Stéphanie Jaubert-Possamai

Crops face attacks from a wide range of pathogens and pests that deploy various parasitic strategies. All aggressors must manipulate major plant functions, such as immune signaling, hormone pathways, metabolism, and development, to successfully establish themselves and complete their life cycle. Here, we compared the tomato plant’s response to three evolutionary distant pathogens (nematodes, aphids and oomycetes) during compatible interactions using a transcriptomic approach. We identified differentially expressed genes and biological processes, and highlighted potential key host hubs associated with successful parasitism. By integrating recent published datasets, we refined our understanding of the global and tissue-specific mechanisms targeted during compatible interactions and, through co-expression analysis, identified clusters showing shared dysregulation patterns enriched in specific GO terms. Finally, model-to-crop translational analysis using the Arabidopsis interactome network, repositioned tomato candidates within larger interaction networks and emphasized the key positions occupied by some of them. Identifying these pivotal tomato targets is crucial to decipher processes underlying parasitism and, consequently, offers new opportunities to develop sustainable multi-pathogen control strategies.

Summary: Tomato ( Solanum lycopersicum ), despite being the most important vegetable crop world‐wide, remains vulnerable to over 200 diseases caused by different pests. Although tomato molecular response to individual stresses is well studied, the gene regulatory network (GRN) representing the crosstalk and trade‐offs of multi‐stress responses remains almost unexplored. We developed GENIAL (Gene rEgulatory Network and topologIcal datA anaLysis) to refine and analyze complex GRNs and TomTom, a knowledge graph that gathers 11 publicly available databases in a unique FAIR (Findable, Accessible, Interoperable, Reproducible) resource. To test GENIAL, we used transcriptomics data from tomato subjected to six distinct pathogens from the literature. GENIAL yielded the identification of transcription factors (TFs) coordinating the specific and multiple pathogen response. Functional validation using the virus‐induced gene silencing system in tomato demonstrated that ETHYLENE RESPONSE FACTOR 16 and TCP DOMAIN PROTEIN 17 act as key TFs in the response to Botrytis cinerea , as silencing of either TF resulted in increased susceptibility. The validation of selected downstream targets allowed the validation of the robustness of the interactions highlighted by GENIAL. This study represents a proof of concept of our framework and can be extended to include other molecular layers and scaled to other questions involving tomato and beyond.

Root-knot nematodes (RKNs) are obligate plant parasites that establish biotrophic interactions with a wide range of host plants, causing major crop losses worldwide. During infection, RKNs secrete effector proteins from their esophageal glands into root tissues via a stylet. These effectors target specific subcellular compartments and manipulate host cell functions to induce the formation of hypertrophied and multinucleated giant cells, which serve as feeding sites essential for nematode development. Here we describe a new RKN-specific effector, EFFECTOR17 (EFF17), conserved in the five most damaging RKN species: Meloidogyne incognita, M. enterolobii , M. arenaria , M. javanica , and M. hapla . In situ hybridization showed that EFF17 genes are specifically expressed in the esophageal glands of various RKN species. Silencing EFF17 in M. incognita reduced its reproduction on Nicotiana benthamiana , demonstrating a key role in parasitism. Yeast two-hybrid and split luciferase assays revealed that tomato and Arabidopsis KINESIN LIGHT CHAIN RELATED (KLCR)/CELLULOSE-MICROUBULE UNCOUPLING (CMU) proteins are host targets of MiEFF17. Arabidopsis klcr mutants developed significantly fewer galls and egg masses. Our findings propose that EFF17 manipulates KLCR function to promote parasitism.

Cross-kingdom RNA interference (ckRNAi) is emerging as a mode of inter-organismal gene regulation, yet mechanistic examples in plant–metazoan interactions remain limited. Here, we demonstrate miRNA-driven ckRNAi in the nematode–plant pathosystem. Root-knot nematodes are among the most destructive plant pathogens, reprogramming root tissues to develop into galls containing multinucleated, hypermetabolic giant feeding cells essential for parasitism. AGO1-associated small-RNA immunoprecipitation (AGO1-RIP) from tomato galls revealed the selective in planta loading of 10 M. incognita miRNAs into host AGO1. Integrating degradome profiling, target prediction, and dual-luciferase reporter assays, we validated miRNA-directed silencing of nine tomato transcripts by four secreted nematode miRNAs. These targets map to major pathway classes involved in immune signaling, metabolic regulation, and cellular reprogramming linked to feeding-site establishment. Functional analyses further show that the nematode-secreted miR-2b is enhances giant feeding cell development. Comparative AGO1-RIP in Arabidopsis thaliana identified a conserved subset of AGO1-loaded nematode miRNAs, including miR-2b and miR-100, consistent with shared small-RNA effectors across hosts. Finally, the overlap between AGO1-loaded miRNA families and helminth secreted small-RNA repertoires supports evolutionary convergence on RNA-based virulence strategies. Collectively, our findings establish miRNA-mediated ckRNAi as a mechanistic component of plant–root-knot nematode interactions and provide a framework for leveraging RNA-based vulnerabilities for nematode control.

Crops face attacks from a wide range of pathogens and pests that deploy various parasitic strategies. All aggressors must manipulate major plant functions, such as immune signaling, hormone pathways, metabolism, and development, to successfully establish themselves and complete their life cycle. Here, we compared the tomato plant’s response to three evolutionary distant pathogens (nematodes, aphids and oomycetes) during compatible interactions using a transcriptomic approach. We identified differentially expressed genes and biological processes, and highlighted potential key host hubs associated with successful parasitism. By integrating recent published datasets, we refined our understanding of the global and tissue-specific mechanisms targeted during compatible interactions and, through co-expression analysis, identified clusters showing shared dysregulation patterns enriched in specific GO terms. Finally, model-to-crop translational analysis using the Arabidopsis interactome network, repositioned tomato candidates within larger interaction networks and emphasized the key positions occupied by some of them. Identifying these pivotal tomato targets is crucial to decipher processes underlying parasitism and, consequently, offers new opportunities to develop sustainable multi-pathogen control strategies.

Global population is rapidly increasing, representing a major challenge for food supply, exacerbated by climate change and environmental degradation. Despite the pivotal role of agriculture, plant health and survival are threatened by various biotic stressors. Although how plants respond to each of these individual stresses is well studied, little is known about how they respond to a combination of many of these bio-aggressors occurring together. To tackle this question, first, we built TomTom, a knowledge graph gathering molecular interactions from nine publicly available databases, including transcription factors- or microRNAs-targets, protein-protein interactions, and functional terms. Then, we selected transcriptomics data of tomato subjected to six distinct pathogens and performed an integrative analysis. We found 5561 candidate genes involved in the multi-stress response of tomato. To study how the response is orchestrated, we mapped those genes in TomTom and extracted a comprehensive gene regulatory network (GRN) composed of 71 transcription factors (TF) and 1786 target genes. By estimating the TF activity, we identified 43 TFs responding either specifically to one or multiple bio-aggressors. GRN analyses with a topological data analysis approach allowed to identify 18 clusters of TFs with similar properties, yielding four main configurations localized in specific regions of the GRN. Finally, we found one NAC and four ERF hubs which cooperatively coordinate the tomato response to multiple pathogens. Our findings allowed to study the complex molecular reprogramming in tomato upon interaction with different biotic agents, providing tools scalable to other questions involving tomato molecular interactions and beyond.

Root-knot nematodes (RKN) from genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression and auxin is known to be a key player in their development. However, little is known how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their microRNA167 regulators, were identified as robust gene/miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-GUS fusions showed the upregulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.

Summary: Root‐knot nematodes (RKNs) are root endoparasites that induce the dedifferentiation of a few root cells and the reprogramming of their gene expression to generate giant hypermetabolic feeding cells. We identified two microRNA families, miR408 and miR398, as upregulated in Arabidopsis thaliana and Solanum lycopersicum roots infected by RKNs. In plants, the expression of these two conserved microRNA families is known to be activated by the SPL7 transcription factor in response to copper starvation. By combining functional approaches, we deciphered the network involving these microRNAs, their regulator and their targets. MIR408 expression was located within nematode‐induced feeding cells like its regulator SPL7 and was regulated by copper. Moreover, infection assays with mir408 and spl7 knockout mutants or lines expressing targets rendered resistant to cleavage by miR398 demonstrated the essential role of the SPL7/MIR408/MIR398 module in the formation of giant feeding cells. Our findings reveal how perturbation of plant copper homeostasis, via the SPL7/MIR408/MIR398 module, modulates the development of nematode‐induced feeding cells.

Root-knot nematodes (RKN) from genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression in targeted root cells, as shown by transcriptomic analyses of galls or giant cells from various plant species. Small non-coding RNAs, and messenger RNAs from tomato ( Solanum lycopersicum ) galls and uninfected roots were sequenced. De novo microRNA prediction in the tomato genome identified microRNAs expressed in galls and uninfected roots. Statistical analyses identified 174 miRNA genes differentially expressed in galls at 7 and/or 14 days post infection (dpi). Integrative analyses combining small non-coding RNA and transcriptome datasets with the specific sequencing of cleaved transcripts identified miRNA targets in tomato galls. Functional analyses of promoter-GUS fusions and CRISPR-Cas9 mutants highlighted the role of the miR167-regulated transcription factor AUXIN RESPONSE FACTOR 8 (ARF8) in giant cell formation.

Root-knot nematodes (RKNs) are among the most damaging pests of agricultural crops. Meloidogyne is an extremely polyphagous genus of nematodes that can infect thousands of plant species. A few genes for resistance (R-genes) to RKN suitable for use in crop breeding have been identified, but virulent strains and species of RKN have emerged that render these R-genes ineffective. Secretion of RKN effectors targeting plant functions mediates the reprogramming of root cells into specialized feeding cells, the giant cells, essential for RKN development and reproduction. Conserved targets among plant species define the more relevant strategies for controlling nematode infection. The EFFECTOR18 (EFF18) protein from M. incognita interacts with the spliceosomal small nuclear ribonucleoprotein D1 (SmD1) in Arabidopsis (Arabidopsis thaliana), disrupting its function in alternative splicing regulation and modulating the giant cell transcriptome. We show here that EFF18 is a conserved RKN-specific effector that targets this conserved spliceosomal SmD1 protein in Solanaceae. This interaction modulates alternative splicing events produced by tomato (Solanum lycopersicum) in response to M. incognita infection. The alteration of SmD1 expression by virus-induced gene silencing in Solanaceae affects giant cell formation and nematode development. Thus, our work defines a promising conserved SmD1 target gene to develop broad resistance for the control of Meloidogyne spp. in plants.