The Bacteriolytic Activity in Transgenic Potatoes Expressing a Chimeric T4 Lysozyme Gene and the Effect of T4 Lysozyme on Soil- and Phytopathogenic Bacteria

Vries, Johann de; Harms, Klaus; Broer, Inge; Kriete, Guido; Mahn, Andreas; Düring, Klaus; Wackernagel, Wilfried

doi:10.1016/s0723-2020(99)80075-7

Cited by 51 publications

(47 citation statements)

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“…Concerns have been raised about the environmental impact associated with genetically modified organisms particularly GM crop. Variable and transient impacts on non-target microorganisms were already shown (De Vries et al, 1999;Lottmann et al, 2000;Dunfield and Germida, 2003;Castaldini et al, 2005;Rasche et al, 2006). We show here that manipulation of glucosinolate content in Brassicaceae could have some repercussions on the microbial community in their close soil environment.…”

Section: Discussionsupporting

confidence: 55%

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

et al. 2009

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A specificity of Brassicaceous plants is the production of sulphur secondary metabolites called glucosinolates that can be hydrolysed into glucose and biocidal products. Among them, isothiocyanates are toxic to a wide range of microorganisms and particularly soil-borne pathogens. The aim of this study was to investigate the role of glucosinolates and their breakdown products as a factor of selection on rhizosphere microbial community associated with living Brassicaceae. We used a DNA-stable isotope probing approach to focus on the active microbial populations involved in root exudates degradation in rhizosphere. A transgenic Arabidopsis thaliana line producing an exogenous glucosinolate and the associated wild-type plant associated were grown under an enriched 13 CO 2 atmosphere in natural soil. DNA from the rhizospheric soil was separated by density gradient centrifugation. Bacterial (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Acidobacteria), Archaea and fungal community structures were analysed by DGGE fingerprints of amplified 16S and 18S rRNA gene sequences. Specific populations were characterized by sequencing DGGE fragments. Roots of the transgenic plant line presented an altered profile of glucosinolates and other minor additional modifications. These modifications significantly influenced microbial community on roots and active populations in the rhizosphere. Alphaproteobacteria, particularly Rhizobiaceae, and fungal communities were mainly impacted by these Brassicaceous metabolites, in both structure and composition. Our results showed that even a minor modification in plant root could have important repercussions for soil microbial communities.

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Section: Discussionsupporting

confidence: 55%

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

et al. 2009

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“…It is conceivable that the released enzyme is rapidly inactivated in soil either by chemical conditions not previously detected when the T4 lysozyme activity was measured in aqueous soil extracts (4) or by adsorption to solid surfaces. If this were the case, strong effects of T4 lysozyme release from the potato plants on the soil bacterial community would not be expected although most of the tested soil bacteria turned out to be sensitive to T4 lysozyme in vitro (4).…”

Section: Discussionmentioning

confidence: 99%

“…The assay was essentially performed as described previously (4) except that the cultures were incubated at 30°C and the cells were harvested at a titer of 4 ϫ 10 8 /ml (late log phase). In the linear range of the optical density (OD) decrease (Ϸ0.6 to 0.3) with time, the negative inclination (⌬OD/⌬t) of turbidity is proportional to the lytic activity (4).…”

Section: Methodsmentioning

confidence: 99%

“…T4 lysozyme is an endoacetylmuramidase which degrades the murein of the bacterial cell wall by cleavage of the ␤(1-4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (17). It is active against gram-negative and gram-positive bacteria (4,16). For ecological evaluation of the use of lysozyme-producing plants in agriculture, a comparison of the microbial community in the rhizosphere of T4 lysozyme-producing and control plants can yield valuable information.…”

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confidence: 99%

“…To obtain more direct data on whether T4 lysozyme is released from the plants into the soil, a direct and more specific test on lysozyme leakage would be desirable. Previously, the release of antigenic material from the transgenic plant roots acting with anti-T4 lysozyme antibodies was obtained in aquacultures of transgenic plants with T4 lysozyme gene, but actual lysozyme activity could not be determined (4). Leakage of T4 lysozyme from the rhizodermis would be feasible because the transgene-encoded T4 lysozyme is fused to the ␣-amylase leader peptide, which leads to the export of the protein into the apoplast (7).…”

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confidence: 99%

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Increased Killing of Bacillus subtilis on the Hair Roots of Transgenic T4 Lysozyme-Producing Potatoes

Ahrenholtz

Harms

Vries

et al. 2000

Appl Environ Microbiol

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Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5-to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growthsustaining effects of plants on bacteria.Plant-microbe interactions often involve plant activities to suppress growth of bacteria in the rhizosphere, e.g., by secretion of bactericidal substances (3, 9, 11). Recently, transgenic plants producing antimicrobial agents have been developed. Particularly plants expressing foreign lysozyme genes, including tobacco (15) and potato (8), were constructed. In potato plants, T4 lysozyme gene expression has been used to protect the plants against the phytopathogenic enterobacterium Erwinia carotovora (8), which causes blackleg and soft rot in Solanum tuberosum (12). T4 lysozyme is an endoacetylmuramidase which degrades the murein of the bacterial cell wall by cleavage of the ␤(1-4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (17). It is active against gram-negative and gram-positive bacteria (4, 16). For ecological evaluation of the use of lysozyme-producing plants in agriculture, a comparison of the microbial community in the rhizosphere of T4 lysozyme-producing and control plants can yield valuable information. In such a study, no significant differences in the aerobic plate counts and functions of beneficial bacteria including indole-3-acetic acid-producing and antagonistic (against E. carotovora and Verticillium dahliae) organisms was found (10). However, the authors point out that isolates of seven antagonistic species were obtained only from the control plants not producing T4 lysozyme (10). To obtain more direct data on whether T4 lysozyme is released from the plants into the soil, a direct and more specific test on lysozyme...

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Effects of Genetically Modified Plants on Soil Microorganisms

Weinert

Meincke

Schloter

et al. 2009

Environmental Microbiology

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The Bacteriolytic Activity in Transgenic Potatoes Expressing a Chimeric T4 Lysozyme Gene and the Effect of T4 Lysozyme on Soil- and Phytopathogenic Bacteria

Cited by 51 publications

References 16 publications

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

Increased Killing of Bacillus subtilis on the Hair Roots of Transgenic T4 Lysozyme-Producing Potatoes

Effects of Genetically Modified Plants on Soil Microorganisms

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