Suppression of allene oxide cyclase in hairy roots of Medicago truncatula reduces jasmonate levels and the degree of mycorrhization with Glomus intraradices

During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35SuidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis.     

Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.     

A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root-soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the K(m) suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.     

Cloning of cDNAs encoding SODs from lettuce plants which show differential regulation by arbuscular mycorrhizal symbiosis and by drought stress

In the present study three cDNA fragments were cloned using degenerate primers for Mn-sod genes and PCR: two showed a high degree of identity with Mn-sods from plants and the third with Fe-sod. Arbuscular mycorrhizal (AM) symbiosis down-regulated their expression pattern under well-watered conditions. In contrast, AM symbiosis in combination with drought stress considerably increased the expression of the Mn-sod II gene and this correlated well with plant tolerance to drought. These results would suggest that mycorrhizal protection against oxidative stress caused by drought may be an important mechanism by which AM fungi protect the host plant against drought.     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

Expression of a xyloglucan endotransglucosylase/hydrolase gene, Mt-XTH1, from Medicago truncatula is induced systemically in mycorrhizal roots

Xyloglucan endotransglucosylase/hydrolases (XTH) are enzymes that catalyze the hydrolysis and transglycosylation of xyloglucan polymers in plant cell walls. Previously, we isolated a cDNA from mycorrhizal roots of Medicago truncatula that is predicted to encode an XTH [van Buuren, M.L., Maldonado-Mendoza, I.E., Trieu, A.T., Blaylock, L.A., Harrison, M.J., 1999. Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis between M. truncatula and G. versiforme. Mol. Plant-Microb. Interact. 12, 171-181.]. Here, we identified the corresponding XTH gene, designated Mt-XTH1. The Mt-XTH1 gene contains four exons separated by three introns and resides on a 15-kb Xba1 fragment adjacent to a second XTH gene designated Mt-XTH2. Mt-XTH2 shares the same exon-intron structure as Mt-XTH1. Exons 2, 3 and 4 and introns 1 and 2 are identical to Mt-XTH1, while exon 1 and intron 3 are divergent, both in sequence and in length. Mt-XTH1 is induced following colonization of the roots by AM fungi but does not respond to changes in phosphate status. Analysis of transgenic roots expressing an Mt-XTH1 promoterColon, two colonsuidA fusion revealed that the Mt-XTH1 promoter directs expression in cells throughout the root system with significantly higher levels of activity in mycorrhizal roots. Mt-XTH1 expression is elevated not only in the regions of the roots colonized by the fungus, but also at sites distal to the infected regions. These expression patterns are consistent with activation in response to a systemic signal.