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First published online November 11, 2005; 10.1105/tpc.105.035410 The Plant Cell 17:3489-3499 (2005) © 2005 American Society of Plant Biologists Arbuscular Mycorrhizal Fungi Elicit a Novel Intracellular Apparatus in Medicago truncatula Root Epidermal Cells before Infection[W]
a Department of Plant Biology, University of Turin and Istituto per la Protezione delle Piante–Consiglio Nazionale delle Richerche, 10125 Turin, Italy 1 To whom correspondence should be addressed. E-mail barker{at}toulouse.inra.fr; fax 33-561-285061.
The penetration of arbuscular mycorrhizal (AM) fungi through the outermost root tissues of the host plant is a critical step in root colonization, ultimately leading to the establishment of this ecologically important endosymbiotic association. To evaluate the role played by the host plant during AM infection, we have studied in vivo cellular dynamics within Medicago truncatula root epidermal cells using green fluorescent protein labeling of both the plant cytoskeleton and the endoplasmic reticulum. Targeting roots with Gigaspora hyphae has revealed that, before infection, the epidermal cell assembles a transient intracellular structure with a novel cytoskeletal organization. Real-time monitoring suggests that this structure, designated the prepenetration apparatus (PPA), plays a central role in the elaboration of the apoplastic interface compartment through which the fungus grows when it penetrates the cell lumen. The importance of the PPA is underlined by the fact that M. truncatula dmi (for doesn't make infections) mutants fail to assemble this structure. Furthermore, PPA formation in the epidermis can be correlated with DMI-dependent transcriptional activation of the Medicago early nodulin gene ENOD11. These findings demonstrate how the host plant prepares and organizes AM infection of the root, and both the plant–fungal signaling mechanisms involved and the mechanistic parallels with Rhizobium infection in legume root hairs are discussed.
Arbuscular mycorrhizae (AM) are highly specialized endosymbiotic associations formed between a restricted group of biotrophic soil fungi (the Glomeromycota) and the large majority of vascular land plants, including most angiosperm and gymnosperm families. Fossil evidence shows that AM symbiosis has existed for >450 million years (Remy et al., 1994
Despite the agronomic and ecological importance of the AM symbiosis, the molecular and cellular events associated with the establishment of the association are poorly understood. This is primarily attributable to the difficulty in culturing these obligate fungi, coupled with the low frequency and lack of synchrony of host infection. Nevertheless, it is now clearly established that, before infection, germinated AM fungi respond to host root exudates by switching to an active presymbiotic growth phase, which leads to intense hyphal ramification (or branching) in the vicinity of the root (Giovannetti et al., 1993
To date, remarkably little is known about the crucial stage of the interaction that follows the initial fungal–plant contact and precedes infection, and in particular the nature of the molecular/cellular dialog that is required for recognition of the fungal partner and successful infection. However, genetic studies performed with several legume genera, such as Pisum, Medicago, and Lotus, have revealed that a small group of plant genes are essential for successful root penetration (reviewed in Parniske, 2004
To facilitate detailed molecular/cellular studies of the AM infection process, an experimental system was recently developed for M. truncatula using Agrobacterium rhizogenes–transformed roots targeted with negative geotrophic Gigaspora germination hyphae (Chabaud et al., 2002
Despite the genetic and molecular analogies that can be made between Rhizobium and AM infection, it is still unclear how and to what extent the plant plays an active role in the AM penetration process (Parniske, 2000
GFP-Labeled Cellular Markers Expressed in Root Tissues of M. truncatula M. truncatula root clones expressing appropriate GFP-labeled markers for monitoring both the plant cytoskeleton and the ER were generated to study the in vivo intracellular responses and remodeling that occur during the preinfection and infection stages of the AM–plant association (see Methods). GFP:Map4-MBD (Marc et al., 1998  ) was chosen to visualize the microtubular cytoskeleton, GFP:Fimbrin1-ABD (Voigt et al., 2005) was chosen to label actin filaments, and GFP-HDEL (Haseloff et al., 1997) was chosen for ER labeling. The localization of the three GFP-based fusions was first evaluated in epidermal cells of control noncolonized roots. Figure 1A illustrates the typical parallel, predominantly oblique arrays of cortical microtubules labeled by the GFP:Map4-MBD fusion protein (Marc et al., 1998), and Figure 1B shows GFP:Fimbrin1-ABD labeling of the bundled actin microfilaments that originate in the perinuclear cytoplasm and spread across the cell. Finally, the GFP-HDEL marker labels the characteristic ER structure, comprising a lace-like network of lamellar and tubular cisternae present throughout the cortical cytoplasm, with a maximum density around the nucleus (Figure 1C). As expected, time-lapse imaging (data not shown) revealed very dynamic structures for both the fine cortical network of actin filaments (Voigt et al., 2005) and the ER (Staehelin, 1997). We conclude that the transgenic M. truncatula root clones expressing these three GFP cellular tags can be used to identify and follow changes in the structure of the epidermal cell cytoskeleton and ER as well as movements of the cell nucleus in the case of the actin and ER tags.
Epidermal AM Infection Is Preceded by Nucleus-Directed Assembly of a Novel Cytoskeletal/ER Apparatus For the studies described in this article, the targeted AM inoculation technique developed for in vitro–cultured M. truncatula roots (Chabaud et al., 2002 ) was modified to permit continuous microscopic observation (see Methods). Microscopic analysis of >100 independent infection events using the three GFP markers and two Gigaspora species (with time-lapse imaging for a number of penetration events) led to the identification of a series of host cellular responses that are systematically associated with epidermal AM fungal infection. These responses, described in detail below, can be subdivided into those that precede fungal penetration and those that are associated with subsequent intracellular hyphal growth. In particular, the epidermal cell nucleus, with two distinct phases of intracellular movement, appears to play a central role in the dynamics associated with the preinfection responses, which include the assembly of a novel cytoskeleton/ER-containing structure. AM root infection initiates from surface appressoria, whose formation is generally indicated by tip growth arrest and associated hyphal swelling. Observation of numerous hyphal contacts with associated appressoria formation suggests that the epidermal cell nucleus rapidly moves toward and positions itself directly below the site of appressorium contact (or ACS). Several time-lapse observations have shown that this initial nuclear movement is generally completed within <2 h. Associated with this first phase of nuclear movement, we have also observed cortical microtubules reorganizing from parallel loop arrays to a network of randomly oriented bundles (Figure 1D). Note that the nuclear position is visible only with the actin and ER GFP tags. During this nuclear repositioning at the ACS and before the initiation of the second phase of nuclear migration, we have observed thick actin bundles radiating from the nucleus toward the ACS (Figure 1E) and large patches of ER assembling below the ACS and around the nucleus (Figure 1F). This is followed by additional cytoskeletal/ER restructuring, which includes the assembly of a dense subconical set of microtubules (Figure 1G) as well as a radial array of actin bundles (Figure 1H). The ER patches assemble into an approximately doughnut-shaped configuration below the ACS (Figure 1I) as the nucleus initiates a migration across the cell lumen away from the appressorium at an estimated speed of 15 to 20 µm/h. This migration is accompanied by the creation of a broad cytoplasmic column linking the nucleus to its initial position below the ACS. Within this column, all three GFP markers reveal the assembly of a striking structure comprising a high-density array of microtubules (Figure 1J), microfilament bundles running parallel to the column (Figure 1K), and a very dense region of ER cisternae (Figure 1L). Three-dimensional imaging further reveals that the dense ER structure is in fact a hollow tube joining the nucleus to the ACS (see Supplemental Figures 1A to 1D online). Estimations from time-lapse imaging suggest that 4 to 5 h are required for the formation of this novel intracellular structure after initial fungal contact and appressorium formation.
Hyphal Penetration Follows the Path Defined by the Transcellular Nuclear Migration
The images shown in Figures 1M to 1O illustrate hyphae that have penetrated, and in certain cases completely traversed, the epidermal cell layer. Once hyphae have crossed the cell, the nucleus is in general no longer positioned at the end of the cytoplasmic column (clearly visible in Figure 1O). This suggests that the nucleus detaches from the end of the column and repositions at the cell periphery once infection has been completed. Although cytoskeletal/ER labeling is still present peripheral to the penetrating hyphae (Figures 1M to 1O), the overall fluorescence intensity of the GFP markers has decreased significantly compared with that observed before infection (cf. with Figures 1J to 1L). We interpret this finding as a dismantling of the preinfection assembly at some stage after hyphal entry. In our experiments, hyphal penetration has been observed both through the outer epidermal cell wall and through the anticlinal wall between adjacent epidermal cells, as described for Lotus japonicus infection (Bonfante et al., 2000
To provide further evidence for a direct relationship between the formation of the cytoskeleton/ER-containing structure and the synthesis of the perifungal membrane, we stained the root with the vital lipophilic fluorescent dye FM 4-64 (Bolte et al., 2004
Together, our results show that the formation of a specific structure comprising microtubules, microfilaments, and ER is preceded by the migrating nucleus within a cytoplasmic column traversing the epidermal cell, and this structure plays a key role in constructing the apoplastic compartment through which the fungal infection hyphae subsequently traverse the epidermal layer. We have designated this novel transient structure the PPA.
PPAs Are Not Formed in Epidermal Cells of Roots Derived from dmi2 and dmi3 Mutants
When transgenic roots derived from the two dmi mutants were targeted with germination hyphae of either Gigaspora gigantea or Gigaspora rosea, the hyphae ramified in the vicinity of the roots and fungal appressoria were formed on the root surface, as described previously for dmi2 (Chabaud et al., 2002 Significantly, detailed microscopic analyses of AM fungal–root contacts, making use of both ER and actin markers, failed to reveal any cellular responses for either dmi mutant indicative of the formation of a PPA structure (such as the assembly of ER cisternae). Likewise, plasma membrane invaginations in epidermal cells underneath ramifying fungal hyphae were never observed in labeling experiments with the vital FM 4-64 marker (Figure 2C). On the other hand, we did observe a frequent positioning of epidermal cell nuclei directly below ramifying hyphae on the dmi2 and dmi3 root surfaces (Figures 2D to 2F). Together, these results suggest that the epidermal cells in roots of dmi2 and dmi3 are still capable of responding to fungal contact with an initial phase of nuclear movement toward the contact site, but there is a subsequent defect in their capacity to initiate PPA formation.
Correlation between PPA Formation and the Transcriptional Activation of the M. truncatula ENOD11 Gene Gigaspora-targeting experiments systematically revealed PENOD11:GFP-HDEL activation in epidermal cells in contact with fungal appressoria (Figure 2G) as well as in a very limited number of adjacent cells. Note that there is little if any fluorescence induction in the majority of the surrounding epidermal cells, suggesting that ENOD11 activation is probably cell autonomous within the epidermal layer. Among the fluorescent epidermal cells examined, a few showed typical PPA structures associated with very intense ER labeling. Figure 2H illustrates one such cell in which the fungus has not yet entered the cell. At a later stage of infection, when the fungus has traversed the epidermal cell, ER fluorescence around the fungus is still visible, but at a significantly lower level (Figure 2I). This finding is in agreement with the results obtained earlier with the 35S-driven GFP-HDEL construct (cf. with Figures 1L and 1O).
In conclusion, these observations suggest that the M. truncatula ENOD11 gene is activated in epidermal cells after fungal appressorium differentiation both before and during PPA formation and probably also during hyphal penetration. It also appears that not all of the cells expressing PENOD11:GFP-HDEL are subsequently infected. Because our time-lapse experiments have shown that
The PPA, a Novel Plant Intracellular Structure Preparing AM Root Infection In this article, we have described in detail the cellular responses elicited in the host plant root associated with the preparation of the epidermal cell layer for penetration by AM fungi. Real-time imaging coupled with GFP tagging of cytoskeletal/ER components has revealed a complex multistep host response that precedes fungal entry and that ultimately leads to the synthesis of the transcellular apoplastic compartment separating the penetrating hypha from the host cytoplasm. Most striking among our findings has been the identification of a novel structure comprising microtubules, microfilaments, and ER, which we termed the PPA. This structure is assembled within a column of cytoplasm created during the progressive migration of the nucleus across the epidermal cell and defines the future path taken by the infection hyphae. The fact that the identical structure is labeled by all three fluorescent markers makes it extremely unlikely that the PPA is an artifact resulting from the use of transgenic roots expressing GFP tags. The reasons for proposing the PPA as a key cellular actor in AM infection are based on the following observations. (1) The formation of the PPA is strictly associated with the transcellular nuclear migration that always initiates from a position directly below the appressorium (Figures 1J to 1L). (2) Successful hyphal infection always follows the transcellular path laid down by the PPA (Figures 1P to 1R). (3) PPA formation and the associated nuclear migration are totally absent in roots derived from the two infection-defective dmi mutants (dmi2-2 and dmi3-1) that we have analyzed. (4) Expression of the infection-related ENOD11 gene can be observed in PPA-containing cells (Figure 2H). Together, these findings lead us to propose that the PPA is a major player in the synthesis of the membrane–matrix interface that surrounds and isolates the infection hypha from the cell cytoplasm. This is supported by the labeling experiments using the vital stain FM 4-64, which revealed a putative membrane structure that colocalizes with the cytoplasmic column and associated PPA (Figure 2B; see Supplemental Figures 1E to 1G online). Additional experiments are now required to study the dynamics of membrane synthesis and the role of other cellular components required for membrane–matrix synthesis, such as the Golgi apparatus. We estimate from the observation of numerous AM infections that 7 to 8 h are required for both PPA formation and hyphal crossing of the epidermis. This relatively short time, coupled with the transient nature of the PPA and the overall low frequency of infections, probably explains why this transient structure has not been observed previously in chemically fixed tissues. The discovery of the PPA suggests that the plant root plays a central role in preparing for and directing AM infection, and the implications of this in terms of cellular signaling between the plant and fungal symbiont are discussed below. An animated movie, presenting the entire AM infection process, in particular the roles of the plant cell nucleus, cytoskeleton, and ER in PPA formation, can be viewed in Supplemental Video 2 online. Four stills illustrating key steps from the animation are presented in Figures 2J to 2M.
At this stage, it is too early to conclude that the intracellular infection mechanism that we have observed for M. truncatula interacting with both G. gigantea and G. rosea can be generalized for all types of AM fungi and for all host plants. Interestingly, studies with the model legume L. japonicus have shown that Nod– Myc– mutants such as Ljsym4-2 are blocked for hyphal infection within the epidermal layer (Wegel et al., 1998
Symbiotic Fungal–Plant Signaling Leading to PPA Synthesis
In the case of Nod factor signal transduction in root hairs, intracellular Ca2+ oscillations are one of the early characteristic cellular responses (Ehrhardt et al., 1996 ), and it has been proposed that the role of the downstream DMI3 calcium- and calmodulin-dependent kinase is to interpret this Ca2+ signature (reviewed in Oldroyd and Downie, 2004). Because dmi3-1 is defective in PPA formation, intracellular Ca2+ signaling could also be an essential component of the PPA activation pathway (Figure 3). If this turns out to be the case, then it will be important to establish whether the AM-elicited Ca2+ signature is identical or not to the Nod factor–elicited intracellular oscillation pattern.
In our scheme, we have positioned the activation of the M. truncatula ENOD11 gene upstream of PPA assembly, because we have observed expression of the PENOD11:GFP-HDEL reporter in epidermal cells contacted by appressoria but without visible PPAs (Figure 2G). Previous experiments have shown that the PENOD11:GUS reporter is not transcribed in a dmi2 mutant background despite the formation of surface appressoria (Chabaud et al., 2002
Recently, it was shown by Kosuta et al. (2003)
Finally, recent studies have revealed that at least two distinct and separable regulatory regions of the ENOD11 promoter are responsible for gene activation during early stages of the Medicago–Sinorhizobium association. The first region is sufficient for upregulating expression in response to Nod factor perception in root hairs, and the second is sufficient for activating gene expression in tissues associated with rhizobial infection (Boisson-Dernier et al., 2005
Does the PPA Play a Role in Other Endocellular Plant–Microbe Interactions?
There is currently no evidence that the equivalent to preinfection thread–containing cortical cells are formed before or during AM root infection; indeed, we have never observed this very characteristic cellular differentiation in cortical cells underlying epidermal AM infection sites. However, it should be borne in mind that in the case of Rhizobium infection, the bacteria are delivered to the developing nodule primordium in the inner cortex (at least in the case of temperate legumes forming indeterminate nodules, such as Medicago). On the other hand, AM fungal development within the root cortex is quite different, with a progressive and continual colonization of the inner cortex associated with combined intercellular hyphal growth and penetration of certain host cells to differentiate arbuscules. In this respect, it is interesting that Blancaflor et al. (2001)
Finally, it is important to ask whether analogies can be made with plant infection by certain hemibiotrophic fungi. For example, in the case of a number of Colletotrichum species, there is an initial biotrophic phase during which penetration hyphae differentiate specialized structures in epidermal cells that are surrounded by an invaginated plant membrane and interface matrix (reviewed in Mendgen and Hahn, 2002
Plant and Fungal Materials Experiments were performed with Agrobacterium rhizogenes–transformed root cultures derived from both wild-type Medicago truncatula Jemalong A17 and two mutants, dmi2-2 (TR26; Sagan et al., 1995 ) and dmi3-1 (TRV25; Sagan et al., 1998), kindly provided by G. Duc (Institut National de la Recherche Agronomique). The AM fungal partners used in this study are Gigaspora gigantea and Gigaspora rosea. Spores of G. gigantea were produced using clover (Trifolium repens) culture on sand, surface-sterilized, and stored at 4°C according to Bécard and Fortin (1988). Spores of G. rosea (DAOM 194757) were produced in vitro using M. truncatula hairy roots instead of Daucus carota hairy roots. G. gigantea has the advantage of possessing a yellowish cytoplasmic autofluorescence (Séjalon-Delmas et al., 1998), greatly facilitating the coordinate monitoring of hyphal position and host intracellular reorganization, and therefore was used preferentially.
GFP-Tagged Cellular Markers Expressed in M. truncatula Roots
Conditions for in Vivo Microscopic Observation of M. truncatula Root Infection by Gigaspora AM Fungi
Confocal Microscopy
Accession Number
Supplemental Data
We thank Richard Cyr (Pennsylvania State University), Dorus Gadella (University of Amsterdam), Jim Haseloff (Cambridge University), and Boris Voigt (University of Bonn) for providing various cytoskeletal/ER GFP tags. Our thanks also to Alain Jauneau for assistance with confocal microscopy, to Annie Dedieu for aid in constructing the PENOD11:GFP-HDEL reporter, to Gerard Duc (Institut National de la Recherche Agronomique, Dijon, France) for the dmi2-2 and dmi3-1 mutants, and to Fernanda Carvalho-Niebel, Etienne-Pascal Journet, and John Esseling (Laboratory of Plant–Microbe Interactions) for useful criticisms of the manuscript. A.G. received a Marie Curie Fellowship for a 3-month visit to the Laboratory of Plant–Microbe Interactions. This work was supported by grants from the Italian Ministry of Education, University, and Research to P.B. (Fondo Investimenti Ricerca di Base Project Plant/Microbe Interactions, Progetti di Rilevanza Nazionale 40% 2003–2005, IPP-Consiglio Nazionale delle Richerche and Centro di Eccellenza per la Biosensoristica Vegetale e Microbica, University of Torino). The San Paolo Company (Torino, Italy) is acknowledged for partly supporting the acquisition of a Leica confocal microscope at the Laboratory of Advanced Microscopy.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: David G. Barker (barker{at}toulouse.inra.fr).
[W] Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.035410. Received June 21, 2005; Revision received September 25, 2005. accepted October 17, 2005.
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3 h, with an estimated speed of 20 µm/h. Finally, the hyphal penetration event depicted in 
