Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

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La biogenèse des galles induites par des phyto-nématodes dépend de l'hyperactivation du cycle cellulaire : études fonctionnelles des nouveaux acteurs du cycle mitotique et de l'endocycle dans les cellules géantes

During the last twenty years, Arabidopsis thaliana has been successfully used as a model plant accelerating discoveries at the molecular and cellular level in numerous plant-pathogen interactions. Arabidopsis is susceptible to a number of pathogens including nematodes, responding to pathogen attack in a similar manner to cultivated plant species. Meloidogyne incognita is a crop parasite causing extensive economic losses of billions of dollars yearly for the agriculture worldwide. Root-knot nematodes induce feeding sites within the host root inducing up to eight giant-feeding cells within the root vascular cylinder by injecting secretions. These giant cells become hypertrophied and contain multiple enlarged nuclei as a result of numerous mitotic events of which cytokinesis is absent or interrupted. Throughout expansion, giant cells become highly polyploid via successive endoreduplication cycles. Thus, a crucial feature for root-knot nematode development is the hyperactivation of the plant host cell cycle in galls. The eukaryotic cell cycle is conserved and characterized by four phases: The S phase (DNA replication) is followed by the M phase (mitosis), cytokinesis and cell division. These are intercalated by the G1 phase (first gap) which connects the end of mitosis to the start of DNA synthesis, and the G2 phase (second gap) which connects the end of DNA synthesis to the start of mitosis. The endocycle is a variant of the cell cycle in which replication occurs without mitosis, resulting in a doubling of cellular DNA content for each endocycle round. The hyperactivation of the cell cycle is essential for the nematode-induced gall biogenesis, and a precise balance between mitotic and endocycle phases is essential for the successful compatible interaction. As a broad question of my PhD, we addressed which the pre-requisites are needed to establish a functional gall induced by root-knot nematodes. More specifically, I essentially focused on the functional studies of particular plant cell cycle genes playing a role in the mitotic and the endocycle during plant-root-knot nematode interaction. To address this question, functional studies of new players in the cell cycle of the plant host, stimulating (E2F genes), inhibiting (ABAP1 and an interactor AIP10 genes) or controlling (WEE1 gene) the cell cycle, were investigated in galls induced in our model host Arabidopsis thaliana. The results obtained demonstrate that the genes here studied are critical for, as well the formation as the maintenance of nematode feeding sites. Overall, our functional study and molecular analysis show that the absence of E2Fa and E2Fb transcription factors will severely affect gall development and nematode reproduction. As well, overexpression of E2Fa and E2Fb will disrupt the cell cycle in galls, validating their importance for nematode feeding site development. We further investigated if nematode induced stress might trigger a checkpoint control during cell cycle progression in galls. Therefore, functional analysis of the WEE1 transcription factor was performed and showed that its absence induced galls prematurely to enter mitosis, thus affecting feeding site development and nematode reproduction. Performing drug treatments and the use of the stress marker SMR7 and a DNA repair promoter reporter line PARP1 suggested the induction of checkpoint activation in galls at G1/S phase of the cell cycle. The data presented in this thesis provided fundamental knowledge for a better understanding of cell cycle control during gall biogenesis as well sideways for plant development. Currently, our results are being managed for application initially in Arabidopsis, and promising strategies will be conducted and extended towards different crop species in order to better control nematode attack. Here, we specifically aimed at generating genetically modified plants by silencing E2Fa genes as an anti-nematode strategy in the field.Lors des vingt dernières années, l’utilisation d’Arabidopsis thaliana en tant que plante modèle a permis d’accélérer les découvertes biologiques dans l’étude de nombreuses interactions plante-pathogènes tant au niveau moléculaire que cellulaire. Arabidopsis réagis de manière similaire aux plantes cultivées face aux attaques de nombreux pathogènes, y compris les nématodes. Meloidogyne incognita est un nématode endoparasite de la racine des plantes causant de lourdes pertes agronomiques estimées à plusieurs milliards de dollars par an. Afin d’induire la formation de sites nourriciers dans la racine de l'hôte, les nématodes à galles. Ceci a pour effet d’induire la formation de cellules géantes à l’origine des nodosités observées sur la racine de l’hôte communément appelées galles. Lors de la formation des sites nourriciers, les cellules géantes ont pour caractéristiques d’être hypertrophiées et poly-nucléés du fait de l’absence ou de l’interruption de la cytokinèse et de nombreux cycles successifs d'endoreduplication. Ainsi, une caractéristique cruciale du développement du nématode est l’hyper activation du cycle cellulaire dans les galles. Le cycle cellulaire eucaryote est conservé et caractérisé par la phase S (réplication de l'ADN), suivie par la phase mitose (M), la cytokinèse et la division cellulaire. Celles-ci sont entrecoupées par la phase G1 (Gap 1) qui relie la fin de la mitose, et la phase G2 (Gap 2) qui relie la fin de la synthèse de l'ADN. L'endocycle est une variante du cycle cellulaire dans laquelle la réplication se produit sans mitose, induisant le doublement du contenu en ADN cellulaire pour chaque endocycle. L’hyper activation du cycle cellulaire est essentielle pour la biogenèse de la galle induite par les nématodes et un équilibre précis entre les phases mitotiques et endocycliques est essentiel pour la réussite du parasitisme. Dans le cadre de ma thèse de doctorat, nous avons étudié les conditions préalables nécessaires à l’établissement d’une galle fonctionnelle induite par M. incognita. Je me suis particulièrement concentrée sur l’étude fonctionnelle de gènes du cycle cellulaire de la plante jouant un rôle dans la mitose et l’endoreduplication lors de l’interaction plante-nématode. Pour répondre à cette question j’ai réalisé des études fonctionnelles de nouveaux acteurs du cycle cellulaire chez Arabidopsis stimulant (E2F), inhibant (ABAP1 et son-interactor) ou contrôlant (WEE1) le cycle cellulaire des galles. Les résultats obtenus démontrent que les gènes étudiés ici sont essentiels pour la formation et le maintien des sites nourriciers des nématodes. Notre étude fonctionnelle et notre analyse moléculaire montrent que l’absence ou la surexpression de facteurs de transcription E2Fa et E2Fb affectent gravement le développement de la galle et la reproduction des nématodes et perturbent le cycle cellulaire des galles, validant ainsi leur importance pour le développement du site d’alimentation des nématodes. Nous avons également cherché à déterminer si le stress induit par les nématodes déclenche un checkpoint control pendant la progression du cycle cellulaire dans les galles. Par conséquent, une analyse fonctionnelle du facteur de transcription WEE1 a été réalisée et a montré que son absence induit une mitose plus précoce dans les sites nourriciers, affectant ainsi le développement des galles et la reproduction des nématodes. L’utilisation de drogues, d’un marqueur de stress SMR7, ainsi que d’une lignée exprimant un gène rapporteur sous le contrôle du promoteur du gène PARP1 (impliqué dans la réparation de l’ADN) ont suggéré l’activation du checkpoint control G1/S dans les galles. Les données présentées dans cette thèse ont apporté des connaissances fondamentales pour une meilleure compréhension du contrôle du cycle cellulaire lors de la biogenèse de la galle, ainsi que lors du développement de la plante

Root-knot nematode-induced gall biogenesis depends on cell cycle hyperactivation : Functional studies of new players of the mitotic and endocycle in giant-feeding cells

Lors des vingt dernières années, l’utilisation d’Arabidopsis thaliana en tant que plante modèle a permis d’accélérer les découvertes biologiques dans l’étude de nombreuses interactions plante-pathogènes tant au niveau moléculaire que cellulaire. Arabidopsis réagis de manière similaire aux plantes cultivées face aux attaques de nombreux pathogènes, y compris les nématodes. Meloidogyne incognita est un nématode endoparasite de la racine des plantes causant de lourdes pertes agronomiques estimées à plusieurs milliards de dollars par an. Afin d’induire la formation de sites nourriciers dans la racine de l'hôte, les nématodes à galles. Ceci a pour effet d’induire la formation de cellules géantes à l’origine des nodosités observées sur la racine de l’hôte communément appelées galles. Lors de la formation des sites nourriciers, les cellules géantes ont pour caractéristiques d’être hypertrophiées et poly-nucléés du fait de l’absence ou de l’interruption de la cytokinèse et de nombreux cycles successifs d'endoreduplication. Ainsi, une caractéristique cruciale du développement du nématode est l’hyper activation du cycle cellulaire dans les galles. Le cycle cellulaire eucaryote est conservé et caractérisé par la phase S (réplication de l'ADN), suivie par la phase mitose (M), la cytokinèse et la division cellulaire. Celles-ci sont entrecoupées par la phase G1 (Gap 1) qui relie la fin de la mitose, et la phase G2 (Gap 2) qui relie la fin de la synthèse de l'ADN. L'endocycle est une variante du cycle cellulaire dans laquelle la réplication se produit sans mitose, induisant le doublement du contenu en ADN cellulaire pour chaque endocycle. L’hyper activation du cycle cellulaire est essentielle pour la biogenèse de la galle induite par les nématodes et un équilibre précis entre les phases mitotiques et endocycliques est essentiel pour la réussite du parasitisme. Dans le cadre de ma thèse de doctorat, nous avons étudié les conditions préalables nécessaires à l’établissement d’une galle fonctionnelle induite par M. incognita. Je me suis particulièrement concentrée sur l’étude fonctionnelle de gènes du cycle cellulaire de la plante jouant un rôle dans la mitose et l’endoreduplication lors de l’interaction plante-nématode. Pour répondre à cette question j’ai réalisé des études fonctionnelles de nouveaux acteurs du cycle cellulaire chez Arabidopsis stimulant (E2F), inhibant (ABAP1 et son-interactor) ou contrôlant (WEE1) le cycle cellulaire des galles. Les résultats obtenus démontrent que les gènes étudiés ici sont essentiels pour la formation et le maintien des sites nourriciers des nématodes. Notre étude fonctionnelle et notre analyse moléculaire montrent que l’absence ou la surexpression de facteurs de transcription E2Fa et E2Fb affectent gravement le développement de la galle et la reproduction des nématodes et perturbent le cycle cellulaire des galles, validant ainsi leur importance pour le développement du site d’alimentation des nématodes. Nous avons également cherché à déterminer si le stress induit par les nématodes déclenche un checkpoint control pendant la progression du cycle cellulaire dans les galles. Par conséquent, une analyse fonctionnelle du facteur de transcription WEE1 a été réalisée et a montré que son absence induit une mitose plus précoce dans les sites nourriciers, affectant ainsi le développement des galles et la reproduction des nématodes. L’utilisation de drogues, d’un marqueur de stress SMR7, ainsi que d’une lignée exprimant un gène rapporteur sous le contrôle du promoteur du gène PARP1 (impliqué dans la réparation de l’ADN) ont suggéré l’activation du checkpoint control G1/S dans les galles. Les données présentées dans cette thèse ont apporté des connaissances fondamentales pour une meilleure compréhension du contrôle du cycle cellulaire lors de la biogenèse de la galle, ainsi que lors du développement de la plante.During the last twenty years, Arabidopsis thaliana has been successfully used as a model plant accelerating discoveries at the molecular and cellular level in numerous plant-pathogen interactions. Arabidopsis is susceptible to a number of pathogens including nematodes, responding to pathogen attack in a similar manner to cultivated plant species. Meloidogyne incognita is a crop parasite causing extensive economic losses of billions of dollars yearly for the agriculture worldwide. Root-knot nematodes induce feeding sites within the host root inducing up to eight giant-feeding cells within the root vascular cylinder by injecting secretions. These giant cells become hypertrophied and contain multiple enlarged nuclei as a result of numerous mitotic events of which cytokinesis is absent or interrupted. Throughout expansion, giant cells become highly polyploid via successive endoreduplication cycles. Thus, a crucial feature for root-knot nematode development is the hyperactivation of the plant host cell cycle in galls. The eukaryotic cell cycle is conserved and characterized by four phases: The S phase (DNA replication) is followed by the M phase (mitosis), cytokinesis and cell division. These are intercalated by the G1 phase (first gap) which connects the end of mitosis to the start of DNA synthesis, and the G2 phase (second gap) which connects the end of DNA synthesis to the start of mitosis. The endocycle is a variant of the cell cycle in which replication occurs without mitosis, resulting in a doubling of cellular DNA content for each endocycle round. The hyperactivation of the cell cycle is essential for the nematode-induced gall biogenesis, and a precise balance between mitotic and endocycle phases is essential for the successful compatible interaction. As a broad question of my PhD, we addressed which the pre-requisites are needed to establish a functional gall induced by root-knot nematodes. More specifically, I essentially focused on the functional studies of particular plant cell cycle genes playing a role in the mitotic and the endocycle during plant-root-knot nematode interaction. To address this question, functional studies of new players in the cell cycle of the plant host, stimulating (E2F genes), inhibiting (ABAP1 and an interactor AIP10 genes) or controlling (WEE1 gene) the cell cycle, were investigated in galls induced in our model host Arabidopsis thaliana. The results obtained demonstrate that the genes here studied are critical for, as well the formation as the maintenance of nematode feeding sites. Overall, our functional study and molecular analysis show that the absence of E2Fa and E2Fb transcription factors will severely affect gall development and nematode reproduction. As well, overexpression of E2Fa and E2Fb will disrupt the cell cycle in galls, validating their importance for nematode feeding site development. We further investigated if nematode induced stress might trigger a checkpoint control during cell cycle progression in galls. Therefore, functional analysis of the WEE1 transcription factor was performed and showed that its absence induced galls prematurely to enter mitosis, thus affecting feeding site development and nematode reproduction. Performing drug treatments and the use of the stress marker SMR7 and a DNA repair promoter reporter line PARP1 suggested the induction of checkpoint activation in galls at G1/S phase of the cell cycle. The data presented in this thesis provided fundamental knowledge for a better understanding of cell cycle control during gall biogenesis as well sideways for plant development. Currently, our results are being managed for application initially in Arabidopsis, and promising strategies will be conducted and extended towards different crop species in order to better control nematode attack. Here, we specifically aimed at generating genetically modified plants by silencing E2Fa genes as an anti-nematode strategy in the field

RT-qPCR analysis of CBW nuclease expression at different developmental stages.

<p>(A and B) CBW was dissected to obtain the gut and carcass, and nuclease expression was then measured in these samples. The bar chart shows that <i>AgraNuc1</i> expression is similar in the gut and carcass of the adult (A) and larvae (B), whereas <i>AgraNuc2</i> and <i>AgraNuc3</i> are highly expressed in the gut only. (C and D) The insect gut was sectioned into the anterior midgut (AMG), posterior midgut (PMG) and posterior gut (PG), and the expression levels of the nucleases in these sections were evaluated. Higher expression of <i>AgraNuc2</i> and <i>AgraNuc3</i> was observed in the PMG of both adults (C) and larvae (D), whereas <i>AgraNuc1</i> expression was similar in all gut sections. <i>Agra-β-actin</i> and <i>Agra-β-tubulin</i> were used as reference genes. The relative expression (UA) was calculated based on the lowest expression value that was obtained. Statistical analyses of the average transcripts expression levels were performed using Tukey’s test with a 0.05% significance level for comparisons between treatments.</p

Nucleases as a barrier to gene silencing in the cotton boll weevil, <i>Anthonomus grandis</i>

<div><p>RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (<i>Anthonomus grandis</i>) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted <i>AgraNuc1</i>, <i>AgraNuc2</i>, and <i>AgraNuc3</i>, and the influences of these nucleases on the gene silencing of <i>A</i>. <i>grandis</i> chitin synthase II (<i>AgraChSII</i>) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the <i>AgraChSII</i> gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that <i>A</i>. <i>grandis</i> midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.</p></div

Analysis of CBW <i>ChSII</i> gene expression after nuclease gene silencing.

<p>Two days after microinjection of the nuclease dsRNA into the CBW body cavity, which silenced the <i>AgraNuc</i> genes, the insect was starved for two days, and 500 ng of <i>AgraChSII</i> dsRNA was orally administered. The insects with silenced nucleases (fourth bar) showed a decrease in <i>AgraChSII</i> transcript expression compared with the control insects (first, second and third bars). RNA extraction, cDNA synthesis and RT-qPCR were performed with the whole insect. dsRNA against <i>gus</i> was used as a negative control, and <i>Agra-β-actin</i> and <i>Agra-β-tubulin</i> were used as reference genes. The relative expression (UA) was calculated based on the lowest expression value that was obtained the average transcripts expression levels were performed using Tukey’s test with a 0.05% significance level for comparisons between treatments.</p

Analysis of CBW nucleases two days after gene silencing by RT-qPCR and dsRNA digestion assay.

<p>(A) Insect microinjection was performed with 500 ng of dsRNA against each nuclease and a mixture of all three dsRNAs (in a total of 1500 ng of dsRNA) and the analysis was performed two days after the microinjection. dsRNA against <i>gus</i> was used as a negative control, and <i>Agra-β-actin</i> and <i>Agra-β-tubulin</i> were used as reference genes. The relative expression (UA) was calculated based on the lowest expression value that was obtained. Statistical analyses of the average transcripts expression levels were performed using Tukey’s test with a 0.05% significance level for comparisons between treatments. The bar chart shows that the expression of the nucleases, including each individual nuclease and all three nucleases together, was silenced. (B) dsRNA (~ 200 bp) was incubated with CBW gut juice (GJ) for 30 minutes at 37°C. GJ was collected two days after RNAi nuclease gene silencing, and 1% agarose gel electrophoresis was performed to analyze dsRNA digestion. GJ was collected from uninjected insects and from injected insects with all three nucleases silenced at once. GJ: Gut Juice, KD: knocked down, WT: wild type, CBW: cotton boll weevil.</p

Primer sequences.

<p>Primer sequences.</p

Biochemical characterization of CBW gut juice.

<p>(A) CBW gut juice (GJ), which is able to degrade both dsRNA, ~ 200bp, and dsDNA, > 5000 bp (as observed), has non-specific nuclease activity. MM: Molecular Marker 1-Kb Plus DNA ladder (Invitrogen); GJ: Gut Juice. Samples were incubated with GJ for 30 minutes at 37°C. (B) The optimal pH for nuclease activity ranges from 5.5 to 6.5, indicating that the nucleases function best at acidic pH.</p