Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.     

Proteins involved in the production and perception of oligosaccharides in relation to plant and animal development.

Chitin oligosaccharides and their derivatives are involved in developmental and defence-related signalling pathways. Major advances include the structural identification of lectins involved in development that bind chitin oligosaccharides and the links between chitin oligosaccharide and hyaluronan synthesis. Also, recent advances in the understanding of the biological role of oligosaccharides are summarised in a model for multistep glycan recognition.     

A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China

We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 degrees C; melanin production capacity; Congo red absorption and symbiotic characteristics. These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called "strainomics") can be used to answer various biological questions. We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties. From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.     

Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium-mediated plant transformation

The usefulness of the E. coli codA gene encoding cytosine deaminase as a conditional toxic gene was explored during various stages of plant development and in different Agrobacterium -mediated transformation protocols. To this end, several independent tobacco lines transgenic for codA were isolated and these were tested for their sensitivity to 5-fluorocytosine (5-FC) at different developmental stages. On media supplemented with 5-FC, seedling proliferation was inhibited. Leaves failed completely to regenerate sprouts on 5-FC-containing medium. However, 40% of the shoots regenerated on non-selective medium still formed roots on rooting medium with 5-FC. In all these assays, control plants were unaffected by up to 1 mg m1⁻¹ 5-FC. Transformation of a codA and nptll -harbouring T-DNA to tobacco leaf discs did not result in any regenerant using a combined 5-FC and kanamycin selection, indicating that codA does not behave as a cell-autonomous marker here. Nevertheless, transformation of the same T-DNA to tobacco protoplasts resulted in some enrichment of codA ⁻ nptll ⁺ calluses using the proper combination of 5-FC and kanamycin for selection. Mixing of codA -containing and codA -lacking tobacco protoplasts revealed that the codA gene may behave as a cell autonomous marker under certain, appropriately chosen conditions, which seems to be in paradox with the total absence of escapes in tissue explant transformation. In all these experiments, 250 µg ml⁻¹ 5-FC was found to be the most optimal for selection. Our results suggest that codA can be successfully used as a negative selectable marker in Agrobacterium -mediated gene targeting protocols of tobacco whereby selection at the shoot regeneration level is the most effective.     

Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital bifunctional reporters in plants

By fusing the genes encoding green fluorescent protein (GFP) and -glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.     

Auxin distribution in Lotus japonicus during root nodule development

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.     

Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital and vital bifunctional reporters in plants

Plant Molecular Biology -