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Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA.

ABSTRACT: Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

SUBMITTER: Krak K

PROVIDER: S-EPMC3346362 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA.

Krak Karol K Janoušková Martina M Caklová Petra P Vosátka Miroslav M Štorchová Helena H

Applied and environmental microbiology 20120309 10

Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two i ...[more]

PMID: 22407684

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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.

Dataset Information

Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA.

Publications

Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA.

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