Identification and functional analysis of plant targets of effectors of the root-knot nematode Meloidogyne incognita

Among plant parasitic nematodes, root-knot nematodes (RKNs) of the genus Meloidogyne have a remarkable ability to infect more than 5,500 plant species, including major crops, posing a serious threat to global agriculture. The increase in RKN populations due to climate change and the limitation of control methods underlines the need for rapid progress in the development of new effective resistance strategies. RKNs orchestrate a complex molecular reprogramming of plant root cells leading to the formation of a permanent feeding structure made of multinucleated and hypertrophied giant cells that provide nutrients to the nematode. Effector proteins produced in oesophageal glands of the RKNs and secreted in planta through a syringe-like stylet, are instrumental in hijacking host cellular processes and enabling giant cell development and maintenance. These effectors target different subcellular compartments, including the nucleus, to modulate key cellular activities. Understanding the molecular functions of nematode effectors and the mechanisms they target in the host plant, is of interest on a fundamental point of view, and also for applied purposes.Using a combination of comparative genomics and transcriptomics, several candidate effectors of Meloidogyne incognita were identified. In situ hybridization confirmed that these effectors are expressed in juvenile salivary glands, supporting their potential for direct secretion into plant tissues. Agroinfiltration of GFP-tagged effectors in Nicotiana benthamiana identified specific effectors targeting the plant cell nucleus and nucleolus, suggesting manipulation of plant nuclear and nucleolar processes. By studying the role of these nuclear effectors using a luminescence-based splicing reporter assay, we were able to identify MiEFF186 as a modulator of alternative splicing. The search for the plant targets of MiEFF186 in tomato roots, using a yeast two-hybrid assay, identified two proteins, CIS2/GDP1 and RRS1. These proteins have been shown to be involved in the modulation of alternative splicing and/or in ribosome biogenesis. Co-localization assays showed that MiEFF186 interacts with CIS2/GDP1 and RRS1 in the plant nucleolus, suggesting that RKNs hijack nucleolar functions to create favorable conditions for the formation of the feeding site. Functional analysis of CIS2/GDP1 using co-immunoprecipitation, VIGS and mutant plants confirmed the critical role of the CIS2/GDP1-MiEFF186 interaction in successful RKN infection. The results obtained for RRS1 showed the potential regulation by MiEFF186 of ribosome biogenesis in the nucleolus.This work provides important insights into the molecular strategies used by RKNs to exploit plant cell machinery, particularly nucleolar functions, to establish and maintain feeding sites. By identifying critical plant targets of nematode effectors, this research opens up new avenues for the development of crop protection strategies aimed at disrupting these molecular interactions to improve resistance to RKNs.