Towards in vivo imaging of early Rhizobium Nod factor responses

The goal in this thesis is to explore the possibility of live imaging of cellular events using fluorescence microscopy in combination with Green Fluorescent Protein (GFP) based reporter constructs in root hairs during theRhizobium-legume interaction. Legumes have the ability to establish anendosymbioticrelationship with soil bacteria of different genera, collectively namedrhizobia, thatresults in nitrogen-fixing root nodules. The formation of root nodules begins when bacteria colonize the root surface and start secreting signal molecules, named Nod factor. These molecules induce responses in root epidermal cells; e.g. [Ca2+] spiking,actinskeleton rearrangement(Mirabella et al. 2002). Ultimately, specific infection structures are formed in root hairs, which guide the bacteria through the epidermis and outer cortical cells towards the newly formed noduleprimoridum(Mylona 1995; Long 1996; Bladergroen et al. 1998; Hadri 1998; Schultze et al. 1998).

The known [Ca ²⁺ ]-responses andactinskeleton alterations in root hairs were used as parameters to explore whether these can be monitored in living root hairs. However imaging approaches using FP based reporter constructs require the availability of efficient transformation procedures. ThereforeAgrobacteriumrhiogenes based transformation ofMedicagotruncatula (Medicago) andLotusjaponicus(Lotus),was optimised ( Chapter 2 ).

Several pilot experiments indicted thattransgenesdid have negative effects on plant/root development. This is in our system of importance since we use an A.rhizogenes root transformation procedure to obtain root hairs expressing the FP reporter constructs. If the negative influence on root growth is too high no transgenic roots will be obtained from the shoot. Therefore it was decided to test which tissue specific and inducible promoters could be used to create a set of promoters to facilitate future imaging studies. Thus several root epidermis specific promoters were analyzed for their suitability ( Chapter 2 ). In Chapter 3,4,5 inducible promoters have been studied that can be activated by a specific treatment by which the level, place and timing of expression can be controlled.

To study the dynamics ofactinin living cells a vitalactinprobe had to be developed. Therefore fusions ofFPswithactin,actinbinding proteins (ABPs), andactinbinding protein domains (ABDs), respectively, were made. Their usefulness was tested inVignamesophyllprotoplasts ( Chapter 6 ). In addition some attempts were made to express the most promising construct in root hairs. In vivo [Ca ²⁺ ] imaging was tested using use the FP based sensorYcam, enabling [Ca ²⁺ ] imaging(Miyawakiet al. 1997).Ycamwas expressed and tested in root hairs ( Chapter 7 ) by using one of the previously tested root epidermis specific promoters (Chapter 2). Finally the potential of the tested approaches is discussed ( Chapter 8)