Periodic Reporting for period 2 - ROOBABAA (Roots and bacteria: Basis of attraction)
Reporting period: 2023-03-01 to 2024-08-31
In order to feed the world population in a sustainable fashion, there is an urgent need to reduce the current levels of fertilizer and pesticide inputs. Much recent research is motivated by the promise of using growth-promoting, disease-suppressive bacteria as transferable, protective agents in agriculture, with the hope of replacing, or at least reducing the use of fertilizer and pesticides. Although many bacteria have been demonstrated to have major growth promoting and protective function under laboratory conditions, this perfomance often does not translate into reliable improvements in the field. Major obstacle are thought to be the soil's physico-chemical complexity and a staggeringly complex biotic environment. However, current methods of describing bacterial association with root also crucially lack in spatial and temporal resolution. The root has to be seen as an assembly of dynamically growing, distinct micro-environments for bacteria. If we don't understand the specific characteristics of these different micro-environments, we will not be able to understand the reasons for success or failure of bacterial colonisation of roots. Therefore, we are working on describing bacterial attraction to roots and their attachment and growth on them by making extensive use fluorescent bacteria that will allow us to monitor of bacterial metabolism and tracing their movements and growth. We are initially pursuing "simple" experiments, featuring one bacterial species on a root. With this, we aim to understand the basis of attraction between bacteria and roots. In a second step, we will extend on our findings with single bacterial species, by putting them into a context of defined bacterial communities. Our work will provide critical insights into fundamental aspects of bacterial colonization that have remained unadressed, due to a lack of efforts and tools that would allow to observed bacteria root interactions at sufficiently high resolution. This will provide a basis for a more predictive design of bacterial agents for use in agriculture.
We have suceeded in designing custom-made, 3D-printed mini-growth chambers that allow for live-observation of root growth and early stages of bacterial colonisation. We have also developed new types of mini-hydroponic systems that allow for more reliable root growth with less stress induction, as well as designs for growth of defined, complex bacterial communities on roots that still alow for easy root extraction and observation. These designs will be shared on open platforms upon publication. We are also applying new CRISPRi screening methods that will allow us to define the bacterial genes required for colonisation. In collaboration with Prof. Feng Zhou (CEMPS, Shanghai), we have identified the root exudates of our endodermal barrier mutants and identify glutamine as a major factor causing the enhanced colonisation in these mutants. Importantly, this led us to propose that absence or transient breakage of endodermal barriers is an important determinant explaining where bacterial colonisation occurs in wild-type plants. Intriguingly, a Pseudomonas protegens mutant for all of its amino acid chemoreceptors was generated by us and found to be unable to accumulate around lateral root emergence sites. This clearly demonstrates that localised amino acid exudation in roots is important for the spatial pattern of bacterial root colonisation. We have also managed to obtain a new plant line that is hypersensitive exclusively to 10-hydroxy fatty acid, a specific bacterial molecule, recognized by the pattern recognition receptor LORE in Arabidopsis. This will now allow us to compare the responses to bacterial colonisation and whether, as hypothesized by us, immune responses to bacteria are dependent on bacterial status, rather than mere bacterial presence.
By the end of the project, we expect to have established that root surfaces represent rapidly evolving micro-niches that operate at the micrometer scale and will have developed tools that allow us to describe how this impacts bacterial colonisation. We will also have obtained an understanding of how this spatial complexity drives bacterial specialization and spatial segregation into distinct communities.