Chitotetraose activates the fungal-dependent endosymbiotic signaling pathway in actinorhizal plant species.

Mutualistic plant-microbe associations are widespread in natural ecosystems and have made major contributions throughout the evolutionary history of terrestrial plants. Amongst the most remarkable of these are the so-called root endosymbioses, resulting from the intracellular colonization of host tissues by either arbuscular mycorrhizal (AM) fungi or nitrogen-fixing bacteria that both provide key nutrients to the host in exchange for energy-rich photosynthates. Actinorhizal host plants, members of the Eurosid 1 clade, are able to associate with both AM fungi and nitrogen-fixing actinomycetes known as Frankia. Currently, little is known about the molecular signaling that allows these plants to recognize their fungal and bacterial partners. In this article, we describe the use of an in vivo Ca2+ reporter to identify symbiotic signaling responses to AM fungi in roots of both Casuarina glauca and Discaria trinervis, actinorhizal species with contrasting modes of Frankia colonization. This approach has revealed that, for both actinorhizal hosts, the short-chain chitin oligomer chitotetraose is able to mimic AM fungal exudates in activating the conserved symbiosis signaling pathway (CSSP) in epidermal root cells targeted by AM fungi. These results mirror findings in other AM host plants including legumes and the monocot rice. In addition, we show that chitotetraose is a more efficient elicitor of CSSP activation compared to AM fungal lipo-chitooligosaccharides. These findings reinforce the likely role of short-chain chitin oligomers during the initial stages of the AM association, and are discussed in relation to both our current knowledge about molecular signaling during Frankia recognition as well as the different microsymbiont root colonization mechanisms employed by actinorhizal hosts.

Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization.

Nitrogen-fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little-researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection-associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing in vivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin-like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D. trinervis outer root tissues. This is accompanied by nucleo-cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen-fixing rhizobia.

Ancestral alliances: Plant mutualistic symbioses with fungi and bacteria.

Within the plant microbiota, mutualistic fungal and bacterial symbionts are striking examples of microorganisms playing crucial roles in nutrient acquisition. They have coevolved with their hosts since initial plant adaptation to land. Despite the evolutionary distances that separate mycorrhizal and nitrogen-fixing symbioses, these associations share a number of highly conserved features, including specific plant symbiotic signaling pathways, root colonization strategies that circumvent plant immune responses, functional host-microbe interface formation, and the central role of phytohormones in symbiosis-associated root developmental pathways. We highlight recent and emerging areas of investigation relating to these evolutionarily conserved mechanisms, with an emphasis on the more ancestral mycorrhizal associations, and consider to what extent this knowledge can contribute to an understanding of plant-microbiota associations as a whole.

The rice LysM receptor-like kinase OsCERK1 is required for the perception of short-chain chitin oligomers in arbuscular mycorrhizal signaling.

The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca2+ ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca2+ signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca2+ spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.

Nuclear Ca2+ signalling in arbuscular mycorrhizal and actinorhizal endosymbioses: on the trail of novel underground signals.

Contents 533 I. 533 II. 534 III. 536 IV. 536 537 References 537 SUMMARY: Root endosymbioses are beneficial associations formed between terrestrial plants and either bacterial or fungal micro-organisms. A common feature of these intracellular symbioses is the requirement for mutual recognition between the two partners before host-regulated microbial entry. As part of this molecular dialogue, symbiosis-specific microbial factors set in motion a highly conserved plant signal transduction pathway, of which a central component is the activation of sustained nuclear Ca2+ oscillations in target cells of the host epidermis. Here, we focus on recent findings concerning this crucial Ca2+ -dependent signalling step for endosymbiotic associations involving either arbuscular mycorrhizal fungi or nitrogen-fixing Frankia actinomycetes, and in particular how this knowledge is contributing to the identification of the respective microbial factors.

The Symbiosis-Related ERN Transcription Factors Act in Concert to Coordinate Rhizobial Host Root Infection.

Legumes improve their mineral nutrition through nitrogen-fixing root nodule symbioses with soil rhizobia. Rhizobial infection of legumes is regulated by a number of transcription factors, including ERF Required for Nodulation1 (ERN1). Medicago truncatula plants defective in ERN1 are unable to nodulate, but still exhibit early symbiotic responses including rhizobial infection. ERN1 has a close homolog, ERN2, which shows partially overlapping expression patterns. Here we show that ern2 mutants exhibit a later nodulation phenotype than ern1, being able to form nodules but with signs of premature senescence. Molecular characterization of the ern2-1 mutation reveals a key role for a conserved threonine for both DNA binding and transcriptional activity. In contrast to either single mutant, the double ern1-1 ern2-1 line is completely unable to initiate infection or nodule development. The strong ern1-1 ern2-1 phenotype demonstrates functional redundancy between these two transcriptional regulators and reveals the essential role of ERN1/ERN2 to coordinately induce rhizobial infection and nodule organogenesis. While ERN1/ERN2 act in concert in the root epidermis, only ERN1 can efficiently allow the development of mature nodules in the cortex, probably through an independent pathway. Together, these findings reveal the key roles that ERN1/ERN2 play at the very earliest stages of root nodule development.

Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca(2+) spiking and NIN gene expression in the actinorhizal plant Casuarina glauca.

Although it is now well-established that decorated lipo-chitooligosaccharide Nod factors are the key rhizobial signals which initiate infection/nodulation in host legume species, the identity of the equivalent microbial signaling molecules in the Frankia/actinorhizal association remains elusive. With the objective of identifying Frankia symbiotic factors we present a novel approach based on both molecular and cellular pre-infection reporters expressed in the model actinorhizal species Casuarina glauca. By introducing the nuclear-localized cameleon Nup-YC2.1 into Casuarina glauca we show that cell-free culture supernatants of the compatible Frankia CcI3 strain are able to elicit sustained high frequency Ca(2+) spiking in host root hairs. Furthermore, an excellent correlation exists between the triggering of nuclear Ca(2+) spiking and the transcriptional activation of the ProCgNIN:GFP reporter as a function of the Frankia strain tested. These two pre-infection symbiotic responses have been used in combination to show that the signal molecules present in the Frankia CcI3 supernatant are hydrophilic, of low molecular weight and resistant to chitinase degradation. In conclusion, the biologically active symbiotic signals secreted by Frankia appear to be chemically distinct from the currently known chitin-based rhizobial/arbuscular mycorrhizal signaling molecules. Convenient bioassays in Casuarina glauca are now available for their full characterization.

A role for the mevalonate pathway in early plant symbiotic signaling.

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.

Remodeling of the infection chamber before infection thread formation reveals a two-step mechanism for rhizobial entry into the host legume root hair.

In many legumes, root entry of symbiotic nitrogen-fixing rhizobia occurs via host-constructed tubular tip-growing structures known as infection threads (ITs). Here, we have used a confocal microscopy live-tissue imaging approach to investigate early stages of IT formation in Medicago truncatula root hairs (RHs) expressing fluorescent protein fusion reporters. This has revealed that ITs only initiate 10 to 20 h after the completion of RH curling, by which time major modifications have occurred within the so-called infection chamber, the site of bacterial entrapment. These include the accumulation of exocytosis (M. truncatula Vesicle-Associated Membrane Protein721e)- and cell wall (M. truncatula EARLY NODULIN11)-associated markers, concomitant with radial expansion of the chamber. Significantly, the infection-defective M. truncatula nodule inception-1 mutant is unable to create a functional infection chamber. This underlines the importance of the NIN-dependent phase of host cell wall remodeling that accompanies bacterial proliferation and precedes IT formation, and leads us to propose a two-step model for rhizobial infection initiation in legume RHs.

High phosphate reduces host ability to develop arbuscular mycorrhizal symbiosis without affecting root calcium spiking responses to the fungus.

The arbuscular mycorrhizal symbiosis associates soil fungi with the roots of the majority of plants species and represents a major source of soil phosphorus acquisition. Mycorrhizal interactions begin with an exchange of molecular signals between the two partners. A root signaling pathway is recruited, for which the perception of fungal signals triggers oscillations of intracellular calcium concentration. High phosphate availability is known to inhibit the establishment and/or persistence of this symbiosis, thereby favoring the direct, non-symbiotic uptake of phosphorus by the root system. In this study, Medicago truncatula plants were used to investigate the effects of phosphate supply on the early stages of the interaction. When plants were supplied with high phosphate fungal attachment to the roots was drastically reduced. An experimental system was designed to individually study the effects of phosphate supply on the fungus, on the roots, and on root exudates. These experiments revealed that the most important effects of high phosphate supply were on the roots themselves, which became unable to host mycorrhizal fungi even when these had been appropriately stimulated. The ability of the roots to perceive their fungal partner was then investigated by monitoring nuclear calcium spiking in response to fungal signals. This response did not appear to be affected by high phosphate supply. In conclusion, high levels of phosphate predominantly impact the plant host, but apparently not in its ability to perceive the fungal partner.

Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone.

The primary objective of this study was to identify the molecular signals present in arbuscular mycorrhizal (AM) germinated spore exudates (GSEs) responsible for activating nuclear Ca(2+) spiking in the Medicago truncatula root epidermis. Medicago truncatula root organ cultures (ROCs) expressing a nuclear-localized cameleon reporter were used as a bioassay to detect AM-associated Ca(2+) spiking responses and LC-MS to characterize targeted molecules in GSEs. This approach has revealed that short-chain chitin oligomers (COs) can mimic AM GSE-elicited Ca(2+) spiking, with maximum activity observed for CO4 and CO5. This spiking response is dependent on genes of the common SYM signalling pathway (DMI1/DMI2) but not on NFP, the putative Sinorhizobium meliloti Nod factor receptor. A major increase in the CO4/5 concentration in fungal exudates is observed when Rhizophagus irregularis spores are germinated in the presence of the synthetic strigolactone analogue GR24. By comparison with COs, both sulphated and nonsulphated Myc lipochito-oligosaccharides (LCOs) are less efficient elicitors of Ca(2+) spiking in M. truncatula ROCs. We propose that short-chain COs secreted by AM fungi are part of a molecular exchange with the host plant and that their perception in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonization.

Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection.

Rhizobial nodulation factors (NFs) activate a specific signaling pathway in Medicago truncatula root hairs that involves the complex interplay of Nodulation Signaling Pathway1 (NSP1)/NSP2 GRAS and Ethylene Response Factor Required for Nodulation1 (ERN1) transcription factors (TFs) to achieve full ENOD11 transcription. ERN1 acts as a direct transcriptional regulator of ENOD11 through the activation of the NF-responsive "NF box." Here, we show that NSP1, when combined with NSP2, can act as a strong positive regulator of ERN1 and ENOD11 transcription. Although ERN1 and NSP1/NSP2 both activate ENOD11, two separate promoter regions are involved that regulate expression during consecutive symbiotic stages. Our findings indicate that ERN1 is required to activate NF-elicited ENOD11 expression exclusively during early preinfection, while NSP1/NSP2 mediates ENOD11 expression during subsequent rhizobial infection. The relative contributions of ERN1 and the closely related ERN2 to the rhizobial symbiosis were then evaluated by comparing their regulation and in vivo dynamics. ERN1 and ERN2 exhibit expression profiles compatible with roles during NF signaling and subsequent infection. However, differences in expression levels and spatiotemporal profiles suggest specialized functions for these two TFs, ERN1 being involved in stages preceding and accompanying infection thread progression while ERN2 is only involved in certain stages of infection. By cross complementation, we show that ERN2, when expressed under the control of the ERN1 promoter, can restore both NF-elicited ENOD11 expression and nodule formation in an ern1 mutant background. This indicates that ERN1 and ERN2 possess similar biological activities and that functional diversification of these closely related TFs relies primarily on changes in tissue-specific expression patterns.

Environmental temperatures, physiology and behavior limit the range expansion of invasive Burmese pythons in southeastern USA.

A well-established population of Burmese pythons resides in the Everglades of southern Florida. Prompted in part by a report that identified much of southern USA as suitable habitat for expansion or establishment of the Burmese python, we examined the plausibility of this snake to survive winters at sites north of the Everglades. We integrated daily low and high temperatures recorded from October to February from 2005-2011 at Homestead, Orlando and Gainesville, Florida; and Aiken, South Carolina, with minimum temperatures projected for python digestion (16 °C), activity (5 °C) and survival (0 °C). Mean low and high temperatures decreased northward from Homestead to Aiken and the number of days of freezing temperatures increased northward. Digestion was impaired or inhibited for 2 months in the Everglades and up to at least 5 months in Aiken, and activity was increasingly limited northward during these months. Reports of overwinter survivorship document that a single bout of low and freezing temperatures results in python death. The capacity for Burmese pythons to successfully overwinter in more temperate regions of the USA is seemingly prohibited because they lack the behaviors to seek refuge from, and the physiology to tolerate, cold temperatures. As tropical Southeast Asia is the source of the Everglades Burmese pythons, we predict it is unlikely that they will be able to successfully expand to or colonize more temperate areas of Florida and adjoining states due to their lack of behavioral and physiological traits to seek refuge from cold temperatures.

A switch in Ca2+ spiking signature is concomitant with endosymbiotic microbe entry into cortical root cells of Medicago truncatula.

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.

Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis.

• The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. • Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. • Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.

Infection-specific activation of the Medicago truncatula Enod11 early nodulin gene promoter during actinorhizal root nodulation.

The MtEnod11 gene from Medicago truncatula is widely used as an early infection-related molecular marker for endosymbiotic associations involving both rhizobia and arbuscular mycorrhizal fungi. In this article, heterologous expression of the MtEnod11 promoter has been studied in two actinorhizal trees, Casuarina glauca and Allocasuarina verticillata. Transgenic C. glauca and A. verticillata expressing a ProMtEnod11::beta-glucuronidase (gus) fusion were generated and the activation of the transgene investigated in the context of the symbiotic associations with the N-fixing actinomycete Frankia and both endo- and ectomycorrhizal fungi (Glomus intraradices and Pisolithus albus, respectively). ProMtEnod11::gus expression was observed in root hairs, prenodules, and nodules and could be correlated with the infection of plant cells by Frankia spp. However, no activation of the gus reporter gene was detected prior to infection or in response to either rhizobial Nod factors or the wasp venom peptide MAS-7. Equally, ProMtEnod11::gus expression was not elicited during the symbiotic associations with either ecto- or endomycorrhizal fungi. These observations suggest that, although there is a conservation of gene regulatory pathways between legumes and actinorhizal plants in cells accommodating endosymbiotic N-fixing bacteria, the events preceding bacterial infection or related to mycorrhization appear to be less conserved.

A nuclear-targeted cameleon demonstrates intranuclear Ca2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors.

Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca(2+) spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca(2+) responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca(2+) spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca(2+) oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca(2+) spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca(2+) concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca(2+) oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.

Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration.

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.

Mechanism of infection thread elongation in root hairs of Medicago truncatula and dynamic interplay with associated rhizobial colonization.

In temperate legumes, endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT). To study IT development in living root hairs, a protocol has been established for Medicago truncatula that allows confocal microscopic observations of the intracellular dynamics associated with IT growth. Fluorescent labeling of both the IT envelope (AtPIP2;1-green fluorescent protein) and the host endoplasmic reticulum (green fluorescent protein-HDEL) has revealed that IT growth is a fundamentally discontinuous process and that the variable rate of root hair invagination is reflected in changes in the host cell cytoarchitecture. The concomitant use of fluorescently labeled Sinorhizobium meliloti has further revealed that a bacteria-free zone is frequently present at the growing tip of the IT, thus indicating that bacterial contact is not essential for thread progression. Finally, these in vivo studies have shown that gaps within the bacterial file are a common feature during the early stages of IT development, and that segments of the file are able to slide collectively down the thread. Taken together, these observations lead us to propose that (1) IT growth involves a host-driven cellular mechanism analogous to that described for intracellular infection by arbuscular mycorrhizal fungi; (2) the non-regular growth of the thread is a consequence of the rate-limiting colonization by the infecting rhizobia; and (3) bacterial colonization involves a combination of bacterial cell division and sliding movement within the extracellular matrix of the apoplastic compartment.

Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota.

Arbuscular mycorrhizas (AM) are widespread, ancient endosymbiotic associations that contribute significantly to soil nutrient uptake in plants. We have previously shown that initial fungal penetration of the host root is mediated via a specialized cytoplasmic assembly called the prepenetration apparatus (PPA), which directs AM hyphae through the epidermis (Genre et al., 2005). In vivo confocal microscopy studies performed on Medicago truncatula and Daucus carota, host plants with different patterns of AM colonization, now reveal that subsequent intracellular growth across the root outer cortex is also PPA dependent. On the other hand, inner root cortical colonization leading to arbuscule development involves more varied and complex PPA-related mechanisms. In particular, a striking alignment of polarized PPAs can be observed in adjacent inner cortical cells of D. carota, correlating with the intracellular root colonization strategy of this plant. Ultrastructural analysis of these PPA-containing cells reveals intense membrane trafficking coupled with nuclear enlargement and remodeling, typical features of arbusculated cells. Taken together, these findings imply that prepenetration responses are both conserved and modulated throughout the AM symbiosis as a function of the different stages of fungal accommodation and the host-specific pattern of root colonization. We propose a model for intracellular AM fungal accommodation integrating peri-arbuscular interface formation and the regulation of functional arbuscule development.