Periodic Reporting for period 1 - Mi-DOG (The “parasitism-readers” of the world’s most damaging plant-parasitic nematode Meloidogyne incognita: new avenues to address food global security.)
Berichtszeitraum: 2020-04-01 bis 2023-03-31
Plant-parasitic nematodes (PPNs) represent a dramatical risk to a wide range of crop plants, by causing considerable losses estimated at several billion euros per year. Understanding how these microscopic worms achieve the pathogenic success is crucial given the significant impact on worldwide food security. The parasitic animals employ sophisticated strategies necessary for host invasion, resulting by the manipulation of important plant processes (i.e development, metabolism, or immunity). This is mostly based on the delivery of hundreds and highly diversified parasitism factors into the host cells – termed “effector proteins”. A majority are originated from dedicated secretory cell factories and play a crucial role in parasitism along the nematode lifecycle. Despite the growing number of studies on functional analyses of effectors, our understanding of how these pathogens cause diseases is far from complete. Studying the details of effector biology promises to be a fundamental task for the design of impactful and sustainable parasite control solutions. Recent efforts coupling large-scale transcriptome and genomic studies in different PPNs have revealed two important points:
1) The existence of concertedly regulated effector gene expression program at specific phases of their life cycle
2) and the presence of promoter motifs found upstream of co-expressed effector genes.
From a collaborative work with French and UK researchers, we also identified a novel promoter motif - termed Mel-DOG box in the root-knot nematode Meloidogyne spp. These upstream non-coding genetic signatures are conceptually analogous but vary in their sequence, illustrating lineage-specific promoter motif in the phylum Nematoda. Together these studies have provided a means to further explore the effector biology in PPNs. In the light of this emerging theme, my objective was to answer the fundamental question:
how parasitism is regulated in Meloidogyne incognita?
I have thus addressed two specific objectives:
• Capture and identify the key components associated to Mel-DOG signature in effectors using a combination of well-established and highly innovative CRISPR mediated methodologies
• Understand their functional role in parasitism in vivo by RNA interference
1) The existence of concertedly regulated effector gene expression program at specific phases of their life cycle
2) and the presence of promoter motifs found upstream of co-expressed effector genes.
From a collaborative work with French and UK researchers, we also identified a novel promoter motif - termed Mel-DOG box in the root-knot nematode Meloidogyne spp. These upstream non-coding genetic signatures are conceptually analogous but vary in their sequence, illustrating lineage-specific promoter motif in the phylum Nematoda. Together these studies have provided a means to further explore the effector biology in PPNs. In the light of this emerging theme, my objective was to answer the fundamental question:
how parasitism is regulated in Meloidogyne incognita?
I have thus addressed two specific objectives:
• Capture and identify the key components associated to Mel-DOG signature in effectors using a combination of well-established and highly innovative CRISPR mediated methodologies
• Understand their functional role in parasitism in vivo by RNA interference
My work for the first year was exclusively devoted to the realization of the Work Package 1 as presented in the DoA to the Grant Agreement. Progress has been delayed considerably as I have been unable to have access to a laboratory for the entire duration due to the Covid 19 pandemic and the transfer of my fellowship to the new host laboratory. Using in silico data from UK and French collaborators, I have participated remotely to a major resource of transcriptomic profiling at specific life-stages of M. incognita. By focusing on effectors, we revealed 1) spatio-temporal expression patterns underlying a precise transcription regulation in parasitism and 2) DNA conserved motif enriched in the promoter of effector genes and 3) roadmap for effector prediction in the genus Meloidogyne. As co-author, these findings have been published in a peer-reviewed international journal MDPI (doi: 10.3390/genes12050771). From this detailed survey, I was next interested to analyse key components associated to Mel-DOG effector gene expression at early- and late stages of infection. Using yeast one-hybrid (Y1H) libraries screening, coupled with data from expression and literature, we identified five major candidates involved in parasitism regulation. To further maximize the likelihood to find trans-regulatory inputs, I aimed to develop an engineered chromatin immunoprecipitation (enChIP-CRISPR) methodology that allow 1) to capture simultaneously these proteins in a recalcitrant organism for genetic transformation and 2) in a natural chromatin context. Although no candidates have not yet identified from this tool, I established a framework that will allow future studies of regulatory components in the root-knot nematode and further open up enChIP-CRISPR technology to other plant-pathogen interactions. However, proteomic characterization by mass spectrometry (EDyP platform, CEA Grenoble) may soon allow the confirmation the enChiP-CRISPR reproducibility by crossing Y1H data. To go deeper in their functional parasitism role, two RNA interference (RNAi) tools have been conducted: in vitro RNAi soaking – to transiently silent of gene targets at early stage (i.e hatched pre-parasitic juveniles) - and Host-Induced Gene Silencing (HIGS) – to stably silent candidates along the nematode lifecycle. The work package will be achieved with pathogenicity assays will be experimentally assessed and will allow overall to confirm the crucial role of candidates in nematode parasitism.
To share the project with a wider audience, I have been particularly attached to disseminate knowledge and results through different communication supports. First year of the fellowship, I had contributed to the publication of a scientific article – towards the search of Mel-DOG motif - as pre-print (BioRxirv) and then in a peer-reviewed international journal (MDPI Genes). These findings have been also continuously communicated through national and international conferences by oral / poster presentations (i.e PhD day, ESN’s Virtual Nematology Conference 2021, 7th International Congress of Nematology 2022). Dissemination activity will be pursued after the end of the project by exposing results through a poster presentation at the international conference (International Congress of Plant Pathology 2023). By joining the UMR MAP academic research unit (Lyon, France), I have been also exposed to its long-standing industrial partnership established with Bayer CropScience. Every year, I have diffused the result outcomes and the innovative research methodologies on its annual scientific outreach newsletter. For dissemination to scholarly community at large, I had benefit to the resources of the host institution (University Claude Bernard Lyon 1) by communicating about biological generalities to study plant-pest interactions with a special emphasis on plant-parasitic nematodes (Fascination of plant day). For general and online audience, I have promptly communicated large scientific interests, events or research findings in the field of plant-microbe interactions on social media (Twitter, Linkedin, Researchgate). Upon completion, I aim to publish two scientific articles in line with the DoA detailing my findings: a peer reviewed protocol article on “The in vitro enChIP-CRISPR tool applied to plant-parasitic nematodes; and an interdisciplinary plant-nematode research article on “Identification & characterization of parasitism-readers in the RKN M. incognita”.
To share the project with a wider audience, I have been particularly attached to disseminate knowledge and results through different communication supports. First year of the fellowship, I had contributed to the publication of a scientific article – towards the search of Mel-DOG motif - as pre-print (BioRxirv) and then in a peer-reviewed international journal (MDPI Genes). These findings have been also continuously communicated through national and international conferences by oral / poster presentations (i.e PhD day, ESN’s Virtual Nematology Conference 2021, 7th International Congress of Nematology 2022). Dissemination activity will be pursued after the end of the project by exposing results through a poster presentation at the international conference (International Congress of Plant Pathology 2023). By joining the UMR MAP academic research unit (Lyon, France), I have been also exposed to its long-standing industrial partnership established with Bayer CropScience. Every year, I have diffused the result outcomes and the innovative research methodologies on its annual scientific outreach newsletter. For dissemination to scholarly community at large, I had benefit to the resources of the host institution (University Claude Bernard Lyon 1) by communicating about biological generalities to study plant-pest interactions with a special emphasis on plant-parasitic nematodes (Fascination of plant day). For general and online audience, I have promptly communicated large scientific interests, events or research findings in the field of plant-microbe interactions on social media (Twitter, Linkedin, Researchgate). Upon completion, I aim to publish two scientific articles in line with the DoA detailing my findings: a peer reviewed protocol article on “The in vitro enChIP-CRISPR tool applied to plant-parasitic nematodes; and an interdisciplinary plant-nematode research article on “Identification & characterization of parasitism-readers in the RKN M. incognita”.
Understanding of how nematode cause plant disease is a major task and challenge of research and beyond in plant-pathogen interactions. This project aimed to push forward the knowledge on effector biology by dissecting gene regulation networks in parasitism. The development of two-tiered approach (Y1H and enChiP-CRISPR) paves the way in 1) the understanding of the crosstalk between chromatin structure and transcription factor binding, two phenomena often studied independently and 2) the generation of valuable data in organisms not transformable such as PPNs. Lastly, the Mi-DOG project will also contribute to developing more accurate strategies against plant-pathogenic nematodes.