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Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices.

ABSTRACT: Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.

SUBMITTER: Mrosk C

PROVIDER: S-EPMC2736893 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices.

Mrosk Cornelia C Forner Susanne S Hause Gerd G Küster Helge H Kopka Joachim J Hause Bettina B

Journal of experimental botany 20090702 13

Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms o ...[more]

PMID: 19574251

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Project description:BACKGROUND: Most vascular flowering plants have the capacity to form symbiotic associations with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the roots where AM fungi colonize the root cortex and form arbuscules within the cortical cells. Arbuscules are enveloped in a novel plant membrane and their establishment requires the coordinated cellular activities of both symbiotic partners. The arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. To determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. Toward this goal we used the Affymetrix GeneChip Medicago Genome Array to document the M. truncatula transcript profiles associated with AM symbiosis, and then developed laser microdissection (LM) of M. truncatula root cortical cells to enable analyses of gene expression in individual cell types by RT-PCR. RESULTS: This approach led to the identification of novel M. truncatula and G. intraradices genes expressed in colonized cortical cells and in arbuscules. Within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. Analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif (LysM)-receptor like kinase, members of the GRAS transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the AM symbiosis. CONCLUSION: Transcript profiling using the Affymetrix GeneChip Medicago Genome Array provided new insights into gene expression in M. truncatula roots during AM symbiosis and revealed the existence of several G. intraradices genes on the M. truncatula GeneChip. A laser microdissection protocol that incorporates low-melting temperature Steedman's wax, was developed to enable laser microdissection of M. truncatula root cortical cells. LM coupled with RT-PCR provided spatial gene expression information for both symbionts and expanded current information available for gene expression in cortical cells containing arbuscules.

Project description:Differences in the plant's response among ecotypes or accessions are often used to identify molecular markers for the respective process. In order to analyze genetic diversity of Medicago truncatula in respect to interaction with the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, mycorrhizal colonization was evaluated in 32 lines of the nested core collection representing the genetic diversity of the SARDI collection. All studied lines and the reference line Jemalong A17 were inoculated with R. irregularis and the mycorrhization rate was determined at three time points after inoculation. There were, however, no reliable and consistent differences in mycorrhization rates among all lines. To circumvent possible overlay of potential differences by use of the highly effective inoculum, native sandy soil was used in an independent experiment. Here, significant differences in mycorrhization rates among few of the lines were detectable, but the overall high variability in the mycorrhization rate hindered clear conclusions. To narrow down the number of lines to be tested in more detail, root system architecture (RSA) of in vitro-grown seedlings of all lines under two different phosphate (Pi) supply condition was determined in terms of primary root length and number of lateral roots. Under high Pi supply (100 µM), only minor differences were observed, whereas in response to Pi-limitation (3 µM) several lines exhibited a drastically changed number of lateral roots. Five lines showing the highest alterations or deviations in RSA were selected and inoculated with R. irregularis using two different Pi-fertilization regimes with either 13 mM or 3 mM Pi. Mycorrhization rate of these lines was checked in detail by molecular markers, such as transcript levels of RiTubulin and MtPT4. Under high phosphate supply, the ecotypes L000368 and L000555 exhibited slightly increased fungal colonization and more functional arbuscules, respectively. To address the question, whether capability for mycorrhizal colonization might be correlated to general invasion by microorganisms, selected lines were checked for infection by the root rot causing pathogen, Aphanoymces euteiches. The mycorrhizal colonization phenotype, however, did not correlate with the resistance phenotype upon infection with two strains of A. euteiches as L000368 showed partial resistance and L000555 exhibited high susceptibility as determined by quantification of A. euteiches rRNA within infected roots. Although there is genetic diversity in respect to pathogen infection, genetic diversity in mycorrhizal colonization of M. truncatula is rather low and it will be rather difficult to use it as a trait to access genetic markers.

Dataset Information

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices.

Publications

Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices.

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