Integrative proteomics and biophysics approach to elucidate signal perception and motility of Phytophthora zoospores during the early stages interaction with plants.

President of the jury :                       

• Bruno FAVERY, Directeur de Recherche, Institut Sophia Agrobiotech, France.

Rapporteurs :                     

• Bernard DUMAS, Directeur de Recherche, CNRS, France.
• Wafa ACHOUAK, Directrice de Recherche, CNRS-CEA-Aix Marseille Univ BIAM/LEMiRE, France.

Examiners

• Agnès ATTARD, Chargée de Recherche, Institut Sophia Agrobiotech, France.
• Carine DOUARCHE, Maître de Conférences, Université Paris-Saclay-CNRS-FAST, France.
• Tijs KETELAAR, Professor, Laboratory of Cell and Developmental Biology, Wageningen University, Netherlands

Thesis Director :

• Eric GALIANA, Directeur de Recherche, Institut Sophia Agrobiotech, France.
• Xavier NOBLIN, Directeur de Recherche, Institut de Physique de Nice, France.

Abstract :

The epidemic spread of plant diseases caused by Phytophthora is primarily based on the dispersal of unicellular, biflagellated zoospores in the soil. Zoospore guidance towards host plants relies on diverse mechanisms such as chemotaxis, electrotaxis, negative geotaxis and rheotaxis. Signals from soil particles and host plants critically influence these motion processes, guiding zoospores during the initial stages of root colonization. However, the mechanisms underlying zoospores perception, resulting in the directed motion toward hosts remain unclear. Critical questions include the nature and the specificity of these sensing mechanisms compared to those used by other soil microorganisms, and the extent to which the dynamic and morphological characteristics of zoospores contribute to their guided motion.

In this context, the first part of this thesis focused on investigating the sensing capabilities of Phytophthora parasitica zoospores by analyzing their plasma membrane protein repertoire through a proteomic approach. Peptides were detected from membrane samples using LC-MS/MS, and related proteins were identified by mapping against the Phytophthora parasitica reference proteome, allowing for detailed characterization and comparison of the membrane profiles of the zoospore cell body and flagella fractions. Given the naturally polarized structure of zoospores, which exhibit two morphologically distinct flagella responsible for oriented motion, we hypothesized a critical role of flagella in sensing mechanisms. Our analysis identified three prominent membrane proteins associated with sensing and motion response mechanisms in zoospores, some of which were specifically localized to the flagella membrane: a sterol-sensing protein, a nucleotide cyclase and a Na+/K+ ATPase. To start a functional analysis in zoospore sensing, immunolocalization, pharmacological and electrophysiological assays were initiated.

The second part of this thesis employed an automated high-content imaging approach to establish a novel method for quantifying diverse characteristics of soil microswimmers, including zoospores, in response to a soil/host factor (potassium gradient). The initial observations focused on the motion responses of zoospores among other species, demonstrating the feasibility to simultaneously distinguish morphologies, trajectories, velocities, and the impacts of the soil factor on a simple synthetic microbial community composed of P. parasitica, Vorticella microstoma, and Enterobacter aerogenes. Following these observations, a detailed biomechanical analysis was conducted to quantify motion metrics, such as velocity, trajectory geometry, and flagellar beating patterns under the same stimulus, revealing specific motion dynamics unique to zoospores compared to other microswimmers. As a result, we found that increasing potassium concentrations disrupt the normal swimming pattern of zoospores, typically characterized by long, straight runs and periodic tumbles. Instead, the zoospores exhibited progressively shorter and more circular trajectories, with reduced velocity and altered flagella beating

These findings integrate biochemical and biomechanical principles to advance the understanding of the fundamental biological process of microbial guidance toward external cues. Elucidating the sensing mechanisms and motion responses of Phytophthora zoospores enhances our understanding of the pre-colonization phase of plant infection, highlighting how these pathogens move toward hosts. This integrated approach offers valuable insights into early infection stages, potentially guiding new plant disease management strategies. 

Keywords : Oomycetes, Plant physio-pathology, Motion dynamics, Signalling, Zoospores

In person or via Zoom: https://inrae-fr.zoom.us/j/7066402506?omn=91079735006