In-Situ hybridization of nodulin mRNAs in root nodules using non-radioactive probes

Bochenek, Birgit; Hirsch, Ann M.

doi:10.1007/bf02668761

Cited by 28 publications

(6 citation statements)

References 7 publications

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“…The primary region of hybridization is in interzone II-III which comprises the proximal end of the infection zone and the distal portion of the nitrogen-fixing region. As with probes directed at total Lb mRNA (Bochenek and Hirsch, 1990), some hybridization is also visible at the cell layer proximally adjacent to the nodule parenchyma.…”

Section: Resultsmentioning

confidence: 91%

“…The washes for Lb isomer-specific binding were done as described for the RNA dot blot using 50°C for the stringent wash. The overall procedure was similar to that described by Bochenek and Hirsch (1990), but in this study 3 -aminopropyltriethoxysilane was used to coat slides for better adhesion of the cryosections to the microscope slides, oligonucleotide probes were used instead of RNA transcripts, and different washing conditions were used. • partial sequence…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies do not address this issue. In situ studies (Bochenek and Hirsch, 1990;de Billy et al, 1991) did not use Lb isomer-specific probes, and time course studies (Barker et al, 1988) used mRNA extracted from total nodules containing a variety of cell types at different developmental stages.…”

Section: Introductionmentioning

confidence: 99%

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Messenger RNA from diverse classes of alfalfa leghemoglobin genes show a similar pattern of spatial expression in symbiotic root nodules

1994

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The spatial expression of specific classes of leghemoglobin mRNA corresponding to divergent classes of alfalfa leghemoglobin (Lb) isomers was analyzed by in situ hybridization of oligonucleotide probes to root nodule cryosections. The aim of this study was to determine whether structural changes in the nodule may contribute to the shift in Lb protein isomer expression observed during root nodule development. The co-localization of the diverse Lb mRNAs suggests that this is unlikely to be the case.Abbreviations: Lb -leghemoglobin, SDS -sodium dodecyl sulfate, SSC-sodium chloride, sodium citrate solution

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Section: Resultsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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Messenger RNA from diverse classes of alfalfa leghemoglobin genes show a similar pattern of spatial expression in symbiotic root nodules

1994

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“…Alfalfa nodules and nodule primordia induced by wild-type R. meliloti strain 41 were fixed, dehydrated, and finally infiltrated and embedded into paraffin. Sections (9 m thick) were hybridized to digoxigenin-labeled antisense or sense RNA probes according to a procedure derived from Bochenek and Hirsch (14). Sections were photographed with a POLYVAR Reichert-Jung microscope by using T-160 Ektachrome Kodak film.…”

Section: Reverse Transcription-pcr (Rt-pcr)mentioning

confidence: 99%

MsPG3 , a Medicago sativa polygalacturonase gene expressed during the alfalfa– Rhizobium meliloti interaction

Muñoz

Coronado

Perez‐Hormaeche

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Polygalacturonase (PG) is one of the most important enzymes associated with plant cell wall degradation. It has been proposed to participate in the early steps of the Rhizobium-legume interaction. We have identified two classes of cDNA fragments corresponding to two classes of PG genes in the Medicago genome. One of this class, represented by E 2 in M. truncatula and Pl 1 in M. sativa, seems to be related to previously characterized plant PG genes expressed in pollen. We have isolated the genomic clone containing the entire gene corresponding to the second class (E 3 ). We showed that MsPG3 is a single gene in the Medicago genome coding for PG. By reverse transcription-PCR, MsPG3 expression was detected in roots 1 day after Rhizobium inoculation. The early induction of the MsPG3, as also seen by in situ hybridization experiments, supports its involvement in the early stages of the Rhizobium-legume infection process. In addition, by analyzing the expression of a MsPG3 promoter-gus construct in Vicia hirsuta-transgenic root nodules, we showed that MsPG3 was expressed in all cells of nodule primordia and in the cells of the invasion zone. By Northern blot, MsPG3 transcripts are not detected in various Medicago tissues, indicating that the function of this gene is related closely to symbiosis. Thus, our results strongly suggest the involvement of MsPG3 gene during meristem formation and͞or in the infection process, probably by facilitating cell wall rearrangement, penetration of the bacteria through the root hair wall, or infection thread formation and release of bacteria in plant cells. MsPG3 represents a class of PG genes, distinct from the pollenspecific genes, and it is the first pectic encoded enzyme demonstrated to be involved in Rhizobium-legume symbiosis.

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“…Altemative labeling and detection strategies relying on enzyme assays can also help to circumvent problems caused by the autofluorescence of the matrix (Edman et al 1988, Bochenek andHirsch 1991). The application of digoxigenin-labeled probes, detected via antibody/alkaline phosphatase conjugates, is not restricted by background signals of plant material (van de Wiel et al 1990, Bochenek andHirsch 1991). This detection strategy consequently allows the reliable differentiation of spores, hyphae and vesicles of Frankia from nodule material (Fig.…”

Section: Reducing the Background Of The Matrixmentioning

confidence: 99%

Whole cell hybridization as a tool to study Frankia populations in root nodules*

Hahn¹,

Zepp²,

Zeyer³

1997

Physiol Plant

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J. 1997. Whole cell hybridization as a tool to study Frankia populations in root nodules. -Physiol. Plant. 99: 696-706.Molecular methods based on DNA or rRNA hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising altemative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole cell hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin-labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes-specific insertion on the 23S rRNA are used. Digoxigenin-labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.

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In-Situ hybridization of nodulin mRNAs in root nodules using non-radioactive probes

Cited by 28 publications

References 7 publications

Messenger RNA from diverse classes of alfalfa leghemoglobin genes show a similar pattern of spatial expression in symbiotic root nodules

Messenger RNA from diverse classes of alfalfa leghemoglobin genes show a similar pattern of spatial expression in symbiotic root nodules

MsPG3 , a Medicago sativa polygalacturonase gene expressed during the alfalfa– Rhizobium meliloti interaction

Whole cell hybridization as a tool to study Frankia populations in root nodules*

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