Construction of Reporter Lines

In order to study the mechanism underlying cytoplasmic bridge formation and root nodulation, a number of marker lines of L. japonicus have been constructed. For the analysis of promoter activity it is often very useful to make use of a fusion construct consisting of the green fluorescent protein (GFP) and (5-glucuronidase (GUS) genes (Quaedvlieg et al. 1998). Using this construct we have obtained various transgenic L. japonicus lines which will be used for analysis of responses to Nod factors. These promoters are either known promoters that were selected based on their known responsiveness to Nod factors or plant hormones. We have fused the GH3 promoter to the GUS-GFP reporter construct in order to analyze the effect of Nod factors on auxin transport in L. japonicus. We have also fused the promoter of ENOD40 of soybean and of L. japonicus {ENOD40-1: Flemetakis et al. 2000) to GUS-GFP. In addition to external addition of Nod factors and plant hormones we also use microtargeting following the strategy of Mathesius et al. (1998).

For the analysis of GFP various microscopic imaging techniques have been employed in our laboratory (Spaink et al. 2000). The most commonly used technique is confocal laser scanning microscopy (CLSM) which enables the four-dimensional (i.e. in space and time) visualization of GFP in living tissues. However, a pitfall that is often encountered is the high level of autofluorescence in various tissues (especially in leguminous plants). This problem can in several cases be solved with using spectral-resolved CLSM (e.g. using a Leica TCS SP system). This system makes a quantitative analysis of GFP in the case of the GH3 promoter possible. However, for weak promoter activity, such as in the case of the ENOD40 promoter, GFP fluorescence cannot be detected above the autofluorescence background using existing methods.

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