TRANSIENT EXPRESSION IN ARABIDOPSIS ROOT CELLS BY MICRO-INJECTION TECHNIQUES
A LEONE, V MIANO and T BLEVE-ZACHEO
Istituto di Nematologia Agraria, CNR, Via Amendola 165/A, 70126 Bari, Italy
Background and objectives
Endoparasitic root-knot nematodes appear to rely on very specific interactions with root cells to establish complex feeding sites (giant cells in galled roots). Induction and maintenance of giant cells involve physiological changes including enlargement of cells, nuclear division, cell wall ingrowth and subcellular membrane and organelle proliferation. Histological observations strongly indicate that these nematodes in some way regulate specific host genes . Recent studies from several laboratories have recognized several promoters as responsive to root-knot nematodes. In a few cases, the minimal promoter regions required for the response have been found, and these are being scanned for the presence of specific nematode-responsive sequences, i.e. DNA fragments required in functional assays such as transgenic plants and transient expression . One of the alternatives to stable transformation for promoter analysis is micro-injection, which offers the opportunity of introducing well-defined amounts of foreign DNA into target plant cells without damaging them. The objective of this work is to test transient expression of putative nematode-responsive elements in Arabidopsis-root-knot nematode interaction.
Results and conclusions
Arabidopsis plants to be tested in this study were germinated in vitro on nutrient medium, infected with Meloidogyne incognita juveniles and maintained at 25°C. 5-day-old galled roots, having developed young giant cells with dense cytoplasm, were the most efficient for micro-injection. This indicates that the age of giant cells represents a key factor in the success of this system. Intracellular injection of Lucifer Yellow demonstrated that giant cells were viable 1 week after injection and did not lose fluorescent tracer. On the basis of these results, two or three giant cells per gall were injected for transient expression. The relatively high number of injections appeared not to disturb the viability of the gall. A viral promoter and the Lemmi9 gene have been found to be activated by nematodes, the latter coding for a homologue of a cotton protein related to desiccation.
Transient expression (dark-blue staining) in giant cells injected either with plasmids carrying the viral promoter-GUS chimaeric constructs or Lemmi9 promoter (pLE2280GUS) was positive in about 10% of the successfully injected cells. These fundamental primary results appear to support previous proposals on the ability of nematodes to control some transcription factors in plant cells. Analyses of a number of deletion series from Lemmi9 and other promoters are in progress. Although transient expression for nematode-dependent promoters is discouragingly low, micro-injection of reporter plasmids may represent a good tool for a fast DNA delivery system in single target cells.
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