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Mutualistic interactions on a knife-edge between saprotrophy and pathogenesis
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Mutualistic interactions on a knife-edge between saprotrophy and pathogenesis Claire Veneault-Fourrey and Francis Martin Saprophytic, ectomycorrhizal (ECM) and pathogenic fungi play a key role in carbon and nutrient cycling in forest ecosystems. Whereas more than 50 genomes of saprotrophic and pathogenic fungi have been published, only two genomes of ECM fungi, Laccaria bicolor and Tuber melanosporum, have been released. Comparative analysis of the genomes of biotrophic species highlighted convergent evolution. Mutualistic and pathogenic biotrophic fungi share expansion of genome size through transposon proliferation and common strategies to avoid plant detection. Differences mainly rely on nutritional strategies. Such analyses also pinpointed how blurred the molecular boundaries are between saprotrophism, symbiosis and pathogenesis. Sequencing of additional ECM species, as well as soil saprotrophic fungi, will facilitate the identification of conserved traits for ECM symbiosis and those leading to the transition from white-rotting and brown-rotting to the ECM lifestyle. Address UMR 1136 INRA-Nancy Universite´ « Tree-Microorganisms Interactions », Ecogenomics of Interactions, Centre INRA de Nancy, 54280 Champenoux, France Corresponding author: Martin, Francis ([email protected])
Current Opinion in Plant Biology 2011, 14:444–450 This review comes from a themed issue on Biotic interactions Edited by Giles Oldroyd and Silke Robatzek Available online 27th April 2011 1369-5266/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2011.03.022
Introduction As they display a wide-range of nutritional strategies, fungi are ubiquitous and able to live in almost all ecological niches. As a consequence of this ubiquity, more and more fungal genomes have been sequenced with an emphasis on questions important to biomedical and bioenergy-related research [1,2] as well as basic science [3]. Comparative genomics combined with systems biology has an enormous potential to improve our understanding of fungal biology as well as fungal–biotic environmental interactions [4]. In natural forest ecosystems characterized by mineral nitrogen and phosphate limitation, ectomycorrhizal Current Opinion in Plant Biology 2011, 14:444–450
(ECM) symbioses play a crucial role in plant nutrient acquisition and hence increase growth of the host-plant [5]. ECM-forming fungi play a key role in belowground biosequestration of carbon, mineral mobilization and organic nutrients degradation. A better understanding of the mycobiont’s role in ECM development and functioning appears to be crucial in ecology for durable forest management and to improve tree productivity. Interestingly, fungal ECM symbionts share their biotrophic lifestyle with obligate fungal plant pathogens, as the latter infect plant cells and feed on them but need to leave plant cells alive. The biotrophic life style is polyphyletic as it appeared several times across evolution [6]. To date, the genome sequencing of two non-phylogenetically related ECM-fungi, Laccaria bicolor (Basidiomycotina, Agaricomycotina, Agaricales, Hydnangiaceae) and Tuber melanosporum (Ascomycotina, Pezizomycotina, Pezizales, Tuberaceae), and their transcriptome profiling [7,8] have raised ectomycorrhizae as a model to study the evolution of eukaryotic symbiosis. RNA-seq technologies have been useful tools to improve their genome annotations and identify post-transcriptional mechanisms as well as aiding in generating a blue-print of the genes expressed during symbiosis [9,10]. With the recent release of hundreds of fungal genomes as well as the development of high-throughput functional analysis, we have an unprecedented opportunity to decipher molecular mechanisms driving ECM-symbiosis and, more generally, biotrophic life style as well as its evolution [11,12]. Based on comparative genomic analysis, we will firstly discuss convergent evolution between biotrophic fungi (mutualistic or pathogenic) through the expansion of genome size and the strategies to avoid triggering of plant defence responses. Secondly, we will discuss the crucial role of nutrient exchanges in the outcome of the interaction and finally, how biotrophic fungi could control the interaction with their host-plant. In this article, we speculate that saprotrophism, symbiosis and pathogenesis may be view as a continuum (Figure 1).
Genome expansion The genomes of the ECM-fungi L. bicolor and T. melanosporum are among the largest in size of fungal sequenced genomes to date, with 65-Mb and 125-Mb respectively [7,8]. Genome-size expansion is largely explained by a proliferation of repeated elements, primarily composed of transposable elements (TE) or the accumulation of their degraded remnants. They represent about 38% and 58% of L. bicolor and T. melanosporum genomes, respectively, whereas most fungal species contain 1 to 15% repetitive DNA in their genomes. www.sciencedirect.com
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Figure 1
PREDICTING CHANGES WITHIN COMMUNITIES
Functional markers to study ECM in situ
FUNGAL LIFE-STYLES: Identification of candidate genes for FUNCTIONAL ANALYSIS.
COMPARATIVE GENOMICS: - Genome evolution - Genome architecture - Molecular signatures of evolution - Metabolic and regulatory pathways White-rot
ECOSYSTEM METADATA: - Geochemical and physical parameters - Biological functions
Brown-rot
POPULATION GENOMICS: - Adaptation signatures of ECM fungi in various ecosystems and environmental conditions - Genotypic polymorphism
Biotrophic
Necrotrophic
Saprotrophic fungi
ECM fungi
Pathogenic fungi
> 30 genome
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Harnessing ‘omics’ for understanding the ecology of ECM fungi. Understanding how saprotrophic, symbiotic and pathogenic fungi achieve their lifestyle is crucial to understand their ecological functions, and their subsequent impact on the fate of plant communities. Several hundred species are active in soils and on-going genomics, metagenomics and metatranscriptomics studies will uncover the functions encoded in their genomes as well as their expressed transcripts. These sequenced genomes will provide baseline genomic information that enables scientists to investigate the interactions between fungal and plant communities.
Genome-size expansion has also been recently observed for obligate pathogens, such as powdery mildew fungi [13], rust fungi (Duplessis et al., unpublished) and the oomycete Hyaloperonospora arabidopsidis [14]. Therefore, biotrophy seems to be associated with convergent genome-size expansion owing to a proliferation of TEs. This may account for genome instability [15]. Comparative analyses of well-assembled and annotated fungal genomes lead to the idea that genome architecture is composed of syntenic regions containing a high-density of conserved genes and constituting the vast majority of the ‘core genome’, separated by non-syntenic regions containing a few genes and prone to rearrangements [3,16,17]. These gene sparse regions with high plasticity contain novel genes or duplicated paralogous genes [3] or clade-specific genes [18] probably required in adaptation to a given environment (variable traits). On the contrary, genomes of ECM and obligate fungal pathogens display uniform distribution of TEs across genome [7,8,13, Duplessis et al., unpublished], suggesting that genome evolution is shaped by other still unknown mechanisms in the biotrophic life-style. www.sciencedirect.com
In addition to transposon expansion, the L. bicolor genome displays expansion of multigene families as well as lineagespecific multigene families with a total of 19,102 proteinencoding genes, whereas the T. melanosporum genome displays only 7496 protein-encoding genes [7,8]. These strikingly different gene repertoires suggest that expansion of multigene families is not mandatory for symbiosis. Gene family evolution and gene duplication have been suggested to be important processes in the generation of evolutionary novelty through neofunctionalization and/or subfunctionalization. In the L. bicolor genome, striking gene family expansions occurred in genes predicted to have roles in protein-protein interactions (e.g. NACHT and ankyrin repeats) and signal transduction mechanisms (Tyrosine-kinase like, RAS small GTPase and WD40) [19,20]. Recently, Meng and Li showed that a viral capsid protein contains a functional nuclear localization signal ‘KRKR’, similar to a domain found in an alpha subunit of guanine nucleotide binding protein from L. bicolor [21]. If this nuclear localization signal is functional in L. bicolor, it might highlight a new mechanism of action for G-protein. In addition, several of these lineage-specific proteins are Current Opinion in Plant Biology 2011, 14:444–450
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upregulated during symbiosis (WD40 domain, G-beta repeat; tyrosine-kinase like; NB-ARC domain, NACHT domain), suggesting a role for these proteins in signaling pathways leading to symbiosis. In particular, recent findings indicate the WD-40 domains may act as hubs in cellular networks as they may interact with diverse proteins, peptides or nucleic acids [22]. Rust fungi that need two different hosts to complete their entire life-style, have genomes that display expansion of lineage-specific genes (Duplessis et al., unpublished) such as the alphakinase family as well as some specific nutrient transporters. Restriction of gene duplication is observed in both T. melanosporum and B. graminis genomes [8,13]. Cuomo et al. [23] have suggested that fungal species lacking the opportunity for gene innovation via duplication may be more constrained in adaptation to changing environments. Absence of gene duplication may thus be view as a hallmark of restricted host specificity within Ascomycetes as both T. melanosporum and B. graminis have narrow host specificity, whereas L. bicolor is a basidiomycete and a generalist mutualistic symbiont able to interact with seedlings and older trees from conifers and hardwood species. Lineage-specific multigene families may contain genes important for fungal adaptation to different ecological niches and may aid in broadening host range/specificity. Recently, Vinces et al. [24] showed that mutations in tandem repeats found in promoters might affect gene expression in yeast allowing rapid response to changing environment. T. melanosporum that is lacking in gene duplication contains the vast majority of its SSR within non-coding regions [25]. It will be interesting to test whether similar mechanisms could regulate T. melanosporum gene expression.
Avoiding plant defence responses Mutualistic and pathogenic biotrophic fungi must have evolved several strategies to avoid host detection and the triggering of basal plant defence responses. Chitin deacetylation versus sequestration. Surface-exposed chitin can be converted to chitosan through the action of fungal chitin deacetylase (CE-4) to avoid host detection. CE4-encoding genes from L. bicolor, T. melanosporum as well as from two rust fungi are induced during plant association [7,8, Duplessis et al., unpublished]. De Jonge et al., have shown that the effector protein Ecp6 from C. fulvum is able to sequester, through its LysM domains, chitin oligosaccharides released by plant chitinases preventing immunity induction [26]. A LysM protein from T. melanosporum is highly regulated during symbiosis, suggesting that this mycobiont might use chitin sequestration to avoid induction of plant defence responses. Reduction in plant-cell wall (PCW) degrading enzymes. The repertoire of carbohydrate-active enzyme (CAZyme) in L. bicolor and T. melanosporum revealed massive gene losses in the enzymes that target plant cell wall components Current Opinion in Plant Biology 2011, 14:444–450
such as cellulose (glycoside hydrolase (GH) 6 and GH7) and xylan backbones [7,8], suggesting that both ECM fungi are unable to use cellulose and xylan as nutritional sources. A lack of PCW-cleaving enzymes has also been observed in the ECM fungus Amanita bisporigera [27] and in obligate plant-pathogens [13,14]. This indicates that intimate associations with plant cells lead to convergent losses of enzymes acting on plant cell wall either because they are not required for fungal growth or more probably to avoid induction of plant-defence responses and thus achieve colonization. However, one striking difference concerns the presence of a cellulose-binding domain CBM1 in both ECM fungi genomes and its absence in biotrophic obligate fungal pathogen genomes [13,14]. The sole CBM1 module found in L. bicolor is linked to an endoglucanase GH5, whereas the T. melanosporum genome contains 2 CBM1 domains, with one fused to a candidate beta-glycosidase related to endoglucanase GH61. Both GH5 and GH61 might display a weak cellulolytic activity and raise the question of cellulose integrity as a signal in symbiosis. Both ECM fungi still have a residual ability to degrade pectin with different enzymes that might facilitate the progression of fungal hyphae within pectin-rich middle lamella during ECM formation. In addition, T. melanosporum genome contains the hemicellulases GH10 and GH43 that are absent in L. bicolor. Instead, L. bicolor contains 12 expansin-encoding genes, hypothesized to disrupt the non-covalent links between hemicellulose strands on cellulose microfibrils. The diverging enzymatic arsenal in both ECM fungi suggests a divergence in the way they deal with root colonization and suggest that T. melanosporum is more ‘aggressive’ towards its host-plant than L. bicolor. This relative aggressiveness of T. melanosporum is confirmed with a high level of expression of tyrosinases, lipases and laccases in planta [8]. In addition, an orthologue of a high-affinity cellodextrin transporter of T. melanosporum is induced in ECM [8]. In the saprotrophic fungus Neurospora crassa, this transporter is required for rapid growth on cellulose [28] suggesting that T. melanosporum may degrade cellulose during symbiosis functioning. CAZyme repertoires of two brown rot fungi Postia placenta [29] and Serpula lacrymans (Dan Eastwood et al., unpublished) are very similar to that of L. bicolor, with reduction of GH sets as well as losses of lignin-degrading peroxidases. Phylogenetic analyses using Bayesian molecular clock suggest that the ancestor of the Agaricomycotina was not an ECM fungi but probably a saprotrophic fungus [30] and the ancestor of the Agaricomycotina Boletales was probably a fungi with a brown rot nutritional mode (Dan Eastwood et al., unpublished). To explain evolution of fungal nutritional strategy towards ECM, the hypothesis in favour proposes that the ancestor might have been a white rot fungus with a large repertoire of CAZyme [1]. Lost of sets of CAZymes probably occurred at the same time that conifers appeared and led to the apparition of www.sciencedirect.com
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brown rot fungi that had lost efficient enzymatic machinery for lignin depolymerization and were unable to degrade cellulose using CAZymes. Loss of degrading enzymes in the brown rot ancestor might have allowed association between fungi and root cells without damaging the latter. In this context, the project to sequence 25 new mycorrrhizal genomes as well as 32 genomes of saprotrophic fungi will be of great interest and should highlight how symbiosis has evolved from saprotrophic ancestors (Figure 1) [12].
Nutrient exchanges The evolution towards mycorrhizal symbiosis did not lead to the loss of gene families involved in primary carbon or nitrogen metabolism [29,30], contrary to what was observed for obligate plant pathogens that lost the nitrate and/or sulfate assimilation ability ([13,14], Duplessis et al., unpublished). However, biotrophy seems to be associated with losses of secondary metabolism key genes [8,13]. Interestingly, silenced nitrate reductase L. bicolor strains are impaired in mycorrhization ability [31], suggesting that symbiotic interaction does not occur when the fungus does not supply nitrogen compounds to the plant. It is thus tempting to speculate that obligate pathogenic fungi might rely on their host for nitrogen supply and that ECM biotrophic fungi might depend on their host for carbohydrate supply. Hartig net of ECM, the zone of nutrients exchange between fungal and plant cells, constitute new sinks for photosynthate as ECM can drain up to half of the photosynthetically fixed carbon [32]. During the establishment of beneficial interactions with plants, a highaffinity sucrose transporter from the endophytic fungus Trichoderma virens is induced and sucrose hydrolysis through fungal invertase is required for root colonization [33,34]. Both ECM fungi genomes contain a similar transporter [13,34], whereas no similar transporters have been yet identified in obligate pathogenic fungi [13, Duplessis et al., unpublished], suggesting that obligate pathogenic fungi are unable to transport plant-derived sucrose. However, the latter contain at least one invertase with the expression induced during infection [13], whereas for most Basidiomycota ECM fungi including L. bicolor, no invertase gene was found in their genomes [35]. By contrast, the Ascomycotina ECM T. melanosporum genome contains one invertase encoding gene, suggesting that T. melanosporum is able to utilize plantderived sucrose during symbiosis [36]. These results suggest that both ECM fungi are able to transport plant-derived sucrose but have distinct ability towards its hydrolysis, T. melanosporum being closer to obligate pathogen in this perspective. These results also suggest that L. bicolor is more dependent on its host for carbohydrate supply than T. melanosporum. We can then speculate that L. bicolor that has a low ability to obtain carbohydrates from the surrounding soil [7] has to find www.sciencedirect.com
plant roots for a carbohydrate supply and consequently should establish ECM with a wide range of hosts. SWEET genes, recently identified in plants as glucose uniporters (importers or exporters) are differentially expressed in plant cells colonized with pathogenic bacteria or fungi [37]. In addition, only 3 of 7 SWEET genes of Arabidopsis thaliana are induced when plant cells are colonized with bacterial strains deficient in type III secretion system. This suggests that bacterial effectors may regulate plant SWEET genes and thus may control glucose efflux towards plant pathogenic bacteria [37]. Interestingly, orthologous SWEET genes of poplar are also induced during ECM development (Brun, Kohler and Martin unpublished results; [32]). It is then tempting to postulate that not yet identified effectors from ECM fungi may play an active role in controlling symbiosis-related glucose efflux.
Do effector-like proteins exist in mututalistic fungi? Biotrophic fungal–plant associations are probably controlled by microbial effectors that may reprogram plant defence responses and cell metabolism to deliver nutrients [38]. Genome sequencing and ‘secretome’ analysis through proteomics enabled large-scale identification of candidate effectors in biotrophic fungi [39]. The L. bicolor and T. melanosporum genomes also display a set of small secreted proteins [7,8]. A dozen of SSPs from L. bicolor are specifically expressed in symbiotic tissues constituting candidate effectors or MiSSP (Mycorrhiza induced Small Secreted Proteins) and their role are currently being investigated. MiSSP7 is imported into the plant cell via endocytosis and migrated to the nucleus altering the transcriptome of the plant cell [40]. This protein is required for the establishment of symbiosis [40]. In addition, Requena et al. showed that an effector-like protein SP7 secreted by the arbuscular mycorrhizal fungus G. intraradices interacts with an ERF pathogenesis-related transcription factor in the plant nucleus [41]. The presence of effector-like proteins thus appears to be important for both pathogenic and mutualistic plant-fungus interactions. The effectors encoded by plant-pathogenic fungi rapidly evolve through diversifying selection [42]. Population genomics based on next-generation sequencing technologies will allow identification of genes under diversifying selection as well as copy number variation (CNV) [43] in the L. bicolor and the T. melanosporum genomes and might help in identifying genes important for symbiosis. In addition to effector-like proteins, mutualistic fungi may use other compounds to dialogue with their host plants, as it has been very recently shown that fungal lipochitooligosaccharide stimulate root branching and arbuscular mycorrhiza formation [44]. Genome-wide analysis of transcription factors of T. melanosporum and functional Current Opinion in Plant Biology 2011, 14:444–450
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analysis using heterologous yeast system point to the use of specific transcription factors during ECM formation [45].
Future prospects The dramatic increase in the available genomes of plantassociated fungi has led comparative and population genomics to the fore-front of fungal biology. Recent studies shed light on the power of such analysis in order to identify new important genetic determinants for fungal pathogenicity (transfer of host specificity through an additional chromosome in Fusarium [46]; identification of new virulence factors when compared the genomes of two smut fungi [42]). The use of such tools for ECM fungi should help to decipher the different molecular toolboxes that allowed symbiosis to evolve. In forest ecosystems, communities of saprophytic, mutualistic and pathogenic fungi are entangled and are major players in plant health. In addition to species diversity, intraspecific diversity also contributes to ecosystem functioning [47]. For the next few years, the most challenging goal in ECM biology and ecology will be to bridge ‘omics’ studies to ecosystem functioning [48] (Figure 1). Resequencing of several L. bicolor and T. melanosporum strains from different geographical accessions and taxonomically related species using Illumina technology will facilitate the identification of adaptive traits important for ECM symbiosis as well as intraspecific diversity in symbiosis gene networks. In addition, genomic and transcriptomic strategies have started to provide new markers to study ECM fungi in situ, for example, the cold-responsive genes in T. melanosporum [49] and nutrients transporters in L. bicolor [50]. Convergent evolution between mutualistic fungi and obligate plant pathogenic fungi is striking and consolidate the idea, previously discussed by Jones and Smith [51] that ECM fungi fall within the saprotrophism–mutualism– parasitism continuum. The availability of tools for genetic transformation of L. bicolor [52] will help in discovering fungal determinants required for symbiosis establishment, as well as one that might have allowed the transition from pathogenicity to symbiosis and/or transition from saprotrophism (brown-rot fungi) to the ECM life-style.
Acknowledgements We would like to thank the members of the ‘Ecogenomics of Interactions’ Laboratory for fruitful discussions. Research in our laboratory is supported by the European Commission within the Project ENERGYPOPLAR (FP7211917), the US Department of Energy – Oak Ridge National Laboratory Scientific Focus Area for Genomics Foundational Sciences (Project Plant– Microbe Interfaces) and the ANR TRANSMUT.
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36. Ceccaroli P, Buffalini M, Saltarelli R, Barbieri E, Polidori E, Ottonello S, Kohler A, Tisserant E, Martin F, Stocchi V: Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum. New Phytol 2011, 189:751-764. 37. Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, Guo WJ, Kim JG, Underwood W, Chaudhuri B et al.: Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 2010, 468:527-532. This paper describes the identification of new plant glucose transporters, so-called SWEET, involved in glucose efflux. Some of these transporters are co-opted by bacterial or fungal pathogens and bacterial symbionts probably to supply nutrients. In addition, type-III bacterial effectors control induction of these transporter genes indicating that effectors may manipulate plant transport and metabolism. 38. Stergiopoulos I, de Wit PJ: Fungal effector proteins. Annu Rev Phytopathol 2009, 47:233-263. 39. Choi J, Park J, Kim D, Jung K, Kang S, Lee YH: Fungal secretome database: integrated platform for annotation of fungal secretomes. BMC Genomics 2010, 11:105-120. 40. Kohler A, Plett JM, Tisserant E, Kemppainen M, Legue´ V, VeneaultFourrey C, Brun A, Pardo AG, Martin F: The impact of the mycorrhizal symbiosis on the transcriptome of Laccaria bicolor and Poplar. Fungal Genet Rep 2011, 58:62. 41. Requena N, Kloppholz S, Kuhn H: Root sweet home: signaling in the arbuscular mycorrhizal symbiosis. Fungal Genet Rep 2011, 58:36. 42. Schirawski J, Mannhaupt G, Munch K, Brefort T, Schipper K, Doehlemann G, Di Stasio M, Rossel N, Mendoza-Mendoza A, Current Opinion in Plant Biology 2011, 14:444–450
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Pester D et al.: Pathogenicity determinants in smut fungi revealed by genome comparison. Science 2010, 330:1546-1548. Comparative genomics of two closely related species Sporisorum reilianum and Ustilago maydis lead to the identification of new virulence genes based on their high divergence when compared to the rest of the genomes. These new effectors are mostly located in small clusters and deletion of these clusters had confirmed their involvement in plant infection. This study highlights the value of next-generation sequencing-based comparative genomics in the study of plant-microbe interactions. 43. Cheeseman IH, Gomez-Escobar N, Carret CK, Ivens A, Stewart LB, Tetteh KK, Conway DJ: Gene copy number variation throughout the Plasmodium falciparum genome. BMC Genomics 2009, 10:353. 44. Maillet F, Poinsot V, Andre O, Puech-Pages V, Haouy A, Gueunier M, Cromer L, Giraudet D, Formey D, Niebel A et al.: Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 2011, 469:58-63. The first biochemical characterization of Myc factors leading to arbuscular symbiosis. Glomus intraradices secretes diffusible symbiotic signals – a mixture of sulfated and non-sulfated lippochitooligosaccharides – that induce arbuscular symbiosis in a wide range of species and that share structural similarities with rhizobial Nod factors. 45. Montanini B, Levati E, Bolchi A, Kohler A, Morin E, Tisserant E, Martin F, Ottonello S: Genome-wide search and functional identification of transcription factors in the mycorrhizal fungus Tuber melanosporum. New Phytol 2011, 189:736-750.
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46. Ma LJ, van der Does HC, Borkovich KA, Coleman JJ, Daboussi MJ, Di Pietro A, Dufresne M, Freitag M, Grabherr M, Henrissat B et al.: Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 2010, 464:367-373. 47. Wilkinson A, Solan M, Taylor AF, Alexander IJ, Johnson D: Intraspecific diversity regulates fungal productivity and respiration. PLoS ONE 2010, 5:e12604. 48. Whitham TG, Difazio SP, Schweitzer JA, Shuster SM, Allan GJ, Bailey JK, Woolbright SA: Extending genomics to natural communities and ecosystems. Science 2008, 320:492-495. 49. Zampieri E, Balestrini R, Kohler A, Abba S, Martin F, Bonfante P: The Perigord black truffle responds to cold temperature with an extensive reprogramming of its transcriptional activity. Fungal Genet Biol 2011, 48:85-91. 50. Lucic E, Fourrey C, Kohler A, Martin F, Chalot M, Brun-Jacob A: A gene repertoire for nitrogen transporters in Laccaria bicolor. New Phytol 2008, 180:343-364. 51. Jones MD, Smith SE: Exploring functional definitions of mycorrhizas: are mycorrhizas always mutualisms? Can J Bot 2004, 82:1089-1109. 52. Kemppainen MJ, Pardo AG: pHg/pSILBAgamma vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA-triggering in the mycorrhizal fungus Laccaria bicolor. Microb Biotechnol 2010, 3:178-200.
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Mutualistic interactions on a knife-edge between saprotrophy and pathogenesis Claire Veneault-Fourrey and Francis Martin Saprophytic, ectomycorrhizal (ECM) and pathogenic fungi play a key role in carbon and nutrient cycling in forest ecosystems. Whereas more than 50 genomes of saprotrophic and pathogenic fungi have been published, only two genomes of ECM fungi, Laccaria bicolor and Tuber melanosporum, have been released. Comparative analysis of the genomes of biotrophic species highlighted convergent evolution. Mutualistic and pathogenic biotrophic fungi share expansion of genome size through transposon proliferation and common strategies to avoid plant detection. Differences mainly rely on nutritional strategies. Such analyses also pinpointed how blurred the molecular boundaries are between saprotrophism, symbiosis and pathogenesis. Sequencing of additional ECM species, as well as soil saprotrophic fungi, will facilitate the identification of conserved traits for ECM symbiosis and those leading to the transition from white-rotting and brown-rotting to the ECM lifestyle. Address UMR 1136 INRA-Nancy Universite´ « Tree-Microorganisms Interactions », Ecogenomics of Interactions, Centre INRA de Nancy, 54280 Champenoux, France Corresponding author: Martin, Francis ([email protected])
Current Opinion in Plant Biology 2011, 14:444–450 This review comes from a themed issue on Biotic interactions Edited by Giles Oldroyd and Silke Robatzek Available online 27th April 2011 1369-5266/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2011.03.022
Introduction As they display a wide-range of nutritional strategies, fungi are ubiquitous and able to live in almost all ecological niches. As a consequence of this ubiquity, more and more fungal genomes have been sequenced with an emphasis on questions important to biomedical and bioenergy-related research [1,2] as well as basic science [3]. Comparative genomics combined with systems biology has an enormous potential to improve our understanding of fungal biology as well as fungal–biotic environmental interactions [4]. In natural forest ecosystems characterized by mineral nitrogen and phosphate limitation, ectomycorrhizal Current Opinion in Plant Biology 2011, 14:444–450
(ECM) symbioses play a crucial role in plant nutrient acquisition and hence increase growth of the host-plant [5]. ECM-forming fungi play a key role in belowground biosequestration of carbon, mineral mobilization and organic nutrients degradation. A better understanding of the mycobiont’s role in ECM development and functioning appears to be crucial in ecology for durable forest management and to improve tree productivity. Interestingly, fungal ECM symbionts share their biotrophic lifestyle with obligate fungal plant pathogens, as the latter infect plant cells and feed on them but need to leave plant cells alive. The biotrophic life style is polyphyletic as it appeared several times across evolution [6]. To date, the genome sequencing of two non-phylogenetically related ECM-fungi, Laccaria bicolor (Basidiomycotina, Agaricomycotina, Agaricales, Hydnangiaceae) and Tuber melanosporum (Ascomycotina, Pezizomycotina, Pezizales, Tuberaceae), and their transcriptome profiling [7,8] have raised ectomycorrhizae as a model to study the evolution of eukaryotic symbiosis. RNA-seq technologies have been useful tools to improve their genome annotations and identify post-transcriptional mechanisms as well as aiding in generating a blue-print of the genes expressed during symbiosis [9,10]. With the recent release of hundreds of fungal genomes as well as the development of high-throughput functional analysis, we have an unprecedented opportunity to decipher molecular mechanisms driving ECM-symbiosis and, more generally, biotrophic life style as well as its evolution [11,12]. Based on comparative genomic analysis, we will firstly discuss convergent evolution between biotrophic fungi (mutualistic or pathogenic) through the expansion of genome size and the strategies to avoid triggering of plant defence responses. Secondly, we will discuss the crucial role of nutrient exchanges in the outcome of the interaction and finally, how biotrophic fungi could control the interaction with their host-plant. In this article, we speculate that saprotrophism, symbiosis and pathogenesis may be view as a continuum (Figure 1).
Genome expansion The genomes of the ECM-fungi L. bicolor and T. melanosporum are among the largest in size of fungal sequenced genomes to date, with 65-Mb and 125-Mb respectively [7,8]. Genome-size expansion is largely explained by a proliferation of repeated elements, primarily composed of transposable elements (TE) or the accumulation of their degraded remnants. They represent about 38% and 58% of L. bicolor and T. melanosporum genomes, respectively, whereas most fungal species contain 1 to 15% repetitive DNA in their genomes. www.sciencedirect.com
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Figure 1
PREDICTING CHANGES WITHIN COMMUNITIES
Functional markers to study ECM in situ
FUNGAL LIFE-STYLES: Identification of candidate genes for FUNCTIONAL ANALYSIS.
COMPARATIVE GENOMICS: - Genome evolution - Genome architecture - Molecular signatures of evolution - Metabolic and regulatory pathways White-rot
ECOSYSTEM METADATA: - Geochemical and physical parameters - Biological functions
Brown-rot
POPULATION GENOMICS: - Adaptation signatures of ECM fungi in various ecosystems and environmental conditions - Genotypic polymorphism
Biotrophic
Necrotrophic
Saprotrophic fungi
ECM fungi
Pathogenic fungi
> 30 genome
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Harnessing ‘omics’ for understanding the ecology of ECM fungi. Understanding how saprotrophic, symbiotic and pathogenic fungi achieve their lifestyle is crucial to understand their ecological functions, and their subsequent impact on the fate of plant communities. Several hundred species are active in soils and on-going genomics, metagenomics and metatranscriptomics studies will uncover the functions encoded in their genomes as well as their expressed transcripts. These sequenced genomes will provide baseline genomic information that enables scientists to investigate the interactions between fungal and plant communities.
Genome-size expansion has also been recently observed for obligate pathogens, such as powdery mildew fungi [13], rust fungi (Duplessis et al., unpublished) and the oomycete Hyaloperonospora arabidopsidis [14]. Therefore, biotrophy seems to be associated with convergent genome-size expansion owing to a proliferation of TEs. This may account for genome instability [15]. Comparative analyses of well-assembled and annotated fungal genomes lead to the idea that genome architecture is composed of syntenic regions containing a high-density of conserved genes and constituting the vast majority of the ‘core genome’, separated by non-syntenic regions containing a few genes and prone to rearrangements [3,16,17]. These gene sparse regions with high plasticity contain novel genes or duplicated paralogous genes [3] or clade-specific genes [18] probably required in adaptation to a given environment (variable traits). On the contrary, genomes of ECM and obligate fungal pathogens display uniform distribution of TEs across genome [7,8,13, Duplessis et al., unpublished], suggesting that genome evolution is shaped by other still unknown mechanisms in the biotrophic life-style. www.sciencedirect.com
In addition to transposon expansion, the L. bicolor genome displays expansion of multigene families as well as lineagespecific multigene families with a total of 19,102 proteinencoding genes, whereas the T. melanosporum genome displays only 7496 protein-encoding genes [7,8]. These strikingly different gene repertoires suggest that expansion of multigene families is not mandatory for symbiosis. Gene family evolution and gene duplication have been suggested to be important processes in the generation of evolutionary novelty through neofunctionalization and/or subfunctionalization. In the L. bicolor genome, striking gene family expansions occurred in genes predicted to have roles in protein-protein interactions (e.g. NACHT and ankyrin repeats) and signal transduction mechanisms (Tyrosine-kinase like, RAS small GTPase and WD40) [19,20]. Recently, Meng and Li showed that a viral capsid protein contains a functional nuclear localization signal ‘KRKR’, similar to a domain found in an alpha subunit of guanine nucleotide binding protein from L. bicolor [21]. If this nuclear localization signal is functional in L. bicolor, it might highlight a new mechanism of action for G-protein. In addition, several of these lineage-specific proteins are Current Opinion in Plant Biology 2011, 14:444–450
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upregulated during symbiosis (WD40 domain, G-beta repeat; tyrosine-kinase like; NB-ARC domain, NACHT domain), suggesting a role for these proteins in signaling pathways leading to symbiosis. In particular, recent findings indicate the WD-40 domains may act as hubs in cellular networks as they may interact with diverse proteins, peptides or nucleic acids [22]. Rust fungi that need two different hosts to complete their entire life-style, have genomes that display expansion of lineage-specific genes (Duplessis et al., unpublished) such as the alphakinase family as well as some specific nutrient transporters. Restriction of gene duplication is observed in both T. melanosporum and B. graminis genomes [8,13]. Cuomo et al. [23] have suggested that fungal species lacking the opportunity for gene innovation via duplication may be more constrained in adaptation to changing environments. Absence of gene duplication may thus be view as a hallmark of restricted host specificity within Ascomycetes as both T. melanosporum and B. graminis have narrow host specificity, whereas L. bicolor is a basidiomycete and a generalist mutualistic symbiont able to interact with seedlings and older trees from conifers and hardwood species. Lineage-specific multigene families may contain genes important for fungal adaptation to different ecological niches and may aid in broadening host range/specificity. Recently, Vinces et al. [24] showed that mutations in tandem repeats found in promoters might affect gene expression in yeast allowing rapid response to changing environment. T. melanosporum that is lacking in gene duplication contains the vast majority of its SSR within non-coding regions [25]. It will be interesting to test whether similar mechanisms could regulate T. melanosporum gene expression.
Avoiding plant defence responses Mutualistic and pathogenic biotrophic fungi must have evolved several strategies to avoid host detection and the triggering of basal plant defence responses. Chitin deacetylation versus sequestration. Surface-exposed chitin can be converted to chitosan through the action of fungal chitin deacetylase (CE-4) to avoid host detection. CE4-encoding genes from L. bicolor, T. melanosporum as well as from two rust fungi are induced during plant association [7,8, Duplessis et al., unpublished]. De Jonge et al., have shown that the effector protein Ecp6 from C. fulvum is able to sequester, through its LysM domains, chitin oligosaccharides released by plant chitinases preventing immunity induction [26]. A LysM protein from T. melanosporum is highly regulated during symbiosis, suggesting that this mycobiont might use chitin sequestration to avoid induction of plant defence responses. Reduction in plant-cell wall (PCW) degrading enzymes. The repertoire of carbohydrate-active enzyme (CAZyme) in L. bicolor and T. melanosporum revealed massive gene losses in the enzymes that target plant cell wall components Current Opinion in Plant Biology 2011, 14:444–450
such as cellulose (glycoside hydrolase (GH) 6 and GH7) and xylan backbones [7,8], suggesting that both ECM fungi are unable to use cellulose and xylan as nutritional sources. A lack of PCW-cleaving enzymes has also been observed in the ECM fungus Amanita bisporigera [27] and in obligate plant-pathogens [13,14]. This indicates that intimate associations with plant cells lead to convergent losses of enzymes acting on plant cell wall either because they are not required for fungal growth or more probably to avoid induction of plant-defence responses and thus achieve colonization. However, one striking difference concerns the presence of a cellulose-binding domain CBM1 in both ECM fungi genomes and its absence in biotrophic obligate fungal pathogen genomes [13,14]. The sole CBM1 module found in L. bicolor is linked to an endoglucanase GH5, whereas the T. melanosporum genome contains 2 CBM1 domains, with one fused to a candidate beta-glycosidase related to endoglucanase GH61. Both GH5 and GH61 might display a weak cellulolytic activity and raise the question of cellulose integrity as a signal in symbiosis. Both ECM fungi still have a residual ability to degrade pectin with different enzymes that might facilitate the progression of fungal hyphae within pectin-rich middle lamella during ECM formation. In addition, T. melanosporum genome contains the hemicellulases GH10 and GH43 that are absent in L. bicolor. Instead, L. bicolor contains 12 expansin-encoding genes, hypothesized to disrupt the non-covalent links between hemicellulose strands on cellulose microfibrils. The diverging enzymatic arsenal in both ECM fungi suggests a divergence in the way they deal with root colonization and suggest that T. melanosporum is more ‘aggressive’ towards its host-plant than L. bicolor. This relative aggressiveness of T. melanosporum is confirmed with a high level of expression of tyrosinases, lipases and laccases in planta [8]. In addition, an orthologue of a high-affinity cellodextrin transporter of T. melanosporum is induced in ECM [8]. In the saprotrophic fungus Neurospora crassa, this transporter is required for rapid growth on cellulose [28] suggesting that T. melanosporum may degrade cellulose during symbiosis functioning. CAZyme repertoires of two brown rot fungi Postia placenta [29] and Serpula lacrymans (Dan Eastwood et al., unpublished) are very similar to that of L. bicolor, with reduction of GH sets as well as losses of lignin-degrading peroxidases. Phylogenetic analyses using Bayesian molecular clock suggest that the ancestor of the Agaricomycotina was not an ECM fungi but probably a saprotrophic fungus [30] and the ancestor of the Agaricomycotina Boletales was probably a fungi with a brown rot nutritional mode (Dan Eastwood et al., unpublished). To explain evolution of fungal nutritional strategy towards ECM, the hypothesis in favour proposes that the ancestor might have been a white rot fungus with a large repertoire of CAZyme [1]. Lost of sets of CAZymes probably occurred at the same time that conifers appeared and led to the apparition of www.sciencedirect.com
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brown rot fungi that had lost efficient enzymatic machinery for lignin depolymerization and were unable to degrade cellulose using CAZymes. Loss of degrading enzymes in the brown rot ancestor might have allowed association between fungi and root cells without damaging the latter. In this context, the project to sequence 25 new mycorrrhizal genomes as well as 32 genomes of saprotrophic fungi will be of great interest and should highlight how symbiosis has evolved from saprotrophic ancestors (Figure 1) [12].
Nutrient exchanges The evolution towards mycorrhizal symbiosis did not lead to the loss of gene families involved in primary carbon or nitrogen metabolism [29,30], contrary to what was observed for obligate plant pathogens that lost the nitrate and/or sulfate assimilation ability ([13,14], Duplessis et al., unpublished). However, biotrophy seems to be associated with losses of secondary metabolism key genes [8,13]. Interestingly, silenced nitrate reductase L. bicolor strains are impaired in mycorrhization ability [31], suggesting that symbiotic interaction does not occur when the fungus does not supply nitrogen compounds to the plant. It is thus tempting to speculate that obligate pathogenic fungi might rely on their host for nitrogen supply and that ECM biotrophic fungi might depend on their host for carbohydrate supply. Hartig net of ECM, the zone of nutrients exchange between fungal and plant cells, constitute new sinks for photosynthate as ECM can drain up to half of the photosynthetically fixed carbon [32]. During the establishment of beneficial interactions with plants, a highaffinity sucrose transporter from the endophytic fungus Trichoderma virens is induced and sucrose hydrolysis through fungal invertase is required for root colonization [33,34]. Both ECM fungi genomes contain a similar transporter [13,34], whereas no similar transporters have been yet identified in obligate pathogenic fungi [13, Duplessis et al., unpublished], suggesting that obligate pathogenic fungi are unable to transport plant-derived sucrose. However, the latter contain at least one invertase with the expression induced during infection [13], whereas for most Basidiomycota ECM fungi including L. bicolor, no invertase gene was found in their genomes [35]. By contrast, the Ascomycotina ECM T. melanosporum genome contains one invertase encoding gene, suggesting that T. melanosporum is able to utilize plantderived sucrose during symbiosis [36]. These results suggest that both ECM fungi are able to transport plant-derived sucrose but have distinct ability towards its hydrolysis, T. melanosporum being closer to obligate pathogen in this perspective. These results also suggest that L. bicolor is more dependent on its host for carbohydrate supply than T. melanosporum. We can then speculate that L. bicolor that has a low ability to obtain carbohydrates from the surrounding soil [7] has to find www.sciencedirect.com
plant roots for a carbohydrate supply and consequently should establish ECM with a wide range of hosts. SWEET genes, recently identified in plants as glucose uniporters (importers or exporters) are differentially expressed in plant cells colonized with pathogenic bacteria or fungi [37]. In addition, only 3 of 7 SWEET genes of Arabidopsis thaliana are induced when plant cells are colonized with bacterial strains deficient in type III secretion system. This suggests that bacterial effectors may regulate plant SWEET genes and thus may control glucose efflux towards plant pathogenic bacteria [37]. Interestingly, orthologous SWEET genes of poplar are also induced during ECM development (Brun, Kohler and Martin unpublished results; [32]). It is then tempting to postulate that not yet identified effectors from ECM fungi may play an active role in controlling symbiosis-related glucose efflux.
Do effector-like proteins exist in mututalistic fungi? Biotrophic fungal–plant associations are probably controlled by microbial effectors that may reprogram plant defence responses and cell metabolism to deliver nutrients [38]. Genome sequencing and ‘secretome’ analysis through proteomics enabled large-scale identification of candidate effectors in biotrophic fungi [39]. The L. bicolor and T. melanosporum genomes also display a set of small secreted proteins [7,8]. A dozen of SSPs from L. bicolor are specifically expressed in symbiotic tissues constituting candidate effectors or MiSSP (Mycorrhiza induced Small Secreted Proteins) and their role are currently being investigated. MiSSP7 is imported into the plant cell via endocytosis and migrated to the nucleus altering the transcriptome of the plant cell [40]. This protein is required for the establishment of symbiosis [40]. In addition, Requena et al. showed that an effector-like protein SP7 secreted by the arbuscular mycorrhizal fungus G. intraradices interacts with an ERF pathogenesis-related transcription factor in the plant nucleus [41]. The presence of effector-like proteins thus appears to be important for both pathogenic and mutualistic plant-fungus interactions. The effectors encoded by plant-pathogenic fungi rapidly evolve through diversifying selection [42]. Population genomics based on next-generation sequencing technologies will allow identification of genes under diversifying selection as well as copy number variation (CNV) [43] in the L. bicolor and the T. melanosporum genomes and might help in identifying genes important for symbiosis. In addition to effector-like proteins, mutualistic fungi may use other compounds to dialogue with their host plants, as it has been very recently shown that fungal lipochitooligosaccharide stimulate root branching and arbuscular mycorrhiza formation [44]. Genome-wide analysis of transcription factors of T. melanosporum and functional Current Opinion in Plant Biology 2011, 14:444–450
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analysis using heterologous yeast system point to the use of specific transcription factors during ECM formation [45].
Future prospects The dramatic increase in the available genomes of plantassociated fungi has led comparative and population genomics to the fore-front of fungal biology. Recent studies shed light on the power of such analysis in order to identify new important genetic determinants for fungal pathogenicity (transfer of host specificity through an additional chromosome in Fusarium [46]; identification of new virulence factors when compared the genomes of two smut fungi [42]). The use of such tools for ECM fungi should help to decipher the different molecular toolboxes that allowed symbiosis to evolve. In forest ecosystems, communities of saprophytic, mutualistic and pathogenic fungi are entangled and are major players in plant health. In addition to species diversity, intraspecific diversity also contributes to ecosystem functioning [47]. For the next few years, the most challenging goal in ECM biology and ecology will be to bridge ‘omics’ studies to ecosystem functioning [48] (Figure 1). Resequencing of several L. bicolor and T. melanosporum strains from different geographical accessions and taxonomically related species using Illumina technology will facilitate the identification of adaptive traits important for ECM symbiosis as well as intraspecific diversity in symbiosis gene networks. In addition, genomic and transcriptomic strategies have started to provide new markers to study ECM fungi in situ, for example, the cold-responsive genes in T. melanosporum [49] and nutrients transporters in L. bicolor [50]. Convergent evolution between mutualistic fungi and obligate plant pathogenic fungi is striking and consolidate the idea, previously discussed by Jones and Smith [51] that ECM fungi fall within the saprotrophism–mutualism– parasitism continuum. The availability of tools for genetic transformation of L. bicolor [52] will help in discovering fungal determinants required for symbiosis establishment, as well as one that might have allowed the transition from pathogenicity to symbiosis and/or transition from saprotrophism (brown-rot fungi) to the ECM life-style.
Acknowledgements We would like to thank the members of the ‘Ecogenomics of Interactions’ Laboratory for fruitful discussions. Research in our laboratory is supported by the European Commission within the Project ENERGYPOPLAR (FP7211917), the US Department of Energy – Oak Ridge National Laboratory Scientific Focus Area for Genomics Foundational Sciences (Project Plant– Microbe Interfaces) and the ANR TRANSMUT.
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36. Ceccaroli P, Buffalini M, Saltarelli R, Barbieri E, Polidori E, Ottonello S, Kohler A, Tisserant E, Martin F, Stocchi V: Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum. New Phytol 2011, 189:751-764. 37. Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, Guo WJ, Kim JG, Underwood W, Chaudhuri B et al.: Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 2010, 468:527-532. This paper describes the identification of new plant glucose transporters, so-called SWEET, involved in glucose efflux. Some of these transporters are co-opted by bacterial or fungal pathogens and bacterial symbionts probably to supply nutrients. In addition, type-III bacterial effectors control induction of these transporter genes indicating that effectors may manipulate plant transport and metabolism. 38. Stergiopoulos I, de Wit PJ: Fungal effector proteins. Annu Rev Phytopathol 2009, 47:233-263. 39. Choi J, Park J, Kim D, Jung K, Kang S, Lee YH: Fungal secretome database: integrated platform for annotation of fungal secretomes. BMC Genomics 2010, 11:105-120. 40. Kohler A, Plett JM, Tisserant E, Kemppainen M, Legue´ V, VeneaultFourrey C, Brun A, Pardo AG, Martin F: The impact of the mycorrhizal symbiosis on the transcriptome of Laccaria bicolor and Poplar. Fungal Genet Rep 2011, 58:62. 41. Requena N, Kloppholz S, Kuhn H: Root sweet home: signaling in the arbuscular mycorrhizal symbiosis. Fungal Genet Rep 2011, 58:36. 42. Schirawski J, Mannhaupt G, Munch K, Brefort T, Schipper K, Doehlemann G, Di Stasio M, Rossel N, Mendoza-Mendoza A, Current Opinion in Plant Biology 2011, 14:444–450
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