The formation of symbiotic associations with arbuscular mycorrhizal (AM) fungi is a phenomenon common to the majority of vascular flowering plants. Here, we used cDNA arrays to examine transcript profiles in Medicago truncatula roots during the development of an AM symbiosis with Glomus versiforme and during growth under differing phosphorus nutrient regimes. Three percent of the genes examined showed significant changes in transcript levels during the development of the symbiosis. Most genes showing increased transcript levels in mycorrhizal roots showed no changes in response to high phosphorus, suggesting that alterations in transcript levels during symbiosis were a consequence of the AM fungus rather than a secondary effect of improved phosphorus nutrition. Among the mycorrhiza-induced genes, two distinct temporal expression patterns were evident. Members of one group showed an increase in transcripts during the initial period of contact between the symbionts and a subsequent decrease as the symbiosis developed. Defense- and stress-response genes were a significant component of this group. Genes in the second group showed a sustained increase in transcript levels that correlated with the colonization of the root system. The latter group contained a significant proportion of new genes similar to components of signal transduction pathways, suggesting that novel signaling pathways are activated during the development of the symbiosis. Analysis of the spatial expression patterns of two mycorrhiza-induced genes revealed distinct expression patterns consistent with the hypothesis that gene expression in mycorrhizal roots is signaled by both cell-autonomous and cell-nonautonomous signals.

Most vascular plants can acquire phosphate from the environment either directly, via the roots, or indirectly, via a fungal symbiont that invades the cortical cells of the root. Here we have identified two cDNA clones (MtPT1 and MtPT2) encoding phosphate transporters from a mycorrhizal root cDNA library (Medicago truncatula/Glomus versiforme). The cDNAs represent M. truncatula genes and the encoded proteins share identity with high-affinity phosphate transporters from Arabidopsis, potato, yeast, Neurospora crassa, and an arbuscular mycorrhizal (AM) fungus, G. versiforme. The function of the protein encoded by MtPT1 was confirmed by complementation of a yeast phosphate transport mutant (pho84). The K(m) of the MtPT1 transporter in this system is 192 microM. MtPT1 and MtPT2 transcripts are present in roots and transcript levels increase in response to phosphate starvation. MtPT transcripts were not detected in leaves. Following colonization of the roots by the AM fungus G. versiforme, both MtPT1 and MtPT2 transcript levels decrease significantly. Down-regulation of phosphate starvation-inducible genes in mycorrhizal roots appears to be a common occurrence and a homologue of a phosphate starvation-inducible purple acid phosphatase is also down-regulated in the mycorrhizal roots. The functional characteristics and expression patterns of the MtPT transporters are consistent with a role in the acquisition of phosphate from the environment but suggest that they may not be involved in phosphate uptake at the symbiotic interface in mycorrhizal roots.

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungal symbiont resides in the root cortical cells where it delivers mineral nutrients to its plant host through branched hyphae called arbuscules. Here, we report a Medicago truncatula mutant, stunted arbuscule (str), in which arbuscule development is impaired and AM symbiosis fails. In contrast with legume symbiosis mutants reported previously, str shows a wild-type nodulation phenotype. STR was identified by positional cloning and encodes a half-size ATP binding cassette (ABC) transporter of a subfamily (ABCG) whose roles in plants are largely unknown. STR is a representative of a novel clade in the ABCG subfamily, and its orthologs are highly conserved throughout the vascular plants but absent from Arabidopsis thaliana. The STR clade is unusual in that it lacks the taxon-specific diversification that is typical of the ABCG gene family. This distinct phylogenetic profile enabled the identification of a second AM symbiosis-induced half-transporter, STR2. Silencing of STR2 by RNA interference results in a stunted arbuscule phenotype identical to that of str. STR and STR2 are coexpressed constitutively in the vascular tissue, and expression is induced in cortical cells containing arbuscules. STR heterodimerizes with STR2, and the resulting transporter is located in the peri-arbuscular membrane where its activity is required for arbuscule development and consequently a functional AM symbiosis.

¶ First co-authors. SummaryTwo rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The ®rst approach is based on a method developed for transformation of Arabidopsis thaliana and involves in®ltration of¯owering plants with a suspension of Agrobacterium. The second method involves in®ltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the in®ltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the¯ower in®ltration method, and from 2.9 to 27.6% for the seedling in®ltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T 2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large-scale insertional mutagenesis. T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.