Lectures 19 and 20: Mycorrhizas –

Reading: Text, Chapter 17. ALSO great website from CSIRO in Austalia http://www.ffp.csiro.au/research/mycorrhiza/index.html http://mycorrhizas.info/index.html

Rodriguez R and Redman R. 2008. More than 400 years of evolution and some plants still canʼt make it on their own. Journal of Experimental Botany 59: 1109-1114.

Parniske M. 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews Microbiology 6: 763-773.

Stinson, K.A. et al. 2006. Invasive plant suppresses the growth of native tree seedlings by disrupting belowground mutualisms. PLOS Biology

Objectives: Understand the importance of mycorrhizas. Know how to recognize ectomycorrhizas, and the types of endomycorrhizas. Know how to prove that a mycorrhizal symbiosis has been formed (differentiating between what you expect with ecto- vs. endomycorrhizas). Know which groups of fungi (phyla, orders, families) form ectomycorrhizas, which form endomycorrhizas.

Keywords: ectomycorrhiza, mantle, Hartig net, endomycorrhiza, vesicle, arbuscule, spore, Glomeromycota.

Study questions: 1) What are the soil and root-associated structures found with VAM? With orchid mycorrhizas? Compare and contrast the structures of VAM, orchid mycorrhizas, and ECMs. Which fungal phyla form each of the three types of mycorrhizas that we have discussed?

2) Are VAM common in plant roots? Explain.

3) VAM appears to be a balanced mutualism – is this true?

4) You wish to prove that Russula mississaugii is a mycorrhizal fungus. How could you convince our class? Use your naked eye, a dissecting microscope, a compound microscope, and radio-isotope study of tree seedlings to prove your point! What are the controls?

5) What are the benefits to the plant and to the ecosystem of mycorrhizas?

6) Why do plant species that depend on mycorrhizas tend to lack root hairs?

7) Garlic mustard is very common around Mississauga and on this campus. Despite the wet weather this year, mushroom biomass and diversity was not high on campus. Also, young tree seedlings may not be competing well to restore the tree canopy as older trees die. Explain a possible role for garlic mustard and speculate on additional factors behind these observations.

8) What are myco-heterotrophs and why are they “cheaters”?

9) What do we mean when we say that mycorrhizas are “dynamic” and form “nutrient networks”?

10) What is Parniskeʼs theory about VAM as the “mother of plant root endosymbioses”? There are two types of evidence – what are they?

PLoS BIOLOGY Invasive Plant Suppresses the Growth of Native Tree Seedlings by Disrupting Belowground Mutualisms

Kristina A. Stinson1, Stuart A. Campbell2, Jeff R. Powell2, Benjamin E. Wolfe2, Ragan M. Callaway3, Giles C. Thelen3, Steven G. Hallett4, Daniel Prati5, John N. Klironomos2* 1 Harvard Forest, Harvard University, Petersham, Massachusetts, United States of America, 2 Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada, 3 Division of Biological Sciences, University of Montana, Missoula, Montana, United States of America, 4 Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, United States of America, 5 Department of Community Ecology, UFZ Centre for Environmental Research, Halle, Germany

The impact of exotic species on native organisms is widely acknowledged, but poorly understood. Very few studies have empirically investigated how invading plants may alter delicate ecological interactions among resident species in the invaded range. We present novel evidence that antifungal phytochemistry of the invasive plant, Alliaria petiolata, a European invader of North American forests, suppresses native plant growth by disrupting mutualistic associations between native canopy tree seedlings and belowground arbuscular mycorrhizal fungi. Our results elucidate an indirect mechanism by which invasive plants can impact native flora, and may help explain how this plant successfully invades relatively undisturbed forest habitat.

Citation: Stinson KA, Campbell SA, Powell JR, Wolfe BE, Callaway RM, et al. (2006) Invasive plant suppresses the growth of native tree seedlings by disrupting belowground mutualisms. PLoS Biol 4(5): e140. DOI: 10.1371/journal.pbio.0040140

Introduction plants, can be negatively affected by AMF [14–16]. Natural- ized exotic plants have been found to be poorer hosts and Widespread anthropogenic dispersal of exotic organisms depend less on native AMF than native plants [17]. They often has raised growing concern over their devastating ecological colonize areas that have been disturbed [2], and disturbances impacts, and has prompted decades of research on the to soil have been shown to negatively impact AMF function- ecology of invasive species [1–3]. Exotic plants may become ing [18]. Furthermore, it has been proposed that the aggressive invaders outside their home ranges for a number proliferation of plants with low mycorrhizal dependency of reasons, including release from native, specialized antag- may degrade AMF densities in the soil [17]. However, a few onists [4], higher relative performance in a new site [5], direct invasive plants proliferate in the understory of mature chemical (allelopathic) interference with native plant per- temperate forests [2], where AMF density is typically high formance [6], and variability in the responses and resistance [19]. The existing mycelial network in mature forest soils may of native systems to invasion [7,8]. Thus, successful invasion in facilitate the establishment of exotic, mycorrhizal-dependent, many cases appears to involve the fact that invasive species recruits [20,21], but this should not be the case for non- are not at equilibrium, and are either freed of long-standing mycorrhizal invaders. If non-mycorrhizal invasive plants biotic interactions with their enemies in the home range, and/ establish and degrade AMF in mature forests, then the effects or disrupt interactions among the suite of native organisms on certain resident native plants could be substantial. they encounter in a new range [9]. Nevertheless, experimental One of the most problematic invaders of mesic temperate data on species-level impacts of exotic plants are still limited forests in North America is Alliaria petiolata (garlic mustard; [10]. One particularly understudied area is the potential for Brassicaceae), a non-mycorrhizal, shade-tolerant, Eurasian invasive plants to disrupt existing ecological associations biennial herb which, like most other mustards, primarily occupies disturbed areas. Garlic mustard is abundant in within native communities [6,10]. Many exotic and native forest edges, semishaded floodplains, and other disturbed plants alike depend upon mutualisms with native insects, sites in its home range [22]. However, this species has recently birds, or mammals for pollination and seed dispersal [11], and become an aggressive and widespread invader of both with soil microbes for symbiotic nutrient exchange [12]. Thus, when an introduced species encounters a new suite of resident organisms, it is likely to alter closely interlinked Academic Editor: Michel Loreau, McGill University, Canada ecological relationships, many of which have co-evolved Received December 5, 2005; Accepted March 1, 2006; Published April 25, 2006 within native systems [6,11]. DOI: 10.1371/journal.pbio.0040140 One such relationship is that between plants and mycor- Copyright: Ó 2006 Stinson et al. This is an open-access article distributed under rhizal fungi [12]. Most vascular plants form mycorrhizal the terms of the Creative Commons Attribution License, which permits unrestricted associations with arbuscular mycorrhizal fungi (AMF) [12], use, distribution, and reproduction in any medium, provided the original author and many plants are highly dependent on this association for and source are credited. their growth and survival [12], particularly woody perennials Abbreviations: AMF, arbuscular mycorrhizal fungi; ANOVA, analysis of variance; and others found in late-successional communities [13]. In REGW, Ryan-Einot-Gabriel-Welsch contrast, many weedy plants, in particular non-mycotrophic * To whom correspondence should be addressed. E-mail: [email protected]

PLoS Biology | www.plosbiology.org 0727 May 2006 | Volume 4 | Issue 5 | e140 Invasive Plant Disrupts Mycorrhizas disturbed areas and closed-canopy forest understory across much of the United States and Canada [23], where it apparently suppresses native understory plants, including the seedlings of dominant canopy trees [22,24]. The mecha- nism underlying garlic mustard’s unusual capacity to enter and proliferate within intact North American forest com- munity has not yet been established. As shown in recent greenhouse experiments, garlic mustard’s impact on native understory flora may involve competitive [25] or allelopathic effects on native plants [26], but it has also been hypothesized that this species interferes with plant–AMF interactions in its invaded range [27]. Members of the Brassicaceae, including garlic mustard, produce various combinations of glucosinolate products [28], organic plant chemicals with known anti-herbivore, anti-pathogenic and allelopathic [29] properties, that may also prevent this non-mycorrhizal plant family from associat- ing with AMF [30]. These phytochemicals may be released into soils as root exudates, as a result of damaged root tissue, or in the form of leaf litter. High densities of garlic mustard in the field correlate with low inoculum potential of AMF, and extracts of garlic mustard leaves have been shown to reduce the germination of AMF spores and impair AMF Figure 1. Experiment 1 colonization of cultivated tomato roots in laboratory settings The influence of field soils that were invaded or uninvaded by Al. [27]. Although not all Brassicaceae are invasive, it is possible petiolata (6 sterilized) on (A) mycorrhizal colonization (Fsugar maple¼ 77.7, that garlic mustard’s successful invasion of understory df ¼ 3,39, p , 0.001; Fred maple ¼ 60.5, df ¼ 3,39, p , 0.001; and Fwhite ash ¼ habitats involves the negative effects of its phytochemistry 116.6, df ¼ 3,39, p , 0.001) and (B) biomass accumulation (Fsugar maple ¼ 57.8, df ¼ 3,39, p , 0.001; F ¼ 61.4, df ¼ 3,39, p , 0.001; and F on the native plant and AMF species it encounters outside its red maple white ash ¼ 70.1, df ¼ 3,39, p , 0.001) of native tree seedlings. Bars represent home range. Others have shown that exotic plants can recruit the mean and standard error. different suites of microbial organisms in their new ranges DOI: 10.1371/journal.pbio.0040140.g001 that can be antagonistic to native plants [6]. However, to our knowledge, no previous studies have directly tested whether species is microbially-mediated, and not the result of soil this species or any other exotic plant disrupts native plant– differences or direct allelopathy. AMF mutualisms within natural communities. Here, we We then conducted additional experiments to confirm that present novel evidence that garlic mustard negatively impacts garlic mustard specifically caused AMF decline in the native the growth of AMF-dependent forest tree seedlings by its soils (Experiment 2–4). We grew seedlings of the same three disruption of native mycorrhizal mutualisms. We further native tree species used in Experiment 1 in uninvaded forest show that, because seedlings of dominant tree species in soils that were conditioned for 3 mo with either garlic mature forest communities are more highly dependent on mustard plants or with one of the three native tree species. AMF than plants that typically dominate earlier successional All three tree species demonstrated significantly lower AMF communities, garlic mustard invasion may disproportionately colonization in soils conditioned by Al. petiolata (0%–10%) damage mature forests relative to other habitats. than in soils conditioned by the native plants (20%–65%; Figure 2A). AMF colonization was similar in unconditioned Results/Discussion (control) soils and soils conditioned with native plants. In addition, growth of the tree seedlings was the lowest in soils We first tested whether native tree seedlings were less able conditioned by garlic mustard (Figure 2B), confirming that to form mycorrhizal associations when grown in forest garlic mustard plants reduce native plant performance by understory soils with a history of garlic mustard invasion interfering with the formation of mycorrhizal associations. than when grown in soils that had not experienced invasions We investigated whether there is a phytochemical basis to (Experiment 1). We found that dominant native hardwood garlic mustard’s observed antifungal effects on AMF in tree species of northeastern temperate forests, Acer saccharum Experiments 3–4. In an earlier study, Vaughn and Berhow (sugar maple), Ac. rubrum (red maple), and Faxinus americana [31] isolated the phytotoxic glucosinolate hydrolysis products (white ash), showed significantly less AMF colonization of allyl isothiocyanate, benzyl isothiocyanate, and glucotropaeo- roots (Figure 1A) and slower growth (Figure 1B) when grown lin from extracts of Al. petiolata root tissues and found in soil that had been invaded by garlic mustard. AMF evidence for their allelopathic effects on certain plants in the colonization was almost undetectable in soil that had been absence of mycorrhizas. These phytochemicals could have invaded by garlic mustard. These reductions were similar to direct effects on plant growth through allelopathy as well as those observed when seedlings were grown in sterilized soil indirect effects via disruption of AMF. To experimentally from both garlic mustard–invaded and garlic mustard–free establish that garlic mustard’s effect on AMF is phytochemi- sites (Figure 1B), strongly suggesting that the mechanism by cally based, we grew native tree seedlings on uninvaded soils which garlic mustard suppresses the growth of native tree to which we added individual aqueous extracts of garlic

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Figure 2. Experiment 2 The effect of soils conditioned with garlic mustard Al. petiolata (gm), sugar maple (sm), red maple (rm), or white ash (wa) on (A) mycorrhizal colonization (Fsugar maple ¼ 31.2, df ¼ 4,49, p , 0.001; Fred maple ¼ 18.2, df ¼ 4,49, p , 0.001; and Fwhite ash ¼ 22.1, df ¼ 4,49, p , 0.001) and (B) increase in biomass (Fsugar maple ¼ 15.1, df ¼ 4,49, p , 0.001; Fred maple ¼ 18.1, df ¼ 4,49, p , 0.001; and Fwhite ash ¼ 13.2, df ¼ 4,49, p , 0.001) of native tree seedlings. Bars represent the mean and standard error. Figure 3. Experiments 3 and 4 DOI: 10.1371/journal.pbio.0040140.g002 The effects of extract of garlic mustard (gm), sugar maple (sm), red maple (rm), white ash (wa), or a water control on (A) mycorrhizal colonization of mustard or each of the native trees species (Experiment 3). native tree seedlings (Fsugar maple ¼ 20.3, df ¼ 4,49, p , 0.001; Fred maple ¼ 19.8, df ¼ 4,49, p , 0.001; and Fwhite ash ¼ 25.4, df ¼ 4,49, p , 0.001 We found that garlic mustard extract was just as effective as [Experiment 3]), (B) increase in biomass of native tree seedlings (Fsugar the living plant at reducing AMF colonization (Figure 3A) and maple ¼ 11.7, df ¼ 4,49, p , 0.001; Fred maple ¼ 14.2, df ¼ 4,49, p , 0.001; growth (Figure 3B) of the native plants. Moreover, exposing and Fwhite ash ¼ 27.9, df ¼ 4,49, p , 0.001 [Experiment 3]), and (C) percent germination of native AMF spores (F ¼ 17.3, df ¼ 4,49, p , 0.001; AMF spores to extract of garlic mustard severely and Glomus and FAcaulospora ¼ 21.8, df ¼ 4,49, p , 0.001 [Experiment 4]). Bars significantly reduced germination rates of those spores represent the mean and standard error. (Experiment 4; Figure 3C). Collectively, our results clearly DOI: 10.1371/journal.pbio.0040140.g003 demonstrate that garlic mustard, probably through phyto- chemical inhibition, disrupts the formation of mycorrhizal rhizal-dependent tree seedlings than less-mycorrhizal-de- associations. Our results thus reveal a powerful, indirect pendent plants. Thus, garlic mustard’s successful mechanism by which an invasive species can suppress the colonization of understory habitat may be attributed in part growth of native flora. to its ability to indirectly suppress woody competitors, and its Because plants vary in their dependency on AMF [32], effect on the native flora may be more detrimental in intact garlic mustard’s disruption of native plant–fungal mutualisms forests than disturbed sites. In addition, the data suggest that should not inhibit the growth of all plants equally, but rather invasion by garlic mustard may have profound effects on the should correlate strongly with the mycorrhizal dependence of composition of mature forest communities (e.g., by repres- species encountered in the invaded range. Specifically, sing the regeneration of dominant canopy trees, and by courser root production, which impedes the nutrient uptake favoring plants with low mycorrhizal dependency such as of typically slow-growing, woody plants such as tree seedlings, weedy herbs). may explain the stronger AMF dependency of certain species In conclusion, our results reveal a novel mechanism by [19,33]. To test whether garlic mustard’s effects correlate with which an invasive plant can disrupt native communities: by AMF dependency, and whether garlic mustard has stronger virtually eliminating the activity of native AMF from the soil negative effects on forest tree seedlings than on other plants, and drastically impairing the growth of native canopy species. we conducted another experiment (Experiment 5) using 16 It is currently unclear precisely which phytochemicals plant species for which we determined AMF-dependency by produced by garlic mustard have the observed antifungal computing the difference in plant growth in the presence and properties, whether and how they interact with other soil absence of AMF. We then tested the impact of garlic mustard microbes, and whether these anti-fungal effects extend to on the AM fungal colonization and growth of each plant other functionally important forest soil fungi such as species as above. All 16 plants were successfully colonized by ectomycorrhizal fungi and saprotrophic fungi. In addition, AMF, and the presence of garlic mustard heavily reduced within the home range, it is not known if evolutionary natural AMF colonization in all plants (Figure 4A). However, the resistance of co-occurring European neighbors may buffer presence of garlic mustard had a much stronger effect on the effects of garlic mustard’s antifungal properties [34–36]. plants that had high mycorrhizal dependency than those with Further research in these directions is needed to better less dependency (Figure 4B). The strongest effects were understand the effects of this invader on natural ecosystems observed for woody species most typically found in forested and the mechanisms involved. In North America; however, sites. These results indicate that the invasion of garlic the disruption of native tree seedling–AMF mutualisms may mustard is more likely to negatively impact highly mycor- facilitate garlic mustard’s invasion into mature forest under-

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garlic mustard, (3) soil without a history of garlic mustard, and (4) sterile soil without a history of garlic mustard. Six-inch pots were filled with a 1:1 mixture of sterilized silica sand and one of the four soil types. To each pot, we added a single seedling (seeds germinated on Turface [Aimcor, Buffalo Grove, Illinois, United States], a clay substrate) of one of the three native overstory tree species (sugar maple, red maple, or white ash) in a complete 4 3 3 factorial design with ten replicates of each treatment combination. The initial wet biomass of each seedling was recorded prior to planting, and dry weights were estimated using a dry–wet regression calculated from twenty extra seedlings. Pots were randomly placed on a greenhouse bench. Plants were watered (400 ml) once per week. Fertilizer was not added. After 4 mo of growth, shoots and roots were harvested, dried at 60 8C for 48 h, and weighed to determine biomass. An approximately 1-g subsample of roots from each seedling was extracted, stained with Chlorazol Black E [37] and analyzed for percent colonization by AMF [38]. Biomass and percent colonization data were analyzed using analysis of variance (ANOVA) for two fixed effects (soil type and species) and their interaction, followed by the Ryan-Einot-Gabriel-Welsch (REGW) multiple-range test. Experiment 2. Using field soil without a history of garlic mustard invasion (see Experiment 1), we grew garlic mustard, sugar maple, red maple, and white ash seedlings in separate 6-in pots (n ¼ 10) to condition the soil to each plant species. After 3 mo of conditioning, shoots and roots were removed. Unconditioned soil served as a control to the four plant-conditioning treatments. We added a single seedling of each of the three tree species to each of the five soil treatments. Pots were randomly placed on a greenhouse bench. Plants were watered (400 ml) once per week, without fertilizer. After 4 mo of growth, plants were harvested, biomass was determined, and percent mycorrhizal colonization of roots was assessed as in Experiment 1. Data were analyzed using ANOVA for two fixed effects (species and soil condition treatment). Means from the three species were pooled, and the effect of conditioning treatment was tested with a single- factor ANOVA followed by the REGW multiple-range test. Experiment 3. To 6-in pots containing field soil without a history of garlic mustard (see Experiment 1), we added a one-time, 100-ml aqueous extract [27] of whole plants of either garlic mustard, sugar maple, red maple, or white ash. A water control was included to give Figure 4. Experiment 5 five treatments. Whole-plant extract was used to account for secondary compounds exuded through roots and leaf litter. After 1 (A) Effect of mycorrhizal dependency on Al. petiolata reduction of AMF wk of exposure to the extract, seedlings of each tree species were colonization. planted in each of these five treatments to give a full factorial design (B) Effect of mycorrhizal dependency on Al. petiolata reduction in plant (extract source 3 tree species) with ten replicates of each treatment growth. Mycorrhizal dependency was calculated separately as the combination. Plants were watered (40 ml) every week, without difference between plant growth in the presence and absence of AMF. fertilizer. After 4 mo of growth, plants were harvested, biomass was Different colors represent plants with different life-history strategies, as determined, and roots were assayed for mycorrhizal colonization as follows: yellow dot, herbaceous colonizers of disturbed edges and bare in Experiment 1. Data were analyzed by two-factor ANOVA. ground; reddish brown dot, herbaceous edge and gap species; blue dot, Experiment 4. Spores from AMF native to the forest sites were woody colonizers of forest edges and gaps; black dot, tree species of obtained using trap cultures (as described in [39], but with a mix of mature forest. Species are labeled as follows (with mean mycorrhizal native plants) of soil samples from the uninvaded locations. We colonization in soil not conditioned by garlic mustard 6 standard error visually collected and separated Glomus and Acaulospora spores from in parentheses): 1 ¼ Ci. intybus (18.5 6 4.1), 2 ¼ Tr. repens (46.7 6 6.3), 3 ¼ these cultures, and compared germination rates of each genus in five Pl. major (28.2 6 3.7), 4 ¼ Ta. officinale (37.3 6 2.5), 5 ¼ S. canadensis treatments: a water agar control and water agar amended with an (48.0 6 6.2), 6 ¼ C. leucanthemum (34.6 6 3.1), 7 ¼ D. carota (40.4 6 6.2), aqueous extract from each of the four plants, as above. Ten randomly 8 ¼ As. syriaca (52.1 6 5.8), 9 ¼ J. virginiana (31.2 6 4.4), 10 ¼ Po. deltoids drawn spores were added into each plate, which was then incubated (63.9 6 4.5), 11 ¼ M. alba (38.6 6 5.9), 12 ¼ Pr. virginiana (28.4 6 4.2), 13 at 18 8C for 10 d. Ten replicate plates were prepared for each of the ¼ Fr. americana (65.9 6 5.3), 14 ¼ Ac. saccharum (46.3 6 3.7), 15 ¼ Ac. ten treatment combinations (two AMF genera 3 five extracts). Plates rubrum (59.5 6 5.7), 16 ¼ Pr. serotina (34.8 6 5.5). were monitored microscopically for spore germination. Percent DOI: 10.1371/journal.pbio.0040140.g004 germination data were analyzed using ANOVA for two fixed effects (extract source and AMF genus), and because of a significant interaction, each AMF genus was then analyzed separately using story and have particularly negative effects on the growth, single-factor ANOVA followed by the REGW multiple-range test. survival, and recruitment of native trees, and the composition Experiment 5. We investigated the effects of garlic mustard on of forest communities. woody and herbaceous plants using the following 16 native plant species: Cichorium intybus, Trifolium repens, Plantago major, and Tarax- acum officinale (dominant herbaceous colonizers of disturbed edges Materials and Methods and bare ground); Solidago canadensis, Chrysanthemum leucanthemum, Daucus carota, and Asclepias syriaca (dominant herbaceous edge and gap Experiment 1. Using a 15-cm–wide corer, we collected soil from species); Juniperus virginiana, Populus deltoides, Morus alba, and Prunus garlic mustard–invaded and nearby garlic mustard–free locations at virginiana (dominant woody colonizers of forest edges and gaps); and each of five forested areas dominated by Acer rubrum L. (red maple), Fr. americana, Ac. saccharum, Ac. rubrum, and Pr. serotina (dominant tree Ac. saccharum Marsh. (sugar maple), Fraxinus americana L. (white ash), species of mature forest). Seedlings of each plant were transplanted and Fagus grandifolia Ehrh. (American beech) near Waterloo, Ontario, into 8-in pots. For each species, growth was compared under the Canada. Invaded and uninvaded sites were randomly chosen within a following soil treatments: (1) soil without a history of garlic mustard 40-m2 plot within each forested area. Soils from the invaded and and inoculated with AMF, (2) soil without a history of garlic mustard, uninvaded areas were pooled separately in the lab and screened to without AMF, and (3) soil with a history of garlic mustard, and remove coarse roots and debris. Half the soil from each pool was then inoculated with AMF. Experimental soil was collected within a sterilized by autoclaving at 120 8C to create four soil treatments: (1) mature-canopy maple forest from locations with and without garlic soil with a history of garlic mustard, (2) sterile soil with a history of mustard. Soils from each location type were then mixed, cleaned of

PLoS Biology | www.plosbiology.org 0730 May 2006 | Volume 4 | Issue 5 | e140 Invasive Plant Disrupts Mycorrhizas all coarse roots and debris, autoclaved, and added to the pots as a 1:1 Acknowledgments mix of soil and silica sand. AMF spores were extracted from field soil collected from sites representing the four different habitats, and We thank T. Denich, V. Grebogi, G. Herrin, P. Hudson, G. Kuenen, J. pooled. The AMF-inoculation treatment consisted of adding 200 Lozi, B. Shelton, P. Stephens, J. Van Houten, and Z. Zhu for technical randomly picked spores to each pot, 2 cm below the surface, and assistance, and P. Antunes, G. De Deyn, and M. Hart for helpful beneath the newly transplanted seedlings. Plants were watered (500 comments on the text. ml) once per week, without fertilizer. They were harvested after 4 mo of growth, dried at 60 8C for 36 h, and weighed to determine biomass. Author contributions. KAS, RMC, and JNK conceived and designed AMF dependency of each plant species was determined by computing the experiments. KAS and JNK performed the experiments. KAS, the difference in plant growth in the presence and absence of AMF, SAC, JRP, BEW, RMC, GCT, SGH, DP, and JNK analyzed the data. JNK i.e., contrast of treatments (1) and (2) [32]. The effects of garlic contributed reagents/materials/analysis tools. All authors wrote the mustard on plant growth and percent colonization of each plant were paper. determined by contrasting treatments (1) and (3). To ask whether any Funding. We thank the Natural Sciences and Engineering Research relationships existed among mycorrhizal dependency, life form, and garlic mustard effects, we performed two regressions: percent Council of Canada, and the Harvard University Bullard Foundation reduction in AMF colonization by garlic mustard on AMF depend- for financial support. ency and percent reduction in plant biomass by garlic mustard on Competing interests. The authors have declared that no competing AMF dependency. interests exist. &

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PLoS Biology | www.plosbiology.org 0731 May 2006 | Volume 4 | Issue 5 | e140 Journal of Experimental Botany, Vol. 59, No. 5, pp. 1109–1114, 2008 doi:10.1093/jxb/erm342 Advance Access publication 10 February, 2008

SPECIAL ISSUE REVIEW PAPER More than 400 million years of evolution and some plants still can’t make it on their own: plant stress tolerance via fungal symbiosis

Rusty Rodriguez1,2,* and Regina Redman2,3 1 US Geological Survey, Seattle, WA 98115, USA 2

University of Washington, Seattle, WA, USA Downloaded from 3 Montana State University, Bozeman, MT, USA

Received 19 June 2007; Revised 25 November 2007; Accepted 30 November 2007 jxb.oxfordjournals.org Abstract dispersal, vegetative growth, and complex physiology either to escape or to mitigate the impacts of stress. All All plants in natural ecosystems are thought to be plants are known to perceive and transmit signals, and symbiotic with mycorrhizal and/or endophytic fungi. respond to stress such as drought, heat, salinity, and disease Collectively, these fungi express different symbiotic (Bohnert et al., 1995; Bartels and Sunkar, 2005). Some lifestyles ranging from parasitism to mutualism. Analy- biochemical processes are common to all plant stress at University of Toronto Library on November 19, 2010 sis of Colletotrichum species indicates that individual isolates can express either parasitic or mutualistic responses including the production of osmolytes, altering lifestyles depending on the host genotype colonized. water movement, and scavenging reactive oxygen species The endophyte colonization pattern and lifestyle ex- (ROS) (Leone et al., 2003; Maggio et al., 2003; Tuberosa pression indicate that plants can be discerned as either et al., 2003). Although there has been extensive research disease, non-disease, or non-hosts. Fitness benefits in plant stress responses, it is not known why so few conferred by fungi expressing mutualistic lifestyles species are able to colonize high stress habitats. However, include biotic and abiotic stress tolerance, growth plant stress research rarely takes into consideration enhancement, and increased reproductive success. a ubiquitous aspect of plant biology—fungal symbiosis. Analysis of plant–endophyte associations in high stress Since the first description of symbiosis (De Bary, 1879), habitats revealed that at least some fungal endophytes several symbiotic lifestyles have been defined based on confer habitat-specific stress tolerance to host plants. fitness benefits to or impacts on host and symbiont Without the habitat-adapted fungal endophytes, the (Lewis, 1985). After >100 years of research it is reason- plants are unable to survive in their native habitats. able to conclude that most, if not all, multicellular life on Moreover, the endophytes have a broad host range earth is symbiotic with micro-organisms. For example, all encompassing both monocots and eudicots, and confer plants in natural ecosystems are thought to be symbiotic habitat-specific stress tolerance to both plant groups. with mycorrhizal and/or endophytic fungi (Petrini, 1996; Brundrett, 2006). Recent studies indicate that fitness Key words: Colletotrichum, fungal endophytes, stress benefits conferred by mutualistic fungi contribute to or tolerance, symbiosis, symbiotic lifestyle. are responsible for plant adaptation to stress (Read, 1999; Stone et al., 2000; Rodriguez et al., 2004). Collectively, mutualistic fungi may confer tolerance to drought, metals, disease, heat, and herbivory, and/or promote growth and Introduction nutrient acquisition. It has become clear that at least some Throughout evolutionary time plants have been con- plants are unable to endure habitat-imposed abiotic and fronted with various abiotic and biotic stresses. Lacking biotic stresses in the absence of fungal endophytes any form of locomotion, plants have depended on seed (Redman et al., 2002b). Since there are several excellent

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 1110 Rodriguez and Redman reviews on mycorrhizal and endophytic fungi (Carroll, cent until plant senescence. This represents an excellent 1988; Read, 1999; Stone et al., 2000; Schardl and ecological strategy for fungi to capitalize on plant Leuchtmann, 2005; Brundrett, 2006), the focus of this nutrients. By already being established in tissues, endo- discussion will be on two aspects of fungal endophyte phytes have immediate access to plant nutrients made biology: symbiotic lifestyle switching (Redman et al., available during plant senescence. 2001) and the recently observed ecological phenomenon Studies on host genotype versus symbiotic lifestyle habitat-adapted symbiosis (HA-symbiosis; Rodriguez expression revealed that individual isolates of some fungal et al., 2008). It is hypothesized that HA-symbiosis allows species could span the symbiotic continuum by expressing plants to establish in high stress habitats. either mutualistic or pathogenic lifestyles in different host plants (Redman et al., 2001). For example, Colletotrichum species are classified as virulent pathogens, yet several Fungal endophytes species can express mutualistic lifestyles in non-disease hosts (Table 1). Mutualistic benefits conferred by Colleto- Unlike mycorrhizal fungi, endophytes reside entirely trichum spp. include disease resistance, growth enhance- within host tissues and emerge during host senescence. Downloaded from ment, and/or drought tolerance (Redman et al., 2001). These fungi comprise a phylogentically diverse group that Although the genetic basis of symbiotic communication is are members of the dikarya (Carroll, 1988; Schardl and not yet known, subtle differences in host genomes have Leuchtmann, 2005; Van Bael et al., 2005; Girlanda et al., profound effects on the outcome of symbiotic interactions. 2006; Arnold and Lutzoni, 2007). While most endophytes For example, commercially grown tomato (Solanum belong to the Ascomycota clade, some belong to the lycopersicum) is known to possess relatively few genetic jxb.oxfordjournals.org Basidiomycota. Although these fungi are often grouped differences between varieties yet is able to express high together, they can be discriminated into different func- levels of phenotypic plasticity (Miller and Tanksley, 1990; tional groups just as has been done with mycorrhizal fungi Tanksley, 2004; Brewer et al., 2007). When C. magna is (Brundrett, 2006). Currently, endophytes can be sub- introduced into different tomato cultivars, the fungus may divided into four classes based on host range, colonization express either mutualistic, commensal, or parasitic life- pattern, transmission, and ecological function (Rodriguez

styles. While parasitic and mutualistic lifestyles are easily at University of Toronto Library on November 19, 2010 et al., in review). Nevertheless, endophytes have been observed, commensal lifestyles are often designated when shown to confer fitness benefits to host plants including no host fitness benefit is observed. However, depending tolerance to herbivory, heat, salt, disease, and drought, on the traits being assessed, the commensal designation and increased below- and above-ground biomass (Bacon may be misleading. For example, C. gloeosporioides was and Hill, 1996; Clay and Holah, 1999; Sahay and Varma, designated a pathogen of strawberry and a commensal of 1999; Redman et al., 2001, 2002b; Arnold et al., 2003; tomato because it conferred no disease protection Waller et al., 2005; Ma´rquez et al., 2007). (Redman et al., 2001). However, C. gloeosporioides

The symbiotic continuum, lifestyle switching, Table 1. Symbolic lifestyle expression of Colletotrichum species and host range versus plant host

Collectively, fungi express several different symbiotic Fungal Disease Non-disease Lifestyle expressed lifestyles that are defined by fitness benefits to plant hosts pathogen hosta hostb and symbionts (Lewis, 1985). The range of symbiotic Disease Drought c d lifestyle expression from mutualism to parasitism has been stress stress described as the symbiotic continuum (Carroll, 1988; C. magna Watermelon Tomato Mutualism Mutualism Johnson et al., 1997; Saikkonen et al., 1998; Schulz C. musae Banana Pepper Mutualism Mutualism et al., 1999; Schardl and Leuchtmann, 2005). Within each C. orbiculare Cucumber Tomato Mutualism Mutualism C. acutatum Strawberry Watermelon Commensalism Mutualism group of fungal symbionts there are isolates and/or species C. gloeosporioides Strawberry Watermelon Commensalism Mutualism that span the symbiotic continuum by expressing different a lifestyles. For example, the endophyte genus Epichloe Species were isolated from disease lesions on the indicated host plants. comprises species that express either mutualistic or b Host plants that are asymptomatically colonized by the respective parasitic lifestyles (Schardl and Leuchtmann, 2005). Colletotrichum spp. Several studies that focused on the isolation of endophytes c Symbiotic lifestyle expressed after asymptomatic colonization. Lifestyles were defined by the ability of each Colletotrichum sp. to from asymptomatic plant tissues indicate that individual confer disease resistance against virulent Colletotrichum pathogens of species express either mutualistic, commensal, or parasitic the non-disease hosts (data from Redman et al., 2001). d lifestyles when re-inoculated back on the original host Symbiotic lifestyle expressed after asymptomatic colonization. Lifestyles were defined by the ability of each Colletotrichum sp. to species (Schulz et al., 1999). This indicates that both confer drought tolerance based on the length of time before wilting after mutualists and pathogens infect plants and remain quies- cessation of watering (data from Redman et al., 2001). Plant–fungal symbiosis 1111 increased plant biomass and conferred drought tolerance Colonization of non-disease hosts by pathogenic Colleto- to tomato plants, and was therefore designated a mutualist. trichum species is asymptomatic and there are no observ- A series of experiments were performed to characterize able differences between colonized and uncolonized plants the genetic basis of fungal symbiotic lifestyles. UV in the absence of stress, unless the endophyte promotes mutagenesis of a virulent isolate (CmL2.5) of C. magna plant growth (Redman et al., 1999, 2002a). Conventional resulted in the isolation of a non-pathogenic mutant (Path-1) views suggest that pathogens either cause disease or induce that was able to colonize host plants asymptomatically host defence systems which terminate the infection process. (Freeman and Rodriguez, 1993). Path-1 conferred several When Colletotrichum species express mutualistic lifestyles fitness benefits to hosts, including disease and drought and confer disease resistance, host defence systems are not resistance, and growth enhancement. Based on these activated unless the symbiotic plants are challenged with fitness benefits, it was concluded that Path-1 was express- a virulent pathogen (Redman et al., 1999, 2002a). Once ing a mutualistic lifestyle in host plants. Additional challenged, the host defence systems activate very rapidly studies involving gene disruption by restriction enzyme- (<24 h) to maximal levels (Redman et al., 1999). mediated integration (REMI) with a selectable plasmid The ability to switch lifestyles brings up some resulted in the generation of non-pathogenic mutants that interesting questions: Downloaded from differed in the ability to confer disease resistance (Fig. 1; Redman et al., 1999). The UV and REMI mutants lost the (i) Is there an evolutionary direction to symbiotic life- ability to switch between lifestyles and were ‘locked’ into styles? Clavicipitaceous endophytes expressing mutu- one lifestyle (either mutualism, intermediate-mutualism, or alisms are hypothesized to have evolved directionally commensal). These results indicate that the ability to from pathogenic ancestors (Schardl and Leuchtmann, jxb.oxfordjournals.org switch between symbiotic lifestyles, at least in this 2005). The situation with at least some other endo- species, is controlled by single genetic loci. phytes appears to be quite different, where the Although the original experiments on lifestyle switching evolution of symbiotic lifestyle appears to lack specific were performed with Colletotrichum species known to be directionality (Arnold and Lutzoni, 2007). Endophytes pathogenic, similar results have been observed with other that can switch lifestyles may represent evolutionary

endophytes from plants in natural habitats (RY Rodriguez, transitions or simply fungi that have achieved a higher at University of Toronto Library on November 19, 2010 unpublished results). What does this mean with regard to degree of ecological flexibility to ensure optimal host specificity? It appears that there are non-hosts that growth and reproduction in a variety of hosts. a fungus is unable to infect and two types of hosts that (ii) Do plants inadvertently participate or possibly in- fungi can colonize: disease hosts that they parasitize and stigate disease processes? Individual fungal isolates non-disease hosts that they asymptomatically colonize. can equally colonize different plants irrespective of the symbiotic lifestyle they express. For C. magna to Restriction Enzyme Mediated Integration express mutualism in one tomato cultivar and parasit- ism in another suggests that disease may reflect miscommunication rather than aggressive pathogenicity. 14,400 Transformants screened on plants

176 nonpathogenic REMI mutants Symbiosis and stress tolerance There are numerous reports of fungal symbionts confer- Four phenotypes elucidated based on ability to colonize and confer disease resistance ring tolerance to stress to host plants, including herbivory, drought, heat, salt, metals, and disease (Bacon and Hill, 1996; Clay and Holah, 1999; Sahay and Varma, 1999; Redman et al., 2001, 2002b; Arnold et al., 2003; Waller REMI Mutant Class A B C D et al., 2005; Ma´rquez et al., 2007; Rodriguez et al., 2007). 100 100 100 0 80 - 100 20 - 65 0 0 It is interesting that the stress tolerance conferred by some Symbiotic Lifestyle Mutualist IM Commensal Abortive endophytes involves habitat-specific fungal adaptations. Fig. 1. Gene disruption (restriction enzyme-mediated integration, For example, within the geothermal soils of Yellowstone REMI) of fungal symbiotic lifestyle loci in Colletotrichum magna. National Park, a small number of plant species reside. One Symbiotic lifestyles reflect the ability of REMI mutants to colonize host plant species (Dichanthelium lanuginosum) has been plants (watermelon) asymptomatically and confer disease resistance against the virulent wild type. REMI mutants were designated either as studied and found to be colonized by one dominant mutualists, intermediate mutualists, or commensals based on disease endophyte (Curvularia protuberata). Curvularia protu- protection, or abortive if they were unable to colonize host tissues. berata confers heat tolerance to the host plant, and neither Although these lifestyle designations reflect quantitative differences, they probably reflect a continuum of symbiotic lifestyles represented the fungus nor the plant can survive separate from one among the mutants. Methods and data are from Redman et al. (1999a). another when exposed to heat stress >38 C (Redman 1112 Rodriguez and Redman et al., 2002b). The ability of the endophyte to confer heat Table 2. Physiological defence activity versus symbotically tolerance requires the presence of a fungal RNA virus conferred disease conferred disease resistance by Colletotri- (Ma´rquez et al., 2007). While the genetic/biochemical role chum magna of the virus in symbiotically conferred heat tolerance is Methods and physiological data are from Redman et al. (1999). not known, it is surmised that the virus is providing Host Peroxidase PAL Lignin biochemical functionality to the fungus and it is not the activitya activityb depositionc virus that directly confers heat tolerance. A comparison of C. protuberata isolates from geothermal and non- 24 h 48 h 24 h 48 h 24 h 48 h geothermal plants revealed that the ability to confer heat Watermelon (E–)d 2.76 3.46 2.27 2.90 – + tolerance was specific to isolates from geothermal plants Watermelon (E+)e 5.77 6.30 2.50 3.70 +++ ++++ (Rodriguez et al., 2008). Therefore, the ability to confer Cucumber (E–) 0.63 1.31 0.02 0.25 – + Cucumber (E+) 1.80 2.34 .27 0.34 +++ ++++ heat tolerance is a habitat-adapted phenomenon. a Another example of habitat-specific fungal adaptation Activity based on a guaiacol/H2O2 assay, and units indicate change 1 1 involves a native dunegrass (Leymus mollis) on coastal in A470 min lg protein. b Activity based on the production of cinnamic acid, and units Downloaded from beaches of Puget Sound, WA (Rodriguez et al., 2008). 1 1 indicate change in A290 min lg protein. Leymus mollis is colonized by one dominant fungal c Qualitative assessment of the absence (–) or presence (+) of lignin endophyte (Fusarium culmorum) that can be isolated from visualized with acidic phloroglucinol. d (E–)¼endophyte (C. magna) free. above- and below-ground tissues and seed coats. Fusa- e (E+)¼endophyte (C. magna) colonized. rium culmorum confers salt tolerance to the host plant which cannot survive in coastal habitats without the jxb.oxfordjournals.org habitat-adapted endophyte. A comparison of F. culmorum strong activation of biochemical processes known to isolates from L. mollis and a non-coastal plant revealed confer resistance (Redman et al., 1999). In the absence of that the ability to confer salt tolerance was specific to pathogen challenge, Path-1-colonized plants do not appear isolates from the coastal plants, indicating that the ability to activate host defence systems. However, when Path- to confer salt tolerance is a habitat-adapted phenomenon 1-colonized watermelon and cucumber seedlings were

(Rodriguez et al., 2008). exposed to a virulent pathogen, peroxidase and phenyl- at University of Toronto Library on November 19, 2010 A comparison of C. protuberata, F. culmorum, and alanine ammonia lyase activity and lignin deposition C. magna isolates further supports habitat-specific adapta- increased within 24 h to levels that non-symbiotic plants tion of endophytes: C. protuberata confers heat but not never achieved (Table 2; Redman et al., 1999). Interest- disease or salt tolerance; F. culmorum confers salt but not ingly, Colletotrichum-conferred disease resistance is local- heat or disease tolerance; and C. magna confers disease ized to tissues that the fungus has colonized, and is not but not heat or salt tolerance (Rodriguez et al., 2008). systemic. The results suggest that the endophyte may be These symbiotically conferred stress tolerances conform acting as a type of biological trigger that activates host to the evolutionary dynamics that must play out in the defence systems. The fact that Colletotrichum spp. different habitats, with fungi adapting to habitat-specific expressing non-pathogenic lifestyles do not activate host stresses and conferring stress tolerance to host plants. This defence in the absence of pathogen challenge may be habitat-specific adaptation is defined as HA-symbiosis, viewed as either suppression of host defences or eluding and it is hypothesized that this allows plants to establish host recognition. However, the dynamics of host defence and survive in high stress habitats. activation suggest that the endophytes are recognized and do not suppress defence systems. In barley, the root endophyte Piriformospora indica Biochemical basis of endophyte-conferred confers disease resistance by a different mechanism. stress tolerance Symbiotic plants are thought to resist necrotrophic root It is fascinating that after 400 million years of evolution pathogens due to increased activity of glutathione– there are plants that require symbiotic associations for ascorbate antioxidant systems (Waller et al., 2005). stress tolerance. There has been an enormous research Unlike Colletotrichum endophytes, disease resistance effort in plant stress physiology that is described in conferred by P. indica appears to be systemic. It is not several excellent books and reviews. Although previous clear if P. indica increases antioxidation systems in the studies have elucidated how plants respond to stress, they absence of pathogens or if other aspects of host rarely consider symbiotic contributions. physiology are involved in resistance. Symbiotically conferred disease tolerance appears to The differences between Colletotrichum spp.- and involve different mechanisms depending on the endo- P. indica-conferred disease resistance may indicate that phyte. For example, the ability of a non-pathogenic a greater diversity of mechanisms may yet be elucidated. Colletotrichum mutant (Path-1 that expresses a mutualism) Regardless, these results warrant a more comprehensive to confer disease resistance is correlated to a rapid and analysis of endophyte-conferred disease resistance. Plant–fungal symbiosis 1113 Symbiotic plasticity and fungal taxonomy biology. Moreover, fungal symbionts may also harbour bacteria and viruses that can have dramatic effects on One deficiency in species designations is a dearth of symbiotic communication. For example, the class functional ecological descriptions, symbiotic lifestyle 2 endophyte C. protuberata (Cp4666D), originally iso- potential, and host ranges. A good example of this issue lated from plants growing in geothermal soils, contains is the fact that C. protuberata is described as a plant a double-stranded RNA (dsRNA) virus that is required for pathogen of several monocots (Farr et al., 1989). Yet, symbiotically conferred heat tolerance (Ma´rquez et al., C. protuberata isolate Cp4666D is a mutualist in Dichan- 2007). In the absence of the virus, Cp4666D asymptom- thelium lanuginosum, conferring heat and drought toler- atically colonizes plants but confers no heat tolerance. ance (Rodriguez et al., 2008). While Curvularia species Therefore, a three-way symbiosis (a virus in a fungus in are not known to have broad disease-host ranges, a plant) is required for thermal tolerance. This was an C. protuberata from the monocot D. lanuginosum is unexpected result and reflects our limited understanding of a mutualist (confers heat tolerance) in the eudicot tomato, symbiotic systems and how they function. More impor- and isolates from non-geothermal plants do not confer heat tantly, it indicates the need to study plants from tolerance (Ma´rquez et al., 2007; Rodriguez et al., 2008). Downloaded from a symbiotic systems perspective to elucidate the contribu- A similar scenario occurs with F. culmorum. Designated tions of all symbionts. as a virulent plant pathogen, F. culmorum causes disease on a variety of crop plants (Farr et al., 1989). However, the F. culmorum isolate FcRed1 from dunegrass is a Summary mutualist in dunegrass and tomato conferring salt toler- jxb.oxfordjournals.org ance, and isolates from non-coastal plants do not confer Both laboratory and field studies have demonstrated that salt tolerance (Rodriguez et al., 2008). These examples at least some plant species in natural habitats require indicate that the current concept of a fungus being fungal endophytes for stress tolerance and survival. Since categorized as either a pathogen, saprophyte, or mutualist colonizing land ;400 million years ago, plants have is inadequate to address the fact that individual species can evolved intragenomic mechanisms to perceive and trans- represent significant ecological plasticity. mit signals, and respond to stress (Bohnert et al., 1995; The ability of ‘pathogenic’ Colletotrichum species to Bartels and Sunkar, 2005), but most plants lack the at University of Toronto Library on November 19, 2010 switch symbiotic lifestyles and express mutualisms pro- adaptive capability to mitigate the impacts of stress vides insight into why these species are so ubiquitous. It (Alpert, 2000). At least some plants depend on inter- has been suggested previously that pathogens may be genomic epigenetic processes provided by symbiotic fungi present in non-disease host plants constituting potential for stress adaptation. The observations described in this inocula for disease. In fact, C. acutatum asymptomatically manuscript raise some fundamental questions in plant colonizes pepper, eggplant, bean, and tomato plants, biology. Why have plants in high stress habitats not which can subsequently provide inoculum for disease evolved intragenomic capabilities for stress adaptation? outbreaks in strawberry plants (Freeman et al., 2001). So, Can plants adapt to stress without symbiotic involvement? at least in this genus, species may move freely between Why are so few plants adapted to high stress lifestyles and hosts, thereby expanding bio-geographic habitats? Answers to these questions will require extensive distribution. It is unlikely that this phenomenon is specific research efforts over the coming decades and are necessary to Colletotrichum as asymptomatic colonization of hosts before ecosystem processes are fully understood. has been reported for other genera such as Fusarium (Bacon and Yates, 2006). Incorporating information on lifestyle expression and Acknowledgements ecological functionalities would allow ecologists to un- This project was made possible by the permission, assistance, and derstand better the role of fungi in ecosystem processes, guidelines of YNP and the UW Cedar Rocks Biological Preserve. geneticists to understand better genome differences be- This work was supported by the US Geological Survey, NSF tween isolates, and mycologists to understand phenotypic (0414463) and US/IS BARD (3260-01C). and ecological plasticity.

References Symbiotic communities Alpert P. 2000. The discovery, scope, and puzzle of desiccation While this discussion has focused on fungal symbionts, it tolerance in plants. Plant Ecology 151, 5–17. is important to point out that plants represent communities Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? of fungi, bacteria, viruses, and/or algae. All of these Ecology 88, 541–549. micro-organisms contribute to the outcome of symbiosis Arnold EA, Mejia LC, Kyllo D, Rojas E, Maynard Z, and hence increase the complexity of studying plant Robbins N, Herre EA. 2003. Fungal endophytes limit pathogen 1114 Rodriguez and Redman damage in a tropical tree. Proceedings of the National Academy Petrini O. 1996. Ecological and physiological aspects of host- of Sciences, USA 100, 15649–15654. specificity in endophytic fungi. In: Redlin SC, Carris LM, eds. Bacon CW, Hill NS. 1996. Symptomless grass endophytes: Endophytic fungi in grasses and woody plants. St Paul, MN: APS products of coevolutionary symbioses and their role in the Press, 87–100. ecological adaptations of grasses. In: Redkin SC, Carris LM, eds. Read DJ. 1999. Mycorrhiza—the state of the art. In: Varma A, Endophytic fungi in grasses and woody plants. St Paul, MN: APS Hock B, eds. Mycorrhiza. Berlin: Springer-Verlag, 3–34. Press, 155–178. Redman RS, Dunigan DD, Rodriguez RJ. 2001. Fungal symbio- Bacon CW, Yates IE. 2006. Endophytic root colonization by sis: from mutualism to parasitism, who controls the outcome, host fusarium species: histology, plant interactions, and toxicity. In: or invader? New Phytologist 151, 705–716. Schulz BJE, Boyle CJC, Sieber TN, eds. Microbial root Redman RS, Freeman S, Clifton DR, Morrel J, Brown G, endophytes. Berlin: Springer-Verlag, 133–152. Rodriguez RJ. 1999. Biochemical analysis of plant protection Bartels D, Sunkar R. 2005. Drought and salt tolerance in plants. afforded by a nonpathogenic endophytic mutant of Colletotri- Critical Reviews in Plant Science 24, 23–58. chum magna. Plant Physiology 119, 795–804. Bohnert HJ, Nelson DE, Jensen RG. 1995. Adaptations to Redman RS, Rossinck MR, Maher S, Andrews QC, environmental stresses. The Plant Cell 7, 1099–1111. Schneider WL, Rodriguez RJ. 2002a. Field performance of Brewer MT, Moyseenko1 JB, Monforte AJ, van der Knaap E. cucurbit and tomato plants infected with a nonpathogenic mutant

2007. Morphological variation in tomato: a comprehensive study of Colletotrichum magna (teleomorph: Glomerella magna; Jen- Downloaded from of quantitative trait loci controlling fruit shape and development. kins and Winstead). Symbiosis 32, 55–70. Journal of Experimental Botany 58, 1339–1349. Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Brundrett MC. 2006. Understanding the roles of multifunctional Henson JM. 2002b. Thermotolerance conferred to plant host and mycorrhizal and endophytic fungi. In: Schulz BJE, Boyle CJC, fungal endophyte during mutualistic symbiosis. Science Sieber TN, eds. Microbial root endophytes. Berlin: Springer- 298, 1581. Verlag, 281–293. Rodriguez RJ, Henson J, Van Volkenburgh E, Hoy M, Carroll G. 1988. Fungal endophytes in stems and leaves: from Wright L, Beckwith F, Kim Y, Redman RS. 2008. Stress jxb.oxfordjournals.org latent pathogen to mutualistic symbiont. Ecology 69, 2–9. tolerance in plants via habitat-adapted symbiosis. International Clay K, Holah J. 1999. Fungal endophyte symbiosis and plant Society of Microbial Ecology in press. diversity in successional fields. Science 285, 1742–1745. Rodriguez RJ, Redman RS, Henson JM. 2004. The role of fungal De Bary A. 1879. Die erschenung symbiose. In: Trubner KJ, ed. symbioses in the adaptation of plants to high stress environments. Vortrag auf der versammlung der naturforscher und artze zu Mitigation and Adaptation Strategies for Global Change 9, cassel. Strassburg: 1–30. 261–272. Farr DF, Bills GF, Chamuris GP, Rossman AY. 1989. Fungi on Sahay NS, Varma A. 1999. Piriformospora indica: a new bi- at University of Toronto Library on November 19, 2010 plants and plant products in the United States. St Paul, MN: APS ological hardening tool for micropropagated plants. FEMS Press. Microbiology Letters 181, 297–302. Freeman S, Horowitz S, Sharon A. 2001. Pathogenic and Saikkonen K, Faeth SH, Helander M, Sullivan TJ. 1998. Fungal nonpathogenic lifestyles in Colletotrichum acutatum from straw- endophytes: a continuum of interactions with host plants. Annual berry and other plants. Phytopathology 91, 986–992. Review of Ecology and Systematics 29, 319–343. Freeman S, Rodriguez RJ. 1993. Genetic conversion of a fungal Schardl C, Leuchtmann A. 2005. The epichloe endophytes of plant pathogen to a nonpathogenic, endophytic mutualist. Science grasses and the symbiotic continuum. In: Dighton J, White JF, 260, 75–78. Oudemans P, eds. The fungal community: its organization and Girlanda M, Perotto S, Luppi AM. 2006. Molecular diversity and role in the ecosystem. Boca Raton, FL: Taylor & Francis, ecological roles of mycorrrhiza-associated sterile fungal endo- 475–503. phytes in mediterranean ecosysems. In: Boyle CJC, Sieber TN, Schulz B, Rommert AK, Dammann U, Aust HJ, Strack D. 1999. eds. Microbial root endophytes. Berlin: Springer-Verlag, The endophyte–host interaction: a balanced antagonism? Myco- 207–226. logical Research 10, 1275–1283. Johnson NC, Graham JH, Smith FA. 1997. Functioning of Stone JK, Bacon CW, White JF. 2000. An overview of mycorrhizal associations along the mutualism–parasitism contin- endophytic microbes: endophytism defined. In: Bacon CW, White uum. New Phytologist 135, 575–586. JF, eds. Microbial endophytes. New York: Marcel Dekker, Inc, Leone A, Perrotta C, Maresca B. 2003. Plant tolerance to heat 3–30. stress: current strategies and new emergent insight. In: di Toppi Tanksley SD. 2004. The genetic, development, and molecular bases LS, Pawlik-Skowronska B, eds. Abiotic stresses in plants. of fruit size and shape variation in tomato. The Plant Cell 16, London: Kluwer Academic Publishers, 1–22. S181–S189. Lewis DH. 1985. Symbiosis and mutualism: crisp concepts and Tuberosa R, Grillo S, Ellis RP. 2003. Unravelling the genetic soggy semantics. In: Boucher DH, ed. The biology of mutualism. basis of drought tolerance in crops. In: di Toppi LS, Pawlik- London: Croom Helm Ltd, 29–39. Skowronska B, eds. Abiotic stresses in plants. London: Kluwer Maggio A, Bressan RA, Ruggiero C, Xiong L, Grillo S. 2003. Academic Publishers, 71–122. Salt tolerance: placing advances in molecular genetics into Van Bael SA, Maynard Z, Rojas E, Mejia LC, Kyllo DA, a physiological and agronomic context. In: di Toppi LS, Pawlik- Herre EA, Robbins N, Bischoff JF, Arnold AE. 2005. Skowronska B, eds. Abiotic stresses in plants. London: Kluwer Emerging perspectives on the ecological roles of endophytic Academic Publishers, 53–70. fungi in tropical plants. In: Dighton J, White JF, Oudemans P, Ma´rquez LM, Redman RS, Rodriguez RJ, Roossinck MJ. 2007. eds. The fungal community: its organization and role in the A virus in a fungus in a plant—three way symbiosis required for ecosystem. Boca Raton, FL: Taylor & Francis, 505–518. thermal tolerance. Science 315, 513–515. Waller F, Achatz B, Baltruschat H, et al. 2005. The endophytic Miller JC, Tanksley SD. 1990. Rflp analysis of phylogenetic fungus Piriformospora indica reprograms barley to salt-stress relationships and geneteic variation in the genus Lycopersicon. tolerance, disease resistance, and higher yield. Proceedings of the Theoretical and Applied Genetics 80, 437–448. National Academy of Sciences, USA 102, 13386–13391. focuS on SymbIoSISREVIEWS

Arbuscular mycorrhiza: the mother of plant root endosymbioses

Martin Parniske Abstract | Arbuscular mycorrhiza (AM), a symbiosis between plants and members of an ancient phylum of fungi, the Glomeromycota, improves the supply of water and nutrients, such as phosphate and nitrogen, to the host plant. In return, up to 20% of plant-fixed carbon is transferred to the fungus. Nutrient transport occurs through symbiotic structures inside plant root cells known as arbuscules. AM development is accompanied by an exchange of signalling molecules between the symbionts. A novel class of plant hormones known as strigolactones are exuded by the plant roots. On the one hand, strigolactones stimulate fungal metabolism and branching. On the other hand, they also trigger seed germination of parasitic plants. Fungi release signalling molecules, in the form of ‘Myc factors’ that trigger symbiotic root responses. Plant genes required for AM development have been characterized. During evolution, the genetic programme for AM has been recruited for other plant root symbioses: functional adaptation of a plant receptor kinase that is essential for AM symbiosis paved the way for nitrogen-fixing bacteria to form intracellular symbioses with plant cells.

9 Aseptate Plant root symbioses with fungi occur in several dif- estimated to be consumed by AM fungi . Therefore, Not containing septae. ferent forms and are referred to as mycorrhiza (from AM symbiosis contributes significantly to global the Greek ‘mycos’, meaning fungus and ‘rhiza’, mean- phosphate and carbon cycling and influences primary Coenocytic ing root). In ectomycorrhiza, which is predominant on productivity in terrestrial ecosystems1. The beneficial Multiple nuclei within the same cell. trees in temperate forests, the fungal partner remains effects of AM are most apparent under conditions of outside of plant cells, whereas in endomycorrhiza, limited nutrient availability. Although the underly- including orchid, ericoid and arbuscular mycorrhiza ing regulatory mechanisms are not understood, the (AM), part of the fungal hyphae is inside. AM is amount of root colonization typically decreases when probably the most widespread terrestrial symbiosis1 nutrients are in abundance. Interestingly, the colo- and is formed by 70–90% of land plant species2 with nization of roots with AM fungi has been observed fungi that belong to a monophyletic phylum, the to lead to an inhibition of bacterial leaf pathogens10. Glomeromycota3,4 (FIG. 1). Symbiotic development Whether such increased resilience to pathogens is a results in the formation of tree-shaped subcellular consequence of improved plant fitness or is due to structures within plant cells. These structures, which specific defence responses that are induced by AM are known as arbuscules (from the Latin ‘arbusculum’, fungi is unknown. meaning bush or little tree) are thought to be the main AM fungi are unusual organisms because of their site of nutrient exchange between the fungal and plant age, lifestyle and genetic make-up; they have existed for symbiotic partners (FIG. 2). AM intimately connects more than 400 million years morphologically unaltered plants to the hyphal network of the fungi, which can be and could therefore qualify as living fossils. They are in excess of 100 metres of hyphae per cubic centimetre considered by many to be ancient asexuals, a character- of soil5. This hyphal network is specialized for nutri- istic that defies the predictions of evolutionary theory. Faculty of Biology, University ent (predominantly phosphate) and water uptake6. In The hyphal network of AM fungi is usually aseptate and of Munich, Großhaderner return for supplying plants with nutrients and water, coenocytic, with hundreds of nuclei sharing the same Straße 2-4, 82152 Planegg- 7,8 Martinsried, Germany. AM fungi obtain carbohydrates from plants . Up to cytoplasm. Likewise, individual spores contain hun- e-mail: [email protected] 20% of the photosynthesis products of terrestrial plants dreds of nuclei and the question of how the different doi:10.1038/nrmicro1987 (approximately 5 billion tonnes of carbon per year) are polymorphic DNA-sequence variants that are present

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a Ascomycota

Basidiomycota

Glomeromycota

Endogone and Mortierella Chytridiales, including Basidiobolus Choanoflagellida Blastocladiales

Zea mays and Chlorella

Stylonychia

Homo sapiens Entomophthorales and Aphrodita

Outgroups

Kickxellales and Ulkenia and Harpellales Thraustochytridium

Mucorales 0.05 Dictyostelium

b Archaeosporales

Geosiphonaceae Archaeosporaceae (Geosiphon) Glomus group Ab (Archaeospora) Ambisporaceae Paraglomerales (Ambispora) Glomerales

Glomus group Aa Paraglomeraceae (Paraglomus)

Glomus sp. W3347

Glomeraceae 1 and 2

Russula Glomus group B Boletus

Aspergillus Penicillium Candida Pacisporaceae Kluyveromyces (Pacispora) Outgroups Basidiomycota and Ascomycota Gigasporaceae Diversisporaceae (Gigaspora and Acaulosporaceae (Diversispora) (Acaulospora) 0.01 Scutellospora)

Diversisporales

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Anastomosis within a single cell are distributed between genomes Fungal cell wall 11–13 A hyphal fusion with a or nuclei is the subject of an ongoing debate . cytoplasmic connection. Although there is no confirmed report of a sexual stage Fungus Fungal cytoplasm Plant cell wall in the life cycle of AM fungi, it is possible that genetic Obligate biotroph anastomosis An organism that is unable to material is exchanged and recombined; 14,15 15 complete a reproductive cycle between hyphae allows the exchange of nuclei Plant PAS in the absence of a living host. but has so far only been observed between hyphae of Fungal plasma closely related fungal strains. It will be interesting to Cytoplasm membrane Mycoheterotrophic determine the level of relatedness that is required for Obtains carbon sources from a fungal symbiont. these fusions to occur. Molecular evidence for recom- bination in AM fungi16,17 has been controversial13. As an important step towards the genetic manipulation PAM of these fungi, transient transformation by particle bombardment has been achieved18. Although spores of AM fungi can germinate in Figure 2 | The arbuscule. Schematic drawing of an the absence of host plants, they are obligate biotrophs, arbuscule, the symbiotic structureNature and Revie arbuscularws | Microbiolog y and therefore depend on a living photoautotrophic mycorrhiza (AM). Each fungal branch within a plant cell partner to complete their life cycle and produce is surrounded by a plant-derived periarbuscular the next generation of spores. In one reported case, membrane (PAM) that is continuous with the plant however, co-culture with Paenibacillus validus led plasma membrane and excludes the fungus from the plant cytoplasm. The apoplastic interface between to the production of secondary and infective spores 19 the fungal plasma membrane and the plant-derived PAM in the absence of a host . is called the periarbuscular space (PAS). Because of the Individual fungal strains exhibit little host specifi- cell-wall synthesizing potential of both the fungal city when grown with different plants under laboratory membrane and the PAM, the PAS comprises fungal and conditions2. Likewise, a single plant can be colonized plant cell-wall material. by many different AM fungal species within the same root1,20. Therefore, on the one hand, the AM symbiosis is thought to show little host specificity at the level of have been cloned. This reprogramming involves the colonization. on the other hand, the biodiversity of formation of a newly discovered prepenetration appa- fungal and plant communities are positively correlated ratus (PPA) by the plant cell in anticipation of fungal with each other21 and host preference seems to play an infection, by which the plant cell dictates the route of important role in natural ecosystems22,23. It is likely that fungal intracellular passage. Moreover, our current these host preferences reflect different fungal strategies knowledge of the function of the AM symbiosis is sum- and levels of functional compatibility24,25 (FIG. 3). High marized, including the nutrient exchange and metabo- specificity has been observed between mycoheterotrophic lite fluxes in AM. Finally, evolutionary aspects of AM plants and their mycosymbionts26. Most AM fungi that fungi and the evolution of the plant genetic programme can be detected in natural ecosystems have not been for symbiosis development are considered. cultured1 and it is possible that many have a more restricted host range than ‘generalists’, such as Glomus AM development mossae or Glomus intraradices, which are intensively The presymbiotic phase. Multiple, successive rounds investigated because they are easily cultured. of spore germination and retraction of nuclei and This review will describe AM development, an area cytoplasm can occur in AM fungi. This exploratory in which substantial progress has been made over the hyphal development changes dramatically in the pres- past few years. Two novel signalling molecules have ence of plant-derived signals (FIG. 4). The stimulatory been identified that alter fungal development or plant effect of plant root exudates on AM fungal hyphae has gene expression. Seven plant genes that are involved been recognized for a long time, but the molecular in symbiotic reprogramming of plant cell development identity of the ‘branching factors’ has only recently been identified. In two landmark papers, strigolac- tones were found to be responsible for the induction ◀ Figure 1 | Arbuscular mycorrhiza (Am) fungi form an independent phylum, the of branching27 and alterations in fungal physiology glomeromycota. a | A phylogenetic tree showing the Glomeromycota in relation to other and mitochondrial activity28. Strigolactones can also main fungal lineages: the Ascomycota and Basidiomycota and the non-monophyletic stimulate spore germination in some AM fungi. 3 Chytridiomycota (green) and Zygomycota (blue) . All tested members of the Strigolactones are short-lived in the rhizosphere Glomeromycota form AM and all AM fungi are members of the Glomeromycota. owing to a labile ether bond that spontaneously b | Phylogenetic relationships between members of the Glomeromycota. Among the four hydrolyses in water. This ephemeral compound forms orders that are currently recognized, the Archaeosporales and Paraglomerales are clearly distinct from the subgroup Glomerales and Diversisporales. The phylogeny and taxonomy a steep concentration gradient, and therefore its per- of AM fungi is still under substantial debate. For example, owing to the significant ception has been suggested to be a reliable indicator of 29 divergence among the Glomeraceae, this family will probably be taxonomically separated the proximity of a host root . Interestingly, the same in the future. The scale bar represents the number of substitutions per site. Panel a modified, class of compounds was identified 50 years ago as a with permission, from REF. 3 (2001) Cambridge University Press. Panel b modified, with potent germination inducer of seeds of the parasitic permission, from REF. 122 (2002) Springer Netherlands. plant genus Striga. The discovery that strigolactones

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Glomus hyphal networks Scutellospora hyphal networks Fungal signalling molecules and plant receptors. There is currently much interest in the molecular identifica- tion of fungal signalling molecules that induce sym- Spore distant Branching from root biosis-specific responses in the host root (collectively hyphae called Myc factors). The existence of such molecules became apparent in experiments in which direct contact between the fungus and plant was prevented by fungus-impenetrable membranes. In these experi- Profuse ments, a symbiosis-responsive Enod11-promoter branching b and GUS ( -glucuronidase) reporter gene fusion in roots anastomosis of Medicago truncatula was activated in the vicinity of close to fungal hyphae32. This Myc factor was found to be a root diffusible molecule that induced transcriptional acti- vation of symbiosis-related genes. Whether the produc- tion of this Myc factor is stimulated by strigolactones Spore close Hyphae bridging is unclear. A weak but significant increase in lateral to or even long distances; within root root initiation was observed when roots were treated few branches with a diffusible factor from AM fungi33. However, it or anastomoses is unknown whether the root-inducing and Enod11- Large infected Small infected inducing molecules are the same. calcium signatures patch patch that were reminiscent of, but clearly distinct from, Nod- factor-induced calcium spiking were recently observed Plant root in root hair cells in the vicinity of, but before contact with, approaching AM hyphae34. Interestingly, calcium Figure 3 | Different hyphal growthNature andRevie branchingws | Microbiology oscillations of lower frequency and amplitude than the strategies in arbuscular mycorrhiza (Am) fungi. Nod-factor-induced calcium spiking can be induced AM fungi have different hyphal growth patterns, by oligo-n-acetylglucosamine35,36. LysM domains have anastomoses and branching frequencies. These been implicated in n-acetylglucosamine binding. Two differences probably reflect different strategies and the different receptor-like molecules that are required for occupation of different niches within the soil. Many chitin perception, both of which have LysM domains in Glomus species form highly branched and anastomosing their extracellular domain, have recently been identified hyphal networks. These networks are more recalcitrant in rice and A. thaliana37,38. These receptor molecules to disturbances of the soil than the mycelia of species of are involved in the induction of resistance responses Scutellospora or Gigaspora, which form longer hyphae and can probably explore more distant regions of the and constitute part of an ancient perception system for soil14,123. Most of the fungal biomass in members of the the detection of microbial-associated molecular pat- Gigasporaceae family is found in the hyphae that are terns (MAMPs). The Nod factor receptors also contain located outside the plant root, whereas in members of LysM domains, which are likely to bind the lipochitoo- the Glomeraceae family, most of the hyphal biomass is ligosaccharide Nod factors39,40. Their close structural inside the root21. relationship indicates that the Nod factor and chitin receptors share a common ancestry. AM fungi have chitin in their cell walls and could be recognized by act as signals for AM fungi has revealed that species the chitin-perception system. In addition to chitin and of Striga exploit a conserved and ancient communica- Nod factor receptors, both A. thaliana and rice contain tion system between symbiotic fungi and their host several LysM-containing receptor kinases of unknown plants29. The recent pioneering discovery of strigolac- function. Whether, in common with rhizobia, AM tones as novel endogenous plant hormones in diverse fungi produce chitooligosaccharides or derivatives angiosperms that range from Arabidopsis thaliana to that function as symbiotic signals which are recognized pea and rice30,31 suggests that the strigolactone percep- by LysM receptor kinases remains to be determined. tion system of Striga is less unique than previously The search for the Myc factor requires a specific assay Nod factors The bacterial symbionts of thought. by contrast, it is probably derived from a system, because plant cells are responsive to a range of 41 legumes (rhizobia) produce general hormonal perception system of angiosperms. MAMPs that trigger related downstream responses, signalling molecules named Whether strigolactones first evolved as endogenous including calcium responses42. Therefore, the challenge Nod factors. They consist of plant hormones or as signals in AM remains an open is to show that candidate Myc factors, such as an as-yet- an N-acetylglucosamine backbone that carries various question. Strigolactone perception by the fungus unidentified small molecule from Gigaspora margarita, strain-specific decorations induces the so-called presymbiotic stage, which is which elicits calcium responses in soybean cells, can including a lipid side chain. characterized by continued hyphal growth, increased induce symbiosis-specific responses43. physiological activity and profuse branching of Calcium spiking hyphae. Plant mutants that are unable to produce The prepenetration apparatus. The paradigm-shifting A sharp periodic increase in 30 calcium concentration around strigolactones are now available in pea and rice , and discovery that the plant cell actively prepares the 44,45 the nucleus of symbiotically can be useful for refined functional analyses of these intracellular environment for AM fungal hyphae stimulated root cells. signalling molecules in AM development. changed our view of the role of the plant cell during

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Striga seedling Spore Mutual recognition ‘presymbiotic phase’

Strigolactone Myc factor Formation of Fungal PPA penetration

Hyphopodium Nucleus Epidermis Calcium spiking Outer cortex Arbuscule Inner

Plant root Plant cortex Endodermis Vascular cylinder

Figure 4 | Steps in arbuscular mycorrhiza (Am) development. Plant roots exude strigolactonesNature Revie whichws | Microbiology induce spore germination and hyphal branching and increase physiological activity in fungal spores and hyphae. Strigolactones also induce seed germination in parasitic plants, such as Striga124. Fungi produce mycorrhiza (Myc) factors that are operationally defined through their ability to induce calcium oscillations in root epidermal cells34 and to activate plant symbiosis-related genes32. AM fungi form special types of appressoria called hyphopodia, which by definition develop from mature hyphae and not from germination tubes125. As a consequence of sequential chemical and mechanical stimulation, plant cells produce a prepenetration apparatus (PPA). Subsequently, a fungal hypha that extends from the hyphopodium enters the PPA, which guides the fungus through root cells towards the cortex. Here, the fungus leaves the plant cell and enters the apoplast, where it branches and grows laterally along the root axis. These hyphae induce the development of PPA-like structures in inner cortical cells45, subsequently enter these cells and branch to form arbuscules. Vesicles, which are proposed to function as storage organs of the fungus, are sometimes, but not always, formed in AM and are present in the apoplast (not shown). New spores are typically synthesized outside of the plant root at the leading tip of individual fungal hyphae. Figure modified, with permission, from REF. 45 (2008) © American Society of Plant Biologists.

infection by biotrophic fungi. The PPA is a subcellular ‘pre-infection thread’ of legumes, which forms in response structure that predetermines the path of fungal growth to rhizobia48 in anticipation of bacterial infection, through the plant cell and is formed 4–5 hours after probably evolved from the PPA (FIG. 5). the formation of a fungal appressorium, also called a hyphopodium. Formation of the PPA is preceded by Plant genes required for AM development a migration of the plant cell nucleus towards the point At least seven genes that are required for both the of anticipated fungal entry. The nucleus then moves AM symbiosis and the root-nodule symbiosis with ahead of the developing PPA, as if to guide its growth rhizobia have been identified in legumes49 (TABLE 1). direction through the cell. The PPA is a thick cyto- These genes encode proteins that are directly or plasmic bridge across the vacuole of the host cell. It indirectly involved in a signal transduction network contains cytoskeletal microtubules and microfilaments, that is required for the development of intracellular which together with dense endoplasmic reticulum accommodation structures for symbiotic fungi and cisternae form a hollow tube within the PPA that con- bacteria by the host cell (FIGS 5,6). The AM phenotype nects the leading nucleus with the site of appressorial of a mutant that is defective in a common symbio- contact44,46 (FIG. 5). only after this ‘transcellular tunnel’ sis gene is characterized by an early block of fungal is completed can the fungal hypha penetrate the host infection in the outer cell layers49. Phenotypic analy- cell. endoplasmic reticulum membranes that decorate sis of M. truncatula symbiotic mutants shows that the tunnel are ideally positioned for the synthesis of the the common SyM genes dMi2 and dMi3 (TABLE 1) Appressorium perifungal membrane. However, the signals that trig- are required for PPA induction44 and that dMi3 is A flattened, hyphal organ that 44 facilitates the penetration of ger the formation of the PPA are unknown. Purely required for a subset of genes to be induced during 46 cells or tissues of other mechanical stimulation of plant cells with a needle can PPA formation . Transcriptome analysis revealed that organisms. induce the nucleus to migrate towards the site of distur- most AM-induced genes are not activated in com- bance47. This might be the initial trigger during AM, as mon sym mutants46,49,50. Similarly, the transcriptional Microfilament this response is independent of the common plant SyM response to Nod factors is largely abolished51. Strong, but flexible, linear polymer of actin subunits and genes dMi2 and dMi3. To induce formation of the An analysis of calcium spiking in L. japonicus in component of the PPA, however, additional chemical cues are probably response to Nod factor revealed that symrk, castor, cytoskeleton. needed to provide specificity. The structurally related pollux, nup85 and nup133 mutants are defective for

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a b Infection thread Peri-infection probably cell-type-specific functionality, in the signal- Fungal cell wall Hyphopodium matrix thread membrane ling network that is defined by the common SYM pro- teins. Moreover, accumulating evidence suggests there is common SyM-independent signalling32,46. TABLE 1 FIGURE 6 Fungal plasma ER Rhizobia and provide an overview over the common membrane cisternae SYM pathway, whereas the following sections describe individual components. Perifungal space Perifungal SYMRK. SyMRK (also known as M. truncatula dMi2 or Weakened membrane 58,59 cell wall Medicago sativa noRK) encodes a receptor-like kinase that has an enzymatically functional kinase domain60. PIT owing to the structure of SYMrK and the symbiotic phenotype of corresponding mutants, this molecule is Plant cell PPA Vacuole wall typically portrayed as the entry point into the common symbiotic signalling pathway. In this model, SYMrK has Tonoplast the potential to directly or indirectly perceive extracel- Plant plasma membrane lular signals from microbial symbionts and transduce this perception event through its intracellular kinase domain. The ligands of the SYMrK extracellular domain have not been identified, however. Interestingly, there are at least Figure 5 | comparison of intracellular accommodation structures in bacterial and three different types of SYMrK in the angiosperm lineage fungal root endosymbioses. a | The prepenetration apparatusNature (PPA) Revie wsis a | cytoplasmicMicrobiology which differ in length and the domain structure of their bridge across the vacuole of a plant cell that forms in anticipation of fungal infection predicted extracellular regions. The shortest type (found (lower cell). The plant cell nucleus migrates ahead of the growing PPA and determines its orientation within the cell. The PPA contains a hollow tube that is formed by in rice) is sufficient for restoring AM in Lotus symrk microtubules and is lined with endoplasmic reticulum cisternae. Only after completion mutants, whereas the full-length extracellular extension of the PPA does a fungal hypha enter the PPA (upper cell). b | A preinfection thread (PIT) of SYMrK only seems to be required during interactions forms ahead of the bacteria-filled infection thread. The PIT can be induced by bacterial with rhizobia61. It is therefore possible that during AM signals alone48 and contains an array of microtubules that resemble the arrangement and root-nodule symbiosis different extracellular lig- within the PPA54. The PIT is unique to the nodulating clade and is likely to have evolved ands bind to different parts of the SYMrK extracellular from the PPA of arbuscular mycorrhiza (AM). A plant-derived perimicrobial membrane domain. SYMrK can be exchanged between M. trunca- encloses the bacteria-filled infection thread and the fungal hypha and prevents microbial tula and Lotus japonicus, and the corresponding comple- contact with the plant cytoplasm. This membrane synthesises cell wall material, which mented Lotus and Medicago symrk mutant roots regain contributes to the composition of the apoplastic interface between the symbiotic their ability to form nodules with Mesorhizobium loti and organisms. Part a modified, with permission, from REF. 44 (2005) American Society of Plant Biologists and REF. 45 (2008) American Society of Plant Biologists. Sinorhizobium meliloti, respectively. This indicates that Part b modified, with permission, from REF. 118 (2000) Elsevier Science. SYMrK is not involved in determining rhizobial recog- nition specificity. even SYMrK from actinorhiza plants that nodulate with Gram-positive bacteria of the genus calcium spiking, whereas CCaMK and CyCLoPS Frankia or non-nodulating eurosid species restores nodu- act downstream52,53. The data obtained for the lation and AM in Lotus symrk mutants61,62. Therefore, AM-induced calcium signatures in M. truncatula SYMrK does not contribute to recognitional specificity, mutants are consistent with those for Nod factor, in and probably does not directly bind to the Nod factor61. that dmi1 and dmi2 do not show this response, whereas dmi3 mutants do34. Mutants that have defective com- CASTOR and POLLUX. Mutants that are defective in mon SyM genes do not form infection threads and, CASToR or PoLLUX (also known as pea SyM8 and with the exception of cyclops mutants, do not initi- Medicago dMi1)63 are also defective in Nod-factor- ate nodule organogenesis55,56. This suggests that the induced calcium spiking64,65. cASTor and PoLLUX (or common SyM gene products are involved in the early DMI1) share similar overall domain structures and high stages of symbiotic signal transduction, which involves sequence similarity65. electrophysiological measurements the generation and decoding of calcium oscillations in of channels that were reconstituted in lipid membranes and around the nucleus and causes the induction of early and yeast-complementation experiments unambiguously symbiosis-related gene expression. consistent with this, showed that these proteins are potassium-permeable some of the predicted protein products are typical signal cation channels (M. charpentier and colleagues, per- transduction components, although the contribution of sonal communication). Importantly, these proteins are the nucleoporins is likely to be indirect (FIG. 6; TABLE 1). much less permeable for calcium, which indicates that There are subtle differences in the AM phenotypes of they are unlikely to be the channels that release calcium common sym mutants in the epidermis, the outer corti- from the storage compartment. Nuclear localization of cal cell layers and the arbuscule-forming cells57 (TABLE 1). the cASTor and PoLLUX proteins is consistent with For example, there is a clear requirement for CCaMK their proposed role as counter-ion channels that com- and CyCLoPS in arbuscule development, whereas pensate for the rapid charge imbalance that is produced arbuscules can develop normally in symrk mutant during calcium spiking (M. charpentier and colleagues, roots. This is indicative of substantial plasticity, and personal communication).

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Table 1 | Overview of common sym mutants and their corresponding phenotypes gene mutants Phenotypes of Lotus mutants Predicted function of gene product Lotus japonicus Medicago Pisum Arbuscular root-nodule calcium (previous truncatula sativum mycorrhiza (Am) symbiosis spiking§ designation) phenotype* phenotype‡ SYMRK symrk59 (sym2) dmi2 (REF. 58) and sym19 Type II Non- No Leucine-rich-repeat receptor Medicago sativa (REF. 126) nodulating|| kinase60 nork CASTOR castor65 (sym4 Unknown Unknown Types II and III Non- No Cation channel and sym71) nodulating|| POLLUX pollux65 (sym23 dmi1 (REF. 127) sym8 Type II Non- No Cation channel and sym86) (REF. 127) nodulating|| NUP85 nup85 (REF. 67) Unknown Unknown Type II Temperature No Putative nuclear pore component (sym24, sym73 temperature sensitive¶ and sym85) sensitive NUP133 nup133 (REF. 66) Unknown Unknown Type II Temperature No Putative nuclear pore component (sym3 and sym45) temperature sensitive# sensitive CCaMK ccamk73 (sym15 dmi3 sym9 Types I, II and III Non- Yes Calcium and calmodulin-dependent and sym72) (REFS 70,71) (REF. 128) nodulating||** protein kinase CYCLOPS cyclops (sym6, ipd3 (REF. 129)§§ Unknown Types II and III Small, non- Yes Unknown protein that features a sym30 and infected nuclear localization signal and a sym82)‡‡ nodules|||| carboxy-terminal coiled-coil domain *Arbuscular mycorrhiza (AM) common sym mutant phenotypes I–III are characterized by impaired epidermal opening (type I), impaired intracellular passage through the outer cell layer (or layers) (type II) and/or impaired arbuscule formation (type III). ‡Based on phenotypes described in REFS 115,130–132. §Based on phenotypes described in REFS 52,53. ||Root-hair swelling and branching occurs after inoculation with Mesorhizobium loti, but neither infection threads nor nitrogen-fixing nodules are formed. ¶More arbuscules and nodules form at 18°C compared with 22°C67. #Few nodules form at 22°C, and almost no nodules form at 26°C66. **Deregulated versions of CCaMK induce spontaneous nodule formation in the absence of rhizobia72,73. ‡‡ K. Yano and colleagues, personal communication. §§Mutants not available. |||| cyclops mutants form non-fixing, small white bumps after inoculation with M. loti (K. Yano and colleagues, personal communication).

Nucleoporins. Two genes that encode proteins with unresolved conundrum is the observation that AM fungi similarity to nucleoporins 85 and 133 are required for induce calcium signatures but no nodules. Perhaps dur- the temperature-dependent initiation of symbiosis66,67 ing AM, ccaMK is not activated to the same extent or in (FIG. 6; TABLE 1). In humans and yeast, both of these pro- the same cell types as during nodulation. Alternatively, teins belong to the same NUP107–160 subcomplex of additional layers of negative regulation might be operating the nuclear pore and are not in contact with substrates to inhibit nodule organogenesis during AM. of the canonical import and export pathways68. However, the transport of proteins that are larger than 75 kDa to the CYCLOPS. cyclops mutants severely impair the infec- inner nuclear envelope is not well understood69. because tion process of bacterial or fungal symbionts, and are Lotus NUP85 and NUP133 act upstream of calcium spik- also defective in arbuscule development49. During root- ing, a plant version of the vertebrate NUP107–160 complex nodule symbiosis, cyclops mutants exhibit specific might be involved in transporting cASTor or PoLLUX defects in infection-thread initiation, but not in nod- or both to the inner nuclear envelope. considering the ule organogenesis (K. Yano and colleagues, personal central role of the nuclear pore in transport processes communication), indicating that cYcLoPS acts in an into and out of the nucleus, it is surprising that nup85 infection-specific branch of the symbiotic signalling and nup133 mutants lack major pleiotropic defects. This network. CyCLoPS encodes a protein with no overall could be explained by either a partial redundancy of plant sequence similarity to proteins with known function, nuclear pore components or functional diversification of but contains a functional nuclear localization signal the symbiotic nucleoporin homologues. and a carboxy-terminal coiled-coil domain. cYcLoPS interacts with ccaMK in yeast and in planta and can be CCaMK. A calcium–calmodulin-dependent protein phosphorylated by ccaMK in vitro. kinase (ccaMK) is essential for AM70,71. The calmod- The signalling network that enables symbiotic ulin-binding domain and calcium-binding eF hand infection by AM fungi is starting to emerge from the motifs of ccaMK allow the protein to sense calcium, analysis of these cloned plant genes. Additional insights which makes it a prime candidate for the response to are expected from the identification of the mutations in calcium signatures that are induced by AM fungi34 or the Petunia arbuscule-development mutants74 and in maize Nod factor that induces calcium spiking. Interestingly, AM mutants74,75. a deregulated version of the protein can trigger sponta- neous nodule formation in the absence of rhizobia72,73, Arbuscule development which indicates that deregulation of ccaMK alone is Arbuscules are the result of coordinated subcellular sufficient to trigger the organogenesis programme. An development of the host plant cell and the AM fungus.

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Nod the M. truncatula phosphate transporter PT4 is present factor Myc factor in the PAM but absent from the plasma membrane77. Extracellular space Ultrastructural analyses have detected molecules in the Hypothetical Myc factor receptor periarbuscular space that are typically found in the plant b Plasma membrane primary cell wall, including -1,4-glucans, non-esterified homogalacturonans, xyloglucans, proteins that are rich in hydroxyproline and arabinogalactan proteins78. Cytoplasm Arbuscules have a shorter lifetime than the host cell (perhaps as short as 8.5 days79), and consequently, a single K+ host cell is thought to be competent for several rounds NFR5 NFR1 SYMRK + CASTOR K of successive fungal invasions. In a recent study, Javot POLLUX and colleagues80 analysed the time-scale of arbuscule Ion channel + Second K activation development in more detail. They found that arbuscules messenger undergo a phase of growth until a certain maximum size is Ca2+ Nuclear reached, after which arbuscule degradation or senescence envelope Calcium spiking is induced and the arbuscular hyphae become separated NUP133, NUP85 from the remaining cytoplasm by septation. Arbuscules Nucleoplasm CaM subsequently collapse over time and ultimately disappear. CCaMK This succession of arbuscules is costly in terms of plant and fungal resources, so why are arbuscules so short- Nuclear pore complex lived? The observation that mutation of the arbuscule- CYCLOPS Nodule specific phosphate transporter PT4 results in premature organogenesis degradation of arbuscules80 suggests that the lifetime of Intracellular arbuscules is influenced by their ability to deliver phos- infection Outer membrane Inner membrane phate and probably other nutrients. This provides the Figure 6 | common symbiosis signalling components for arbuscular mycorrhiza plant with a means to maintain efficient arbuscules and (Am) and root-nodule symbiosis. Perception of AM fungalNature or rhizobia-derived Reviews | Microbiology penalize inefficient arbuscules with early degradation. signals triggers early signal transduction, which is mediated by at least seven shared conceptually, this mechanism allows the plant not only components. The symbiosis receptor kinase SYMRK acts upstream of the Nod factor- to discriminate between efficient and inefficient fun- 34 and Myc factor-induced calcium signatures that occur in and around the nucleus . gal species but also to remove potientally ‘good’ fungal Perinuclear calcium spiking involves the release of calcium from a storage symbionts that are attached to a poor phosphate source. compartment (probably the nuclear envelope) through as-yet-unidentified calcium This concept allows fungal clones and species to compete channels. The potassium-permeable channels CASTOR and POLLUX might compensate for the resulting charge imbalance. The nucleoporins NUP85 and for arbuscule formation, which allows succession in an NUP133 are required for calcium spiking, although their mode of involvement is established root system. The spatial distribution of nutri- currently unknown. The calcium–calmodulin-dependent protein kinase (CCaMK) ents in the soil will change over time, and well-connected forms a complex with CYCLOPS, a phosphorylation substrate, within the nucleus. hyphae replace ‘non-providers’. Thus, a limited arbuscule Together with calmodulin, this complex might decode the symbiotic calcium lifetime allows constant renewal and rewiring of the signatures (K. Yano and colleagues, personal communication). Upstream of the hyphal network and allows connections to be made to common pathway, the Nod factor receptor kinases NFR1 and NFR5 are specifically the most efficient providers (FIG. 3). AM is a living fos- 39 required for Nod factor perception . It is possible that similar receptors are involved sil, and therefore mechanisms to specifically promote in Myc factor perception. Lotus japonicus protein nomenclature is used (see TABLE 1 beneficial symbiotic fungi, and to counter-select against for the names of common SyM gene orthologues of other species). inefficient ‘parasitic’ fungi, might have contributed to the long-term evolutionary stability of this symbiosis. A special role in the development of arbuscules has been The fungal hyphae branch repeatedly to produce the ascribed to lysophosphatidylcholine (LPc). LPc is a tree-shaped arbuscule structure. The exact structure that normal product of phospholipid metabolism and was is formed can vary depending on the fungal and host recently described as a signalling molecule that activates genotype2. The branches of the fungi are excluded from the expression of phosphate-transporter genes, includ- the host cytoplasm by a plant-derived periarbuscular ing the potato gene PT3 (REF. 81). This type of phosphate membrane (PAM). Nutrients, and perhaps signals, are transporter was found to be required for arbuscule main- exchanged across the symbiotic interface between the fun- tenance80. A phosphate-containing molecule, such as LPc, gus and the plant (which constitutes the PAM, the fungal might be a cell autonomous molecular measure for how plasma membrane and the periarbuscular space that exists much phosphate is made locally available to the plant. between these two membranes (FIGS. 2,7))76. The trans- Arbuscule development is accompanied by plastid porters that mediate metabolite exchange at the interface proliferation and the formation of a plastidial network in between the plant and the fungus are of key biotechnologi- close physical contact with the arbuscule82. The plastid is cal interest, and some candidate transporter genes have involved in numerous biosynthetic activities, including been cloned, although only the PT4 transporter has been the production of apocarotenoids that specifically accu- specifically localized to the PAM77. The PAM is continu- mulate in AM roots83. Given the involvement of hormones ous with the plant plasma membrane, but has a distinct in almost all plant developmental processes, it is thought protein composition, as revealed by the observation that that hormones have key roles during the development of

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Plant plasma Arbuscule Soil membrane Plant cytoplasm Fungal cell wall Sucrose Hexose Periarbuscular Fungal monosaccharide Fungal hypha space ERH transporter Hexose

Chitin NO Trehalose 3 Acetyl coenzyme A Glycerol Glycogen NH4 Fungal Glycogen Plant cytoplasm Ammonium cell transporter wall Gln TAG TAG

NH4 Urea Arg Arg Amino acid transporter

Poly-P Poly-P

Periarbuscular membrane Pi Fungal plasma Phosphate Phosphate transporter (fungus) membrane transporter (plant) Pi Plant cell wall

Figure 7 | metabolic fluxes and long-distance transport in arbuscular mycorrhiza (Am). Plant-derived carbon is transported to the fungus through the two membranes at the symbiotic interface. This carbon is first released into the Nature Reviews | Microbiology periarbuscular space (PAS), probably in the form of sucrose, then cleaved into hexoses and taken up by AM fungi through transport across the fungal plasma membrane. Within the fungal cytoplasm, hexoses are converted into glycogen granules and triacylglycerol (TAG) lipid droplets, which serve as suitable units for long-distance transport through the hyphal network. Nutrients that are acquired by the fungus from the soil and are delivered to the plant cell have to cross the fungal plasma membrane, be transported long distance to the intraradical hyphae (IRH), including the arbuscules, and subsequently reach the plant cytoplasm across the fungal plasma membrane and the plant periarbuscular membrane (PAM). Phosphate is imported by fungal phosphate transporters (cloned from Glomus intraradices87 and Glomus versiforme86) that are present in extraradical hyphae (ERH). Phosphate is transported towards the root and IRH in the form of polyphosphate granules, which reside in membrane-enclosed vesicles. The negative charge of these granules makes them likely transport vehicles for metal ions and arginine. Phosphate is released from polyphosphate granules within IRH. Plant transporters that are involved in phosphate transport across the PAM have been cloned and characterized80,101–103, whereas the fungal phosphate transporters that are responsible for the release of phosphate from IRH are still unknown. Nitrogen is taken up by ammonium88, nitrate or amino-acid transporters in ERH. In AM fungal hyphae, nitrogen is mainly transported as arginine106. Within the IRH, nitrogen is released from arginine as urea and either transported to the plant directly or after cleavage to ammonium. Figure modified, with permission from REF. 8 (2003) American Society of Plant Biologists, REF. 90 (2005) Blackwell Publishing and Nature REF. 106 (2005) Macmillan Publishers Ltd. All rights reserved.

AM. This is still a developing area of research, but abscisic Carbon metabolism. our understanding of the meta- and jasmonic acid have emerged as potential regulators bolic functions of the AM and AM fungi has been of AM84,85. boosted by the development of axenic culture systems and the ability to restrict fungal and plant tissue to AM Function separate compartments, together with isotope labelling Nutrient uptake and transport in the extraradical and in situ NMr (metabolism and transport in AM has mycelium. Fungal hyphae explore the soil substratum, but been reviewed previously90,91). The plant can control different AM fungi seem to use different strategies to do the flux of sucrose directed to the root, including the so (FIG. 3). The fungal hyphal network is ideally positioned fungus. Jasmonic acid has been proposed to be involved to efficiently take up nutrients and water from the soil, but in the regulation of sink strength of AM roots92. Sucrose only a few fungal transporters that are involved in this that is delivered to the AM root is cleaved either by process, including those that transport phosphate86,87, symbiosis-induced sucrose synthases93 or invertases94. ammonium88 and zinc89, have been cloned. because in vivo NMr studies indicate that AM fungi obtain diffusion is too slow, nutrients are moved in a pack- hexoses from the plant and convert them into lipids and aged form between the extraradical and the intraradical glycogen for long-distance transport8,95. A member of the mycelium (FIG. 7). novel clade of hexose transporters that was identified from

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the symbiotic organ of the glomeromycotan Geosiphon fossil record of early land plants from the rhynie chert pyriformis might also be expressed in the fungal interface in Scotland provided ‘rock-solid’ evidence that typical membrane of the arbuscule96. In a typical AM, no carbon AM fungal structures, such as arbuscules and spores, transport from the fungus to the plant was detected97. were already present 400 million years ago108,109. The However, mycoheterotrophic plants that associate with high level of organization in these fossils and the wide AM fungi are likely to receive carbon from the fungus26, distribution of AM in all branches of the phylogenetic and it is proposed that photosynthetic sporophytes of tree of plants suggest that AM might have been present species of Huperzia deliver carbon to mycoheterotrophic in a common ancestor and perhaps was instrumental in gametophytes through shared fungal networks98. This the initial colonization of land. Interestingly, the rhynie would require carbon transporters that work in the efflux chert fossils contain an impressive range of other fun- direction, which have not yet been reported in fungi. gal endophytes, including potential parasites110, that Alternatively the mycoheterotrophic plant must obtain provoked the formation of structural ‘defences’ by the carbon by efficiently digesting fungal hyphae, similar to plant, which reveals the ancient nature of symbiosis and the orchid symbiosis. defence programmes in land plants. Molecular-clock estimates of the age of members of the Glomeromycota Phosphate. Improved phosphate uptake is the main bene- differ by several hundred million years111,112, but raise the fit of the AM symbiosis99,100. The extensive hyphal network possibility that they evolved before land plants. In this of AM fungi influences the physicochemical properties of context, it is interesting that the glomeromycotan fungus the soil and directly or indirectly contributes to the release G. pyriformis forms a symbiosis with photosynthetic of phosphate from inorganic complexes of low solubility6. (and nitrogen-fixing) species of nostoc cyanobacteria Fungal phosphate transporters that are expressed in the and that similar fungus–bacteria interactions might extraradical mycelium are probably involved in the uptake have preceded the AM symbiosis113. consistent with an of phosphate from the substratum86,87. Polyphosphate ancient fungal-uptake mechanism for bacteria, most AM granules are used as transport vehicles to move phosphate fungi harbour endosymbiotic bacteria, including Gram- (and possibly arginine and trace elements) to the host root. negative Burkholderia species114 and uncharacterized Symbiosis-induced plant phosphate transporter genes Gram-positive species113, indicating multiple independent have been identified in different plant species (reviewed uptake events of symbiotic bacteria. in REF. 101), and accumulating evidence suggests a role for at least a subset of the corresponding proteins in symbiotic A conserved ancient genetic programme for AM phosphate transport80,102,103. Fusions of potato PT3 or M. The presence of AM in the earliest land plants raises the truncatula PT4 promoters to GUS targeted expression possibility that the underlying genetic programme is specifically to arbuscule-containing cells, which is con- conserved among extant AM-forming plants115. Indeed, sistent with results from laser-capture microdissection of phylogenetic and functional conservation of common tomato arbuscules, in which transcripts of five isoforms SyM genes, at least in the angiosperm lineage, has were detected in arbuscule-containing cells104. recently been described61,116. evidence from legumes indicates that the common symbiosis genes are required Nitrogen. AM fungi can accelerate decomposition for the formation of the intracellular accommodation and directly acquire nitrogen from organic material105. structure PPA44. This suggests that these genes are com- A fungal amino-acid transporter46 and an ammonium ponents of an ancient and conserved programme that transporter that might be involved in nitrogen uptake by evolved before the divergence of the angiosperms and extraradical hyphae have been cloned88. Long-distance was retained in most lineages because of the selective transport to the plant probably proceeds mainly through advantages conferred by AM. arginine106,107 (FIG. 7). Nitrogen is released in a carbon-free form (probably ammonium) to the plant106, although AM is the ancestor of bacterial root endosymbioses. The the ammonium transporters in the symbiotic interface discovery that some nodulation-defective legume mutants membranes have not yet been identified. are also defective in AM development revealed a genetic link between bacterial and fungal symbiosis, which has led Evolution of plant root endosymbiosis to the hypothesis that the root-nodule symbiosis evolved All members of the Glomeromycota phylum require a from AM functions. Now that common symbiosis genes photosynthetic partner to complete their life cycle. Not have been cloned and functionally characterized from a single member of this lineage has escaped from this different nodulating and non-nodulating angiosperm dependency, which suggests that the ancestral fungus species, we can draw a more detailed picture of the events was already an obligate biotroph. This extreme specializa- that led to the evolution of the root-nodule symbiosis. tion of an entire fungal clade is unique, as members of all The combined results suggest that the common sym- phylogenetically comparable lineages — the ascomycetes, biosis programme evolved in the context of AM and basidiomycetes and the non-monophyletic zygomycetes was recruited for the bacterial root-nodule symbiosis115. and chytridiomycetes (in the classical sense; for a more The identified genes are all required for induction of the recent classification see REF. 4) — inhabit a wide range intracellular accommodation programme, a common of ecological niches and include plant and animal patho- feature of both bacterial and fungal root endosymbiosis gens and symbionts, as well as free-living saprophytes. (FIG. 5). Most common SyM genes are conserved in over- AM is indeed an ancient symbiosis, and the excellent all domain composition between legumes and rice.

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A notable exception is SYMrK, which exhibits remarkable AM fungi, it has been suggested that both parasitic and variation in its extracellular domain composition across symbiotic fungi rely on partially overlapping intracellular angiosperms. Full-length SYMrK is consistently found in accommodation programmes of the plant118. However, all tested members of the eurosid clade, whereas all other L. japonicus mutants that are defective in common tested angiosperms contain shorter types that lack one of symbiosis genes support completion of the life cycle of a the leucine-rich repeats (Lrrs), or one Lrr and a long leaf rust fungus, including the formation of intracellular amino-terminal extension. A central role of SYMrK in the haustoria119. So far, plant genes that support biotrophic evolutionary events that led to nodulation was revealed fungal pathogens are largely unknown, and therefore the by the observation that only full-length SYMrK can fully molecular components of such a shared programme have complement Lotus symrk mutants and restore nodulation, been elusive. whereas shorter versions are sufficient only for AM61. This finding suggests that exon acquisition in an ancestor Conclusions and outlook of the eurosids was associated with functional adaptation of over the past few years, a novel and unexpected devel- SYMrK and provided the basis for bacterial triggering opmental capacity of plant cells has been discovered of the common symbiosis programme by bacteria61. In that is essential for the intracellular uptake of AM fungi. a hypothetical scenario, SYMrK evolution was a prereq- Plant genetics will continue to be a major tool in the uisite for an intracellular symbiosis with nitrogen-fixing identification of genes that are required for AM develop- bacteria that was manifested by the formation of intracel- ment and function. It is expected that in the near future, lular infection threads. Such an early bacterial symbiosis the chemical structure of the fungal Myc factor that might not have been associated with nodule organo- triggers the symbiotic responses of the root will be pub- genesis115. A situation that mimics such early bacterial lished, which will help us to identify the cognate plant endosymbiosis is found in the hit1/har1 double mutant of receptors. To unlock the potential of AM for sustainable L. japonicus, which develops abundant infection threads agriculture, we must identify the key molecular players. in the absence of nodule formation117. equally importantly, we must investigate the natural Phylogenetic and functional analysis of the symbiosis variation for AM function and responsiveness within receptor kinase gene SyMRK has revealed functional and biodiversity collections of important crop plants120 and structural polymorphism across angiosperms, which between different fungal lineages. The long-term aim is suggests that this gene had a key role in the evolution of to identify or design crop–fungus combinations with the nitrogen-fixing root-nodule symbiosis on the basis optimized AM performance, which would be instrumen- of a pre-existing AM genetic programme. tal in reducing the application of fertilizer and energy because many of the important plant pathogenic input, a goal that is mandatory in a world of depleting fungi share an intracellular biotrophic lifestyle with non-renewable resources121.

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