Activation and mode of action of the Type 6 secretion system of Azoarcus olearius BH72 during plant-growth promoting interaction with rice plants and other crop varieties
Climate change and the current global hunger are two challenging subjects for our society and facing those challenges requires the further development of sustainable agricultural methods. In this regard, the interactions between plants and bacteria can play an important role: On the one hand, bacteria can act as pathogens, on the other hand they can promote plant growth and health. Understanding the dynamics between plants and their associated bacteria can help establishing new strategies in the agricultural sector. One example of such plant-growth promoting bacteria is Azoarcus olearius BH72, a well-studied endophytic gram-negative bacterial strain which was first isolated from the roots of Kallar grass in Pakistan. Under gnotobiotic conditions, this strain does not only colonize the interior of Kallar grass roots, but also roots of several rice plants, without activating any observable defense mechanisms. As diazotrophic strain, A. olearius BH72 is able to reduce atmospheric nitrogen to ammonium which can be provided to the host plant.
In the genome of A. olearius BH72, gene clusters for two Type VI secretion systems (T6SS) could be found, T6SS-1 and T6SS-2, which can inject proteins into other organisms. Several aspects indicate the crucial role of T6SS-1 in the endophytic lifestyle of A. olearius BH72: T6SS-1 is only assembled under conditions of biological nitrogen fixation and activated only in the presence of root exudates of the rice plant Oryza sativa cv. IR36. Moreover, mutants without functional T6SS-1 cannot colonize rice roots as well as the wild-type.
In my PhD project, I aim to investigate the role of the Type VI secretion system further in regard to the interaction with rice plant and other crops. This project is divided into two aspects:
- The rice root exudates of Oryza sativa IR36 seem to activate T6SS-1 which leads to the hypothesis that a component in the exudates might trigger the activation. Which chemical compound acts as trigger and is this process host-specific?
For the identification of these components, a reliable screening system for the activation of secretion apparatus is required. Previously, the A. olearius mutant BHimpVipAsfGFP was used for that purpose. The activation of T6SS-1 in this mutant is visible as green fluorescent foci under the fluorescence microscope. In order to screen the exudates for trigger molecules, the mutant, which was grown under nitrogen-fixing conditions, was incubated in accumulated root exudates. The screening of the exudates with this mutant lead to the conclusions that the component in question is likely to be very stable as heating and freezing the exudates did not affect the occurrence of fluorescent foci. However, for the negative control, bacteria incubated in plant medium without root exudates, fluorescent foci were visible as well, this method needs to be optimized.
In this PhD project, the screening method for the activation of T6SS will be optimized. Furthermore, the root exudates will be fractioned by High Performance Liquid Chromatography (HPLC). The fractions which lead to T6SS-1 activation will then further be analyzed via tandem mass spectrometry.
- The Type VI secretion system injects so-called effector proteins into the target organisms which could be either an opponent or a symbiotic partner. What function do the effector proteins in A. olearius BH72 have?
By now, the effector proteins of Azoarcus olearius BH72 were poorly studied. In my PhD project, I will focus on the biochemical characterization of the proteins by comparing the effector proteins with similar protein classes and the conduction of enzyme assays. Furthermore, the effect of these proteins on the host plant will be studied by expressing the proteins of interest in plant cells and observation of the effect on the cell. However, not only a single plant cell, but also the neighboring cells will be studied by using modern techniques such as MERFISH (Multiplexed Error-Robust Fluorescence in situ Hybridization) and PHYTOMap (Plant Hybridization-based Targeted Observation of gene expression Map).