Arabidopsis Thaliana Colonization By GFP-labelled Bacillus Thuringiensis

Bacillus thuringiensis is a rod-shaped, spore-forming and Gram-positivebacterium, which is the most in-depth studied and the most widely used microbialpesticides. It can form parasporal crystal during spore-forming, which is toxic tomany insects. However, compared with chemical pesticides, there are somedisadvantage in Bt biological agents, such as narrow spectrum, low toxicity, and soon, which lead to its high cost, and great limit the further application. Therefore,improving the ability of colonization of Bt is a usful way to enrich the resistancemechanism of plants against insects. However, the mechanisms of colonization,migration and propagation of Bt in plants are still poorly understood. So, themechanisms of colonization, migration and propagation of Bt in plants were carriedout in this study.To study the colonization of Bacillus thuringiensis in Arabidopsis, a plasmid(pCM20), which carries a functional gfp gene sequence, was introduced into thewild-type stain Bt subsp. israelensis LLP29and a plasmid-cured stain Bt HD73^Cry－.The recombinants were found to express green fluorescent protein (GFP) duringvegitative growth. When cultured in the non-selective medium for50generations,70%of Bt HD73^Cry－were still harbored GFP, and,60%of LLP29when culcured for33generation. The physiological characteristics of the marked strains did notsignificantly change by the transformation of the plasmid. It provides a goodmaterial for further ecological and toxicological studies for Bt.The dynamic distribution of Bt in Arabidopsis thaliana roots, stems and leaves,with gnotobiotic and non-UV system, was measured after the plants were spayed andimmersion with GFP-tagged HD73^Cry－. It was showed that the GFP-tagged HD73Cry－can stable colonize in the roots, stems and leaves of A. thaliana. They existedinside the plants with the form of vegetative, however, existed more than20dayswith the form of spore ouside the plants.Fluorescence microscopy and laser scanning confocal microscope were alsoused to study the distribution of GFP-tagged HD73^Cry－in Arabidopsis. It was showed that the GFP-tagged HD73^Cry－can not only colonize and form biofilm onthe surface of roots and stems, but also enter into the roots, stems and leafs by someunknown ways, and multiply and colonize in the fibrous tissue of the cell gap. Theywere consistent with the results determined by dilution method of plate counting. Itwas observed interestingly that GFP-tagged HD73^Cry－was prolific inside theapoptosis tissue of Arabidopsis, which was indicated that Bt, as the carcasse ofinsect, could reproduct and disseminate in the withered tissue of plants. It might beone of the important reasons that Bt could be widely distributed.It was alsosuggested in this study that HD73^Cry－can colonize in the internal and external ofArabidopsis roots, stems and leaves, and forming biofilm on the surface of the rootsand stems under gnotobiotic and non-UV conditions.It will be great benefit not onlyon Bt's ecological meanings, but also on the new idea of plant pest biocontrol. Withthe technique of molecular engineering, it will provide important theoretical andapplicative value for newly toxic and broad-spectrum biopesticides.