and Environment, University of Sydney, Biomedical Building C81, 1 Central Avenue, Australian Technology Park, Eveleigh, NSW 2015, Australia

Perennial plants need to cope with changing environments and pathogens over their lifespan. Infections are compartmentalised by localised physiological responses, and multiple apical meristems enable repair and regrowth, but genes are another crucial component in the perception and response to pathogens. In this opinion article we suggest that the mechanism for dynamic pathogen-specific recognition in long-lived plants could be explained by extending our current understanding of plant defence genes. We propose that, in addition to physiological responses, tree defence uses a threepronged genomic approach involving: (i) gene numbers, (ii) genomic architecture, and (iii) mutation loads accumulated over long lifespans. A changing pathogen environment To survive and be successful all life forms need to defend themselves against invading pathogens. Plants are sessile and need to respond to pathogens in situ – which would appear to be a particular problem for long-lived trees. Trees provide food, fibre, and biofuels, and are an essential part of our environment. However, as long-lived perennials, trees are exposed to rapidly evolving pathogens, and a static set of defence genes in a host genome of an individual tree is likely to be overcome by changing pathogen populations. A tree hundreds of years old will have faced very different pathogen genomes at different stages of its life [1–3]. How then do trees maintain their vigour in the face of sustained and changing pathogen attack over time? How does a tree that lives for decades or centuries respond effectively to changing microbe populations? Tree physiology provides some answers. The capacity to isolate diseased tissue into woody compartments [4], and to dispense with leaves or roots via induced abscission, minimises infection spread [5]. These processes are coupled with multiple meristematic (plant stem cell) zones that allow repair and new growth to compensate for any sections excised due to infection [6]. Systemic acquired resistance (SAR), involving salicylic acid-based priming of plant Corresponding author: Tobias, P.A. ([email protected]). Keywords: R-genes; gene clusters; transposable elements; mutations. 1360-1385/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants. 2014.01.011

defences, provides a further level of protection [7]. Defence genes, however, are major contributors to successful long life, and plants retain large numbers of diverse resistance genes (R-genes) (Box 1) as well as other defence response genes. Here we propose that tree defence, over a long lifespan, is based on a three-pronged genomic approach to counter pathogen variation over time: (i) gene numbers and diversity, (ii) genomic architecture, and (iii) mutation loads due to lifespan. Many and varied defence genes Plant resistance genes (R-genes) are important for specific recognition of pathogens and are present in large numbers in plant genomes [8] (Table 1) They are known to undergo diversifying selection, thereby providing the flexibility to respond to rapidly evolving pathogens for the next plant generation [9,10] (Box 2). In fact, diversifying selection has been identified in several plant defence gene families including R-genes, guardees, apoplastic proteases, and chitinases [11–13] (Box 2). For short-lived species and seedling progeny of long-lived plants, diversifying selection provides an opportunity to adapt to variation within and between pathogens. Trees are inheritors of a large and diverse array of defence genes that arise due to genetic recombination and diversifying selection. Annotated sequences from woody plants provide recent quantitative evidence of defence gene numbers, and indicate that long-lived trees (apple, cocoa, grape, poplar, and rubber) maintain proportionately larger numbers of the nucleotide-binding site leucine-rich repeat (NBS-LRR) class of R-genes than do the short-lived plants Arabidopsis thaliana, papaya, sorghum, castor oil plant, tomato, common bean, or maize (Table 1) [14–20]. It is suggested that a higher frequency of resistance genes in trees may provide better defence capacity [14,17]. Perennials face ongoing pathogen challenges, perhaps reflected in their accumulation of R-gene sequences. There are however exceptions: rice for example, has proportionately larger numbers of Rgenes than other short-lived plants, potentially as a result of intensive artificial selection [18,21]. Predicted R-gene numbers are largely derived from data-mining of newly sequenced plant genomes using homologous sequences. The estimates may therefore not accurately represent true biological gene numbers and should be interpreted with caution. Indeed the numbers of R-genes for short-lived and long-lived specimens presented in Table 1, although interesting, are perhaps less important than the fact that all plants maintain such a high frequency of these defence Trends in Plant Science xx (2014) 1–4

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Box 1. R-genes: an important class of defence genes

Box 2. Diversifying selection

Resistance genes (R-genes) are a major class of defence genes involved in plant responses to pathogens [8]. Pathogens deliver effector molecules that attenuate host defence responses. R-genes encode proteins, predominantly containing nucleotide binding site and leucine-rich repeat (NBS-LRR) domains, that specifically recognise microbial effector molecules or effector-modified host proteins [50]. Recognition initiates effector-triggered immunity (ETI) that blocks pathogen spread. ETI can involve programmed cell death, antimicrobial accumulation, upregulation of pathogenesisrelated proteins and systemic acquired resistance (SAR), whereby a whole plant broad defence-response is elicited through previous localised pathogen exposure [51,52].

Diversifying selection is tested by looking at rates of nonsynonymous (Ka) versus synonymous (Ks) nucleotide substitutions. Non-synonymous substitutions, where an amino acid is substituted, can reduce or remove function, and are therefore presumed to be biologically detrimental. The Ka:Ks ratio is therefore expected to be less than one. A ratio greater than one indicates an evolutionary advantage to non-synonymous mutation and is termed diversifying selection [10]. Several studies have found evidence of diversifying selection in regions of R-genes [9,10]. Other defence response genes are also identified as undergoing this mode of selection, including chitinases [13,55], apoplastic proteases [12], and guardee molecules that interact with effectors initiating R-gene response [9,11].

genes. Around 1% of all protein coding genes in long-lived woody trees are NBS-LRR R-genes. Studies of relatively long-lived invertebrates, such as sea urchins and snails, indicate that having a large number of diverse pathogen recognition genes provides an effective mechanism for defence [22,23]. Further immune-response diversity in these invertebrates appears to be generated by alternative gene splicing as well as post-translational modifications. Many plant gene transcripts undergo alternative splicing. Sixty-one percent of A. thaliana transcripts are known to be alternatively spliced [24]. Little is known of alternative splicing of genes in trees, but analysis of recently sequenced tree genomes (Phytozome; http://www.phytozome.net/) suggest that transcripts from coding genes in Eucalyptus grandis (28%), apple (16%), and poplar (77%)

undergo alternative splicing. A mechanism for diversity therefore exists at the transcript level, with further potential in post-translational modification of defence gene products, as suggested for the invertebrate sea urchins [22]. In addition, when gene numbers, alternative splicing, and post-translational modification are all combined with diversity afforded through molecular complexing of proteins encoded by different R-genes, defence capacity multiplies. Studies of pathogen-challenged rice and tomatoes have identified the pairing of proteins for pathogen blocking [25], as well as modulating of host responses depending on the number and composition of complexed R-gene encoded proteins [26]. The Human ENCODE project highlighted the inadequacy of a strict interpretation of Crick’s ‘one gene–one protein’ central dogma of molecular biology [27]. The numbers and variety of transcripts, including alternatively spliced mRNA, other RNA products, and transposable elements (TEs) (Box 3), indicate that the genomic sequence of an organism is simply a starting point for determining its gene product repertoire [27]. It would seem then that the multiplying effect of gene numbers, diversity, splicing variants, and post-translational modifications, in response to pathogens, is likely to contribute to an effective and adaptable defence mechanism during the lifetime of a tree.

Table 1. Predicted NBS-LRR resistance gene numbers in longlived woody plants and short-lived herbaceous plants Plants

Predicted NBS-LRR resistance gene numbers

Woody plants (long-lived) 618 Hevea brasiliensis Rubber tree 992 Malus X domestica Apple 402 Populus trichocarpa Poplar 297 Theobroma cacao Cocoa 305 Vitis vinifera Grape Herbaceous plants (short-lived) 178 Arabidopsis thaliana Thale cress 54 Carica papaya Papaya 535 Oryza sativa Rice 125 Phaseolus vulgaris Common bean 121 Ricinus communis Castor oil plant 266 Solanum lycopersicum Tomato 211 Sorghum bicolor Sorghum 129 Zea mays Maize 2

Percentage of predicted proteincoding loci

Refs

0.9%

[16]

1.7%

[15]

1.0%

[14,17]

1.0%

[53]

1.2%

[19]

0.7%

[17]

0.2%

[16]

1.3%

[17]

0.5%

[18]

0.4%

[20]

0.8%

[19]

0.6%

[54]

0.4%

[15]

Genomic architecture – gene clustering and TEs The tight clustering of defence-response genes, and Rgenes in particular, is a frequently observed phenomenon in plant genomes [28]. Tandem duplication of genes, gene conversions, and unequal crossing-over during DNA recombination may account for some of this clustering, but the phenomenon also extends to genes that simply share functional as opposed to sequence attributes [29]. Clustering allows cotranscribed gene expression and is sometimes associated with genes whose products operate within a metabolic pathway [30], as well as in defence [31]. Box 3. Transposable elements (TEs) TEs are regions of DNA that are mobile within the genome. They have the ability to excise and insert themselves in different genomic regions through direct (cut and paste) or RNA-mediated mechanisms. They were first proposed by Barbara McClintock in the 1940s as an explanation for reversible pigment changes in corn kernels, and were termed ‘jumping genes’ [56]. Large regions of eukaryotic DNA in plants are made up of TEs that are often methylated to reduce transcription [37].

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Opinion There is currently no adequate explanation for clustering of non-paralogues (genes of different origin), except for a form of selection pressure within specific genomes, because there is apparently little conservation of these clusters across organisms [28]. The presence of multiple colinear R-genes, which can be in regions up to 1.5 Mb in size, provides a potential target for random mutations as well as providing a framework for mispairing and recombination during meiosis [31]. Unequal crossing-over during meiosis is known to favour regions of tandem repeats [32]. In a study of secondary metabolic pathways involved in stress responses in plants, a higher proportion of transposable elements (TE) occurred within these gene cluster regions than in other chromosome regions, suggesting an involvement in gene transposition and mutations [29]. TEs have been shown to be activated in response to stress [33,34], and TE insertions are associated with somatic diversity in rice [35]. Interestingly, a recent study identified a high proportion of introns retained in alternatively spliced cotton genes that also contained TEs [36]. Indeed, TEs comprise such a large proportion of plant genomes (for example, 85% of the maize genome) that they have long been thought to be involved in the evolution of defence-related genes [37,38]. If mutations are likely to be more frequent in long-lived organisms (see below), perhaps the clustering of defence genes makes them targets for TEmediated generation of diversity? The very fact that these genes are not ‘fixed’ within plant genomes, as evidenced by diversifying selection studies (Box 2), suggests that the genomic architecture facilitates targeted mutations. More mutations in long-lived trees Klekowski [39] suggested in 1988 that long-lived plants fit a model of the ‘chemostat effect’ observed in bacterial colonies maintained for multiple generations, in which neutral mutations increase linearly over time. Plant apical meristems undergo around five mitotic divisions of initials (plant stem cells) per growing season. Based on this figure, and on the estimated frequency of 10 4 mutations (per locus per generation or year), Klekowski predicted that a tree aged 200 years has 10% mutant initials, a 500-year-old tree has 25%, and so on. A detailed method for measurement of mutations in tree canopies that treats branches as stem cell lineages has been proposed, although no experimental confirmation has yet been published [40]. However, a recent transcriptome study has identified putative genetic variation between branches of a single Eucalyptus melliodora specimen where herbivory resistance was present in only one branch [41]. If confirmed, this might represent genetic evidence that mutations within a single long-lived plant can provide an evolutionarily adaptive advantage during its lifespan. Variations such as this arising from meristematic mutations have long been identified phenotypically by gardeners as ‘sports.’ Earlier studies by Klekowski into chlorophyll deficiency mutations indicate that Rhizophora mangle (mangrove) and Pinus sylvestris (pine) trees accumulate mutations at a rate of 1.5  10 3, an approximately 10-fold greater rate per generation than that recorded for annual plants [42]. Recent molecular evolution studies have noted reduced mutation rates in woody plants, and have also identified a

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significant negative correlation between plant height and synonymous substitution rates [43,44]. A proposed mechanistic hypothesis is the declining rate of mitosis as plants age [44]. Investigations of genes involved in DNA repair have found active mechanisms of non-homologous end-joining and homologous recombination in P. sylvestris [45]. Active telomerase activity has been identified in other gymnosperms, suggesting that strategies to amend deleterious mutations and ameliorate age-related stress are employed in trees [46]. Despite declining mutation rates and efforts at DNA repair in long-lived trees, the lifetime accumulation of mutations is likely to be significant [42]. The accumulation of numerous, and variant, mutant initials at disparate sites of meristematic growth (roots, shoots, trunk) provides an interesting model for evolutionary change within a single, long-lived organism. Cell proliferation, when beneficial mutations arise, could lead to adaptive plasticity [41,47] and, perhaps, an additional mode of dynamic pathogen perception over a lengthy lifespan. Concluding remarks and future directions Some non-woody plants, such as palms, can also be longlived, but they lack the potential to accumulate mutations from lateral meristems. The date palm, Phoenix dactylifera, a long-lived monocotyledon, has 144 predicted Rgenes (NBS-LRR), only 0.3% of predicted protein coding genes [48]. Although the three-pronged genomic approach proposed in this article applies to long-lived woody plants, only the first two strategies – defence gene numbers and genomic architecture – are available to monocotyledons owing to the lack of vascular and cork cambium. Date palm resistance gene numbers are more synonymous with herbaceous perennials; even so, the mechanism for evading pathogens is clearly successful. The basis for this success is an area for further speculation and research. It is now understood that there are ‘shades of grey’ in defining immune responses across organisms [49]. The vertebrate antibody, once considered a superior adaptive response, is now regarded as only one of many effective and variant mechanisms of immunity across all organisms. Trees can reform from multiple meristematic cells, around a largely dead structural framework, allowing adaptation to a changing environment at the physical and, we propose, genomic level. This level of adaptation reflects an ongoing ‘arms race’ of co-evolution between host and pathogen within an individual rather than transgenerationally, as in short-lived plants. Future research should focus on understanding plant genomic architecture and how this relates to mutations and evolutionary change, as well as the diversity afforded by variant splicing and complexing of defence gene products. Trees have traditionally been difficult to study because of their size and lifespan, and many aspects of tree defence responses still require investigation. With recent genomic and transcriptomic advances we are now better equipped than ever to begin unravelling the intriguing questions of long-term survival. Determining the interacting mechanisms of defence gene products, genomic architecture, and lifetime accumulation of mutations could provide the basis for understanding the successful immunological responses of long-lived trees. 3

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Opinion Acknowledgements The authors would like to thank Dr Sham Nair, Dr Carsten Kulheim, and Professor Peter Sharp for their comments and contributions to this text.

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