Functional analysis of the role of sphingolipids in Agrobacterium tumefaciens-mediated plant transformation

• Applicant: Privatdozent Dr. Frank Waller
• Subject Area: Plant Physiology ; Plant Biochemistry and Biophysics ; Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems ; Plant Breeding and Plant Pathology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 562480832

The soil-born bacterium Agrobacterium tumefaciens infects a wide range of plant species and can cause crown gall disease. It transfers bacterial DNA together with effector proteins into host cells, where the T-DNA (transfer-DNA) is stably integrated into the plant genome. Integration of a T-DNA and subsequent expression of encoded oncogenes results in cell proliferation and tumor formation. The ability to transfer DNA into the host genome has made A. tumefaciens an important tool for research and genetic engineering of plants. However, many economically important plants remain recalcitrant to transformation, and it is poorly understood why some plant species can be easily transformed and others not. This is largely due to our insufficient knowledge of the host cell molecular requirements for efficient transformation. We observed in a series of independent experiments that the transformation efficiency of Agrobacterium is significantly affected in Arabidopsis plants with an altered sphingolipid composition. Sphingolipid species, such as Glucosylceramides and Glycosylinositolphosphorylceramides (GIPCs) are predominantly localized in nanodomains of the plant plasma membrane. Previous studies in Arabidopsis have shown that presence or absence of specific sphingolipids can affect the function of membrane-localized receptors and channels important for different signal transduction processes. Sphingolipids could therefore impact different phases of the Agrobacterium transformation, e.g. by influencing plasma membrane localized receptors triggering defense responses, or the interaction of the bacterial type IV secretion system before or during T-DNA transfer through the plasma membrane. This project therefore aims to identify those plant sphingolipid species which influence Agrobacterium transformation efficiency and to characterize the role of these lipids during the different stages of transformation. To identify the effect of different sphingolipid profiles on transformation efficiency, we will use a well-established in vivo transformation efficiency assay. We will test our collection of Arabidopsis mutant lines affected in sphingolipid composition, and a range of pharmacological and temperature treatments. This will be accompanied by state-of-the-art sphingolipid profiling using high pressure liquid chromatography followed by mass spectrometry (UPLC-MS/MS). Having identified the relevant sphingolipid species, we will analyze, in which phase of the transformation these lipids are involved. Using the in vivo test system, we will test bacterial growth, attachment and virulence gene expression, parallel to analyzing the plant defense response and the subcellular lipid composition. The characterization of sphingolipid-related host cell processes for agrobacterial transformation is expected to improve our understanding of the mechanisms underlying plant transformation and agrobacterial pathogenicity and can be applied in biotechnological approaches.

• DFG Programme: Research Grants