Use Of The Cre-loxP Recombination System In Interactions Between Rhizobium/Agrobacterium And Host Plants

Conversion of nitrogen gas into ammonia (nitrogen fixation) by symbioticrhizobia in the nodule of legumes plays an important agricultural role. Variousrhizobial strains possess a functional type three protein secretion system (T3SS),which secretes effectors (T3effectors). Rhizobial T3effectors have been originallycharacterized as nodulation outer proteins (Nops) secreted into culture supernatants.Depending on the host plant, T3effectors either positively or negatively influencenodulation and nitrogen fixation. In the first part of this thesis, the Cre-loxP DNArecombination system was chosen to examine delivery of T3effectors into host cells.DNA encoding the secretion signal peptide (first50N-terminal amino acids) of agiven T3effector was fused to the cre recombinase gene. The Nop:Cre fusion proteinswere then expressed in Rhizobium sp. strain NGR234and Mesorhizobium lotiMAFF303099, respectively. Constructed transgenic Lotus japonicus MG20plantswith a loxP-flanked DNA cassette were inoculated with the rhizobia expressing theNop-Cre fusion proteins. It was expected that Nop:Cre fusion proteins are delivered into legume cells during symbiosis and that excision of the loxP-flanked sequence inthe L. japonicus is a measure for T3effector delivery into host cells. However,Cre-loxP recombination was not detected in transgenic L. japonicus transformed witha loxP cassette when calli were inoculated with agrobacteria carrying a binary vectorwith the35S:cre sequence (cre gene expression under the control of the cauliflowermosaic virus35S promoter). Furthermore, Western blot analysis revealed that noexpected bands were detected in apigenin-induced culture supernatants of NGR234derivatives expressing Nop:Cre proteins, indicating that fusion proteins were secretedat very low levels or not at all. Similar results were also obtained for Nop:Cre fusionproteins with a prolonged signal peptide (first150N-terminal amino acids of Nopproteins). Data of nodulation tests indicated that most rhizobial strains expressingNop:Cre fusions successfully induced nodules on L. japonicus transformed with theloxP cassette. However, PCRs with nodule DNA as a template revealed no indicationsfor Cre-loxP recombination events. When a plasmid expressing a given Nop:Crefusion was mobilized into a NGR234derivative carrying a loxP:nifQp:gusA:Ω:loxPcassette in the genome, the loxP cassette was excised by the expressed Nop:Cre fusionprotein, indicating that the fusion protein is functional. However, delivery of T3effectors of NGR234into host cells of L. japonicus could not be tested by the usedCre-loxP system, possible due to toxic effects of Cre in this legume plant.In mature nodules of legumes, rhizobia differentiate into bacteriods, wherenitrogen fixation takes place. However, it is still unclear whether and how bacteroidsdedifferentiate into free-living rhizobia during nodule senescence. In Chapter2of thisthesis, attempts were made to label bacteria that differentiated into the bacteroid stage.The Cre-loxP system under the control of a bacteroid specific promoter was appliedwith the goal to genetically tag developed bacteroids. DNA containing the bacteroidspecific nifQ promoter fused to the Cre recombinase gene and flanked by two loxPsites was cloned into a broad-host-range vector, which was mobilized into strainNGR234. It was expected that the nifQ promoter activates expression of Cre duringthe bacteroid stage. This would result in excision of the whole cassette, resulting in a single remaining loxP site on the plasmid. Surviving bacteroids in senescent nodulescould be genetically tagged and the rate of redifferentiation from bacteroids into thefree-living stage could be determined. Data of nodulation tests with various legumehost plants showed that NGR234, once differentiated into the bacteroid stage, doesnot carry the received plasmid anymore (plasmid instability). Therefore, attemptswere made to integrate the cre-loxP cassette into the genome of NGR234byhomologous recombination with a suicide vector construct. However, once thecre-loxP cassette was cloned into the y4mN site of NGR234, the cre gene wasactivated and the DNA cassette excised. Future work is required to attenuate the Creactivity and to insert a transcriptional terminator upstream of the nifQ promoter.Agrobacterium tumefaciens mediated transformation of tobacco (Nicotianatabacum) is an efficient method to study gene expression in a heterologous geneticbackground. In Chapter3of this thesis, Cre-loxP recombination system was appliedto detect T-DNA transfer by two A. tumefaciens leaf cells harboring different binaryvectors. The cre gene under the control of the CaMV35S promoter was cloned intoone vector, and a loxP cassette into another vector. A mixture of A. tumefaciens, inwhich each cell contained either a cre-or a loxP-vector, was co-infiltrated intotobacco leaves. After two days, excision of loxP-flanked DNA was detected by PCRand used as an estimate for co-transformation events. Strongest excision (>50%) wasobserved when the loxP cassette was cloned into vector pPZP112and cre intopISV2678. The established method can be used to assess the co-transformationefficiency of tobacco cells in future studies.