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Determination of novel molecular cross-signalling mechanisms between bacteria and plants leading to enhanced crop production

Final Report Summary - PROAGROBAC (Determination of novel molecular cross-signalling mechanisms between bacteria and plants leading to enhanced crop production.)

*Summary description of the project objectives*
Using the plant endophytic Serratia plymuthica as a model organism, together with state of the art technologies, this IIF project aimed at investigating the relationships between bacterial signal-driven regulatory networks and their influence on the host plant these bacteria colonise and which leads to plant growth promotion and the protection of crops against bacterial and fungal infections which can result in significant economic losses. The project aimed at investigating some of the molecular mechanism behind these interactions. The ultimate aim, beyond the life-span of this project, is to enable the design of the appropriate environmentally friendly seed inoculants, using optimised bacterial consortiums based on their signalling mechanisms, to maximise plant growth, stress tolerance and resistance to infections.

*The work performed, main results and conclusions from PROAGROBAC*
The determination of the draft genome sequence of bacterial plant isolate G3 of S. plymuthica has enabled the in silico identification of genes coding for key regulatory networks and biocontrol-related traits. Some of their biological functions have been determined through the construction of sets of mutants, followed by phenotypic analysis. This has lead to a more detailed characterisation of the integrated regulatory networks such as: (i) the quorum sensing (QS) signalling systems; (ii) the stationary-phase sigma factor RpoS; (iii) LysR and TyrR-type transcriptional regulators and (iv) the post-transcriptional Rsm system from G3. Assays using gene reporter fusions combined with mass-spectrometry have unravelled the existence of a close integration of these regulatory networks to modulate beneficial bacteria-plant interactions. These include antifungal activity and plant growth promotion abilities via the fine-tuning of the biosynthesis of the antibiotic pyrrolnitrin (PRN), the plant auxin indole-3-acetic acid (IAA) and QS signal molecules N-acylhomoserine lactones(AHLs) at multiple levels. Furthermore the use of confocal laser microscopy (CLSM) revealed the impact of these regulatory networks on in vitro biofilm formation and bacterial colonisation in planta.
The role of indole acetic acid (IAA) production in bacterial physiology was investigated using a global comparative proteomic strategy between the parent G3 and mutants unable to make IAA. A total of 61 proteins were found to be altered in abundance in the mutant strain compared to the parental organism. These proteins are implicated in multiple bacterial physiology processes including primary metabolism, signal transduction, gene regulation and the stress response, indicating that IAA can have a role as global pleiotropic regulator in bacteria in addition to the induction of plant growth promotion.
The cross-kingdom bacteria-plant signalling mechanisms using AHL QS signal molecules were investigated through plant phenotypic analysis, and bioreporter assay in Arabidopsis transgenic plant lines and confocal microscopy using specific fluorescent dyes after treatment with AHLs. The impact of AHLs in other plants was also studied. The results revealed that QS signal molecules can be perceived by a number of plant species modulating a variety of plant physiological processes such as seed and spore germination, alteration of root architecture, growth, and stress tolerance etc. In addition, H2O2 and IAA signalling pathways were activated after AHL treatment. Further proteomic analysis following AHL treatment of rice roots identified 81 proteins altered in abundance involved in different processes such as photosynthesis, the steady-state level of ROS (reactive oxygen species), hormone biosynthesis, response to hormone, stress/defence, amino acid metabolism and signal transduction. These studies revealed further insights into the mechanisms of cross-kingdom signalling to modulate plant growth promotion and stress tolerance.

*Socio-economic impact and the wider social implications of the project*
PROAGROBAC has highlighted the importance of the integration of the bacterial signal-driven regulatory networks, which are widespread in many plant-associated bacteria, in the fine tuning the beneficial bacteria-plant interactions. Better understanding of the cross-kingdom communication mechanisms may therefore facilitate the design of new environmentally friendly microbial seed inoculants to improve crop yield and tolerance to biotic and abiotic stresses. This could be achieved by influencing the behaviour of these integrated regulatory networks to maximise beneficial plant-microbe interactions leading to a reduction in the use of chemical fertilisers and pesticides and the risks they pose to human health and environment which will improve agricultural sustainability through better adaptation to climate changes, and contribute to the reduction of poverty and malnourishment (UN Millennium Development Goals). Additionally, since some of these regulatory systems are present in many other organisms of importance to medicine, industry and agriculture, these findings could be extrapolated to more distant fields such as medicine and the food industry.

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