No abstract

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

SummaryThe 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.

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), and this unique beneficial fungal-plant association is believed to have played a major role in the early colonization of land plants. AM fungi penetrate and colonize the root, forming highly differentiated symbiotic structures known as arbuscules, which are the principal sites of metabolic exchange between the two organisms (reviewed in Harrison, 2005). Concomitant extraradical hyphal development allows the fungus to supply important nutrients, including phosphate, to the host, while in return receiving carbohydrates from the plant. The AM symbiosis also confers resistance to the plant against biotic and abiotic stresses.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;B...