Characterisation of the role of small non-coding RNAs and inter-organism silencing in plant-gall nematode interactions

President of the jury :                       

• Dr. Christine COUSTAU, DR1, ISA

Rapporteurs :                     

• Dr. Muriel VIAUD, DR2, BIOGER
• Dr. Germano CECERE, DR1, Institut Pasteur                              

Examiners

• Pr. Amy BUCK, Professor, Edinburg University
• Dr. Matthias BENOIT, CR, LIPME
• Dr. Guillaume MOISSIARD, CR, LGDP

Thesis Directors :

• Dr. Stéphanie JAUBERT, DR2, ISA
• Dr. Bruno FAVERY, DR1, ISA

Abstract :

Root-knot nematodes of the genus Meloidogyne are among the most destructive plant pathogens. They exhibit a remarkably broad host range, infecting more than 3,000 plant species, including major crop plants, and cause global agricultural losses amounting to several billions of dollars annually. These plant-parasitic nematodes induce the formation of root galls by triggering the dedifferentiation of parenchyma cells into specialized, multinucleated, and hypermetabolic giant cells that function as essential feeding cells throughout the nematode life cycle. The formation of these giant feeding cells results from a massive reprogramming of host gene expression in response to nematode-secreted effector molecules, a molecular mechanism that is conserved across their broad host spectrum.
Inter-kingdom RNA interference (RNAi) has recently emerged as a novel mode of communication between organisms, involving the secretion of small non-coding RNAs by pathogens to regulate host gene expression through the hijacking of the host RNAi machinery, and more specifically Argonaute (AGO) proteins. However, the contribution of small non-coding RNAs from root-knot nematodes, and more broadly from plant-parasitic nematodes to the establishment of infection remains largely unknown.
To investigate their potential role in host gene regulation during feeding site establishment, we performed an AGO1-associated RNA immunoprecipitation (AGO1-RIP) from galls of Solanum lycopersicum infected with Meloidogyne incognita. Mapping of the resulting reads to the genomes of both species confirmed the loading of 11 M. incognita miRNAs onto host AGO1 proteins. We subsequently identified plant transcripts targeted by these nematode-secreted miRNAs by combining degradome sequencing, computational target prediction, and in vivo validation of miRNA-mediated cleavage using a Dual-Luciferase silencing reporter.
To assess how this inter-kingdom RNAi mechanism may contribute to the broad host range of root-knot nematodes, AGO1-RIP experiments were also conducted in two additional model plant species, Arabidopsis thaliana and Medicago truncatula, representing the Brassicaceae and Leguminosae families, respectively. We identified miR-2 and miR-100 as being secreted and loaded onto AGO1 in all three host species tested, highlighting their potential importance in infection establishment. Analysis of target site conservation across homologous transcripts in different host plants revealed conserved cellular pathways targeted by these miRNAs, suggesting shared infection mechanisms among diverse plant species.
An evolutionary parallel is drawn between root-knot nematodes, plant-parasitic nematodes, and helminths, raising the question of evolutionary convergence in the families of miRNAs secreted by these parasites. Collectively, these findings provide the first evidence of inter-kingdom RNA interference between a plant-parasitic nematode and its host plants through the identification of Meloidogyne-secreted miRNAs. This study paves the way for future functional characterization of secretion pathways, identified target transcripts, and the impact of cross-kingdom RNAi on the establishment and maintenance of nematode feeding sites, ultimately enabling the development of novel strategies to control one of the most devastating pathogens of modern agriculture.

Keywords :

Root-knot nematode, RKN, tomato, S. lycopersicum, microRNA, phytopathogen, RNA interference, cross-kingdom silencing, parasitism, giant cells, gene reprogramming, biotic interactions