# Gene silencing targeting uk1_LRR or clathrin in the experimental vector Euscelidius variegatus modifies insect colonization by "flavescence dorée" phytoplasma

Nathalie Arricau-Bouvery, Marie-Pierre Dubrana, Sybille Duret, Xavier Foissac, Sylvie Malembic-Maher

► To cite this version:

Nathalie Arricau-Bouvery, Marie-Pierre Dubrana, Sybille Duret, Xavier Foissac, Sylvie Malembic-Maher. Gene silencing targeting uk1_LRR or clathrin in the experimental vector Euscelidius variegatus modifies insect colonization by "flavescence dorée" phytoplasma. Microbiological Research, 2026, 305, pp.128416. 10.1016/j.micres.2025.128416. hal-05448108

HAL Id: hal-05448108

https://hal.inrae.fr/hal-05448108v1

Submitted on 8 Jan 2026

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# Gene silencing targeting uk1_LRR or clathrin in the experimental vector *Euscelidius variegatus* modifies insect colonization by "flavescence dorée" phytoplasma

Nathalie Arricau-Bouvery* , Marie-Pierre Dubrana, Sybille Duret, Xavier Foissac, Sylvie Malembic-Maher

Univ. Bordeaux, INRAE, BFP, UMR 1332, Villenave d'Ornon F-33140, France

## ARTICLE INFO

**Keywords:**

Plant pathogenic bacteria

Leafhopper

Transmission mechanism

RNAi

Cellular invasion

Adhesin

## ABSTRACT

The "flavescence dorée" (FD) phytoplasma is transmitted from grapevine to grapevine by the leafhopper *Sca-phoides titanus*. In experimental conditions, this phytoplasma is also transmitted by the leafhopper *Euscelidius variegatus* to broad bean in which it multiplies and induces symptoms. To be transmitted to plants, phytoplasmas must invade different cell types of their insect vectors. The process of cellular endocytosis involves both bacterial and eucaryotic factors such as adhesins and receptors. In the present study, it is shown that entry of fluorescent beads coated with the adhesin VmpA of the FD phytoplasma into cultured *E. variegatus* cells depends on the putative receptor Uk1_LRR and clathrin of the insect. *In vivo* experiments have shown that silencing of uk1_LRR gene increased the colonization of *E. variegatus* by the FD phytoplasmas without effect on the plant transmission. On the contrary, silencing of clathrin gene significantly reduced the colonization of *E. variegatus* and the transmission to broad bean.

## 1. Introduction

Phytoplasmas are cell wall-lacking bacteria belonging to the class *Mollicutes*. Despite a reduced genome and an intracellular life style, the members of the genus 'Candidatus Phytoplasma' are characterized by their ability to colonize hosts belonging to two different kingdoms, plants and insects (Hogenhout et al., 2008). Thus, phytoplasmas can infect several families of hemipteran insects (Trivellone et al., 2019) and are associated with severe symptoms in a wide range of cultivated and ornamental plants (Bertaccini, 2022; Namba, 2019). Once introduced into a plant, by insect transmission or grafting, they are restricted to the sieve elements of the phloem even though their effectors can migrate into various tissues of the host plant (Tomkins et al., 2018). Contrary to plant, phytoplasmas perform a persistent circulative propagative cycle in insect that implies the invasion of various cell types (Koinuma et al., 2020). For transmission to the plant, phytoplasmas must pass through the cells of the midgut and salivary glands to join saliva, and multiply efficiently in the insect body (Bosco and D'Amelio, 2009; Koinuma et al., 2020). Thus, phytoplasma invasion of intestinal cells is an initial and

critical step in insect colonization, and entry into salivary gland cells is critical for inoculation into the plant. Phytoplasma surface proteins that interact with various cytosolic components of plant cells and/or with the surface of insect cells therefore play a very important role in the life cycle of these bacteria.

"Flavescence dorée" (FD) is a severe grapevine disease that affects European vineyards and is associated with the presence of phytoplasmas belonging to the 16SrV-C and 16Sr-D subgroups of 'Ca. Phytoplasma' genus. Reduction of yield, mandatory surveillance of the vineyards, removal of infected grapevines and repeated treatments with insecticides induce high economic and environmental costs (Jeger et al., 2016; Oliveira et al., 2019). In vineyards, the leafhopper *Scaphoideus titanus* efficiently transmits FD phytoplasmas from infected grapevines to grapevines and thus causes epidemics (Chuche and Thiéry, 2014; Gonella et al., 2024a, 2024b). The leafhopper *Euscelidius variegatus* belongs to the same Deltocephalinae sub-family of the Ciacadellidae family as *S. titanus*. Experiments that led to transmission of FD phytoplasmas to broad beans by *S. titanus* (Caudwell et al., 1970) allowed to have an experimental plan with *E. variegatus* as experimental vector of

<footnote>* Corresponding author.</footnote>

<footnote>*E-mail addresses:* nathalie.bouvery@inrae.fr (N. Arricau-Bouvery), marie-pierre.dubrana-ourabah@inra.fr (M.-P. Dubrana), sybille.duret@inrae.fr (S. Duret), xavier.foissac@inrae.fr (X. Foissac), sylvie.malembic-maher@inrae.fr (S. Malembic-Maher).</footnote>
FD phytoplasmas to broad bean in confined greenhouse (Caudwell et al., 1972). Due to the important role of surface exposed proteins of phytoplasmas during the colonization of the insect (Ding et al., 2022; Rashidi et al., 2015; Suzuki et al., 2006), researches focused on two FD phytoplasma surface proteins, Imp and VmpA.

The Imp and VmpA proteins are subject to strong selective pressure (Konnerth et al., 2016; Malembic-Maher et al., 2020), suggesting an adaptation process to insect hosts. Imp interacts more efficiently in vitro with proteins extracted from the gut and salivary gland of FD phytoplasma insect vectors than with proteins from insects' non-vectors (Trivellone et al., 2019). In the midgut of *S. titanus* and *E. variegatus*, Imp interacts with four and five proteins, respectively (Galetto et al., 2025). RNAi silencing of the insect natterin and legumain genes induced a significant reduction in phytoplasma multiplication in insects (Galetto et al., 2025). VmpA is a variable membrane protein predicted to be addressed to the phytoplasma surface via the Sec-dependent pathway and ultimately anchored to the membrane via a C-terminal trans-membrane segment (Renaudin et al., 2015). VmpA acts as a FD phytoplasma adhesin, binding to cells in culture and to the perimicrovillar membrane of the insect vector *E. variegatus* (Arricau-Bouvery et al., 2018). The VmpA protein interacts with glycoconjugates, notably mannose and N-acetylglucosamine, present on the cell surface of the insect vector (Arricau-Bouvery et al., 2021). A protein containing leucine-rich repeats (LRRs) was found to be a likely receptor for the insect vector *E. variegatus* (Canuto et al., 2023). This protein is mainly composed of four LRR domains and its gene expression was higher in the midgut than in the salivary glands and ovaries. Inhibition of uk1_LRR expression in *E. variegatus* cultured cells induced a decrease of adhesion of VmpA-coated beads. These results suggest that Uk1_LRR could be a putative receptor of VmpA implicated in the invasion of gut cells by FD phytoplasmas leading to the colonization of the insect vector.

After adhesion to insect cells, FD phytoplasmas use clathrin-mediated endocytosis to enter insect cells (Arricau-Bouvery et al., 2023). Clathrin-mediated endocytosis is a complex process that involves different proteins to form clathrin-coated pits and vesicles (Johnson, 2024). Among the adaptor proteins (AP) that bind clathrin during the formation of clathrin-coated pits, the AP2b was found in protein complexes interacting in vitro with VmpA (Arricau-Bouvery et al., 2023). Clathrin-mediated endocytosis is frequently studied using drugs, like chlorpromazine, whereas nystatin is used to study caveolae-mediated endocytosis (Glebov, 2020). Demonstration of the entry of FD phytoplasmas using clathrin-mediated endocytosis was obtained on cultivated insect cells (Arricau-Bouvery et al., 2023) and the role of clathrin-mediated endocytosis for insect colonization and transmission of phytoplasmas to the plant in vivo was examined here. Gene silencing targeting clathrin was shown to be efficient after ingestion of double strand RNA by *E. variegatus* (Arricau-Bouvery et al., 2023). The present work aimed to measure the impact of the expression silencing of uk1_LRR and clathrin genes using RNAi on the entry of FD phytoplasmas via VmpA into insect cells, and evaluate the effect on the phytoplasma transmission to the plant.

# 2. Materials and methods

## 2.1. Insects, phytoplasmas and cell lines

Healthy *E. variegatus* leafhoppers originally collected on grasses in Villenave d'Ornon, France, in 2003, were reared in cages on broad beans *Vicia faba* var. Aquadulce and oats *Avena sativa* from seeds purchased from Castros Gerand (Carbon Blanc, France) and Jardiland (Villenave d'Ornon, France), respectively, at 25°C in greenhouse under a photoperiod of 16 h light / 8 h dark. The FD phytoplasma strain FD92 was originally transmitted to broad bean by infected *S. titanus* sampled from FD-infected vineyards in south-west France in 1992 (Angelini et al., 2001) as previously described (Caudwell et al., 1970). FD phytoplasmas were then continuously maintained in broad beans by *E. variegatus*

transmission, as described by Caudwell et al. (1972).

The *E. variegatus* Euva cell line was established from *E. variegatus* embryos, as described previously for Ciha-1 cells (Duret et al., 2010). Briefly, the eggs were sterilized with a bleach solution followed by 70 % ethanol. After rinsing, the eggs were ground in a modified culture medium consisting of 350 mL Schneider's Drosophila medium (Invitrogen, Carlsbad, United States), 100 mL of Grace's insect cell culture medium (Invitrogen), 50 mL of heat-inactivated foetal bovine serum (Eurobio, Les Ulis France) and 2 mL of G-5 supplement (Invitrogen). Euva cells were grown at 25°C. After development of the first colonies and establishment of the cell line, the cells were passed by trypsinization every week with an additional change of medium during the week using the modified culture medium without the G-5 supplement. *Drosophila melanogaster* S2 cells (Invitrogen) were grown at 25°C in Euva culture medium and passed twice weekly.

## 2.2. Internalization assays with VmpA-His<sub>6</sub>-coated beads

VmpA-His<sub>6</sub> proteins were obtained as previously described (Arricau-Bouvery et al., 2018). Briefly, proteins were expressed in *Escherichia coli* BL21 Star (DE3) cells containing the pET28-VmpA-His<sub>6</sub> plasmid and purified on a His-select nickel affinity gel packed column (Sigma, Saint-Louis, United States) according to the manufacturer's protocol. Yellow-green, carboxylate-modified fluorescent microspheres (4 × 10<sup>9</sup> beads, 1 µm diameter, Invitrogen) were covalently coated with 9 nmol recombinant VmpA-His<sub>6</sub> proteins plus 1 nmol BSA according to the supplier's instructions. Coated beads were kept at 4°C for one or two days before use. Coating of fluorescent beads was verified using immunofluorescence observations and by measuring remaining uncoated proteins as previously described (Arricau-Bouvery et al., 2018).

For internalization inhibition assays, Euva cells from culture passages 156–200 and S2 cells from passages 18–22, depending on the assay, were cultivated on coverslips in 24-well plates (Falcon, NY, United States), as described previously (Duret et al., 2014). The endocytosis inhibitors chlorpromazine (CPZ, Sigma), cytochalasin D (CytoD, Sigma) and nystatin (Nyst, Sigma) were added to S2 cells and Euva cells for 1 h at concentrations of 10 µM for CytoD, 60 µM for Nyst, and 15 µM, 30 µM and 60 µM for CPZ in a final volume of 400 µL. Treated cells were then incubated with 100 µL of VmpA-His<sub>6</sub>-coated beads in the presence of the inhibitors for 18 h. For each experiment, 20 fields were randomly observed per condition, and three independent assays were performed and pooled. Extracellular beads were differentiated from intracellular beads as described in 2.3. The relative number of intracellular beads per cell in the different conditions was calculated by dividing the number of beads measured in each condition by the mean of bead numbers in the control condition (without endocytosis inhibitor indicated by “0” in Fig. 2).

Other experiments were conducted with cells transfected with dsRNA as described in 2.4. and then incubated with VmpA-His<sub>6</sub>-coated beads three days later, as described above. For each experiment, 20 fields were randomly observed per condition (see below), and three independent assays were performed.

## 2.3. Immunofluorescent staining and microscopic observations

After incubation with VmpA-His<sub>6</sub>-coated beads, S2 and Euva cells were washed in PBS, fixed with 4 % paraformaldehyde (Electron Microscopy Science, Hatfield, United Kingdom) for 15 min, then washed 3 times in PBS. The cells were then incubated for 30 min with rabbit polyclonal anti-VmpA antibodies (diluted 1:7000, Covalab, Bron, France). After three washes, insect cells were incubated for 30 min with goat anti-rabbit IgG conjugated to Alexa 568 (Thermo Fisher Scientific, Waltham, United States) at a dilution of 1:200. This staining allowed to identify VmpA-His<sub>6</sub>-coated outer beads. After washing, nuclei were stained for 5 min in water with DAPI (1 mg.mL<sup>-1</sup>, Sigma). Cells were mounted in Ibidi mounting medium (Ibidi, Gräfelfing, Germany) and
imaged using a Zeiss AxioImager epifluorescent microscope with a 1.4 numerical aperture objective. The filters for excitations and emissions, which are sequentially individually used, were BP 377/50 and BP 447/60 for DAPI, BP 420/35 and BP 472/30 for yellow-green microspheres, and BP 562/40 and LP 593 for Alexa 568, respectively. For Z-stack acquisition, images were acquired every 0.4 µm from the bottom to the top to cells and Z-projections were performed before counting of intracellular and extracellular VmpA-His<sub>6</sub>-coated beads with the free software package Fiji of ImageJ-win64 (http://imagej.nih.gov/ij/) (Schindelin et al., 2012).

For salivary gland labelling, dsRNA-treated *E. variegatus* infected with FD phytoplasmas were dissected 28 days after the insects had ingested dsRNA and been anaesthetized. Salivary glands were fixed in 4 % paraformaldehyde containing 1 % Triton X-100 overnight at 4°C, then washed 3 times in PBS. Next, salivary glands were incubated for 1 h with rabbit polyclonal anti-VmpA antibodies (diluted 1:7000). After three washes in PBS, salivary glands were incubated for 1.5 h with Alexa 568-phalloidin (Invitrogen, diluted 1:40) and Alexa 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) at a dilution of 1:200. Then, nucleic acids were stained after a 5 min wash in water with DAPI (Sigma). The salivary glands were mounted in Ibiid mounting medium and observed using a Zeiss LSM 880 confocal laser-scanning microscope with an objective with a numerical aperture of 1.4. The resolution was improved with an Airyscan detector. Excitation wavelengths, which are sequentially individually switched on, are 405 nm, 488 nm and 561 nm for DAPI, Alexa 488 and Alexa 568, respectively. The same emission filter (BP 420–460 nm + LP 500 nm) was used for the 4 channels. For each experiment, 10 organs were observed per condition.

<caption>**Table 1**</caption>

Sequence of primers used in this study. The primers GST1_F257, GST1_R369, 3Fbl and 3R1 were designed by the authors listed below the table, and the other primers were designed for this work.

<table><thead><tr><th>Aplication</th><th>Primer name</th><th>5'-3' sequence</th><th>Target gene</th><th>Species target / plasmid</th><th>Product size (n)</th><th>PCR efficiency (%)</th></tr></thead><tbody><tr><td rowspan="5">dsRNA synthesis</td><td>Ev-ClatHCH1-T7-F2</td><td>TAATACGACTCACTATAGGGGTGTTGTTGAGGGACTGGACA</td><td>Clathrin heavy chain</td><td>Euscelidius variegatus</td><td>429</td><td>-</td></tr><tr><td>Ev-ClatHCH1-T7-R2<br>AP2b-T7-F2</td><td>TAATACGACTCACTATAGGGGTGCGAGAACATCTGGAACTG<br>TAATACGACTCACTATAGGGGTCCCTTCCAAAAAGCTCTCC</td><td>AP2b adaptor complex</td><td>Euscelidius variegatus</td><td>416</td><td>-</td></tr><tr><td>AP2b-T7-R2<br>db_LRR_15247_T7p_F</td><td>TAATACGACTCACTATAGGGAAGCCAACATAGCCCAAGTG<br>TAATACGACTCACTATAGGGGGACGACTGCGACTGCCACTAC</td><td>uK1_LRR</td><td>Euscelidius variegatus</td><td>483</td><td>-</td></tr><tr><td>db_LRR_15247_T7p_R<br>eGFP-ds-T7F1<br>eGFP-ds-T7R1<br>GST1_F257c<sup>a</sup></td><td>TAATACGACTCACTATAGGGGCGGGTTGCCTGACAGATCTAG<br>TAATACGACTCACTATAGGGGCAAGGAGGACGGCAACATCC<br>TAATACGACTCACTATAGGGGCAACTCCAGCAGGACCATG<br>CCAAGGACCCCAAGAAGCGA</td><td>GFP</td><td>pA-GFP-W</td><td>323</td><td>-</td></tr><tr><td>GST1_R369c<sup>a</sup><br>EvTubB_F1</td><td>TGGCGCTCCTCCAAACATCA<br>CGCCAGAGGTCTCAAGATG</td><td>Glutathione S-transferase</td><td>Euscelidius variegatus</td><td>113</td><td>96</td></tr><tr><td>EvTubB_R2<br>EvClatH1_F3</td><td>ATCTCGTCCATGCCCTCGC<br>AGGCCACACATTTGAGCAAG</td><td>Tubulin β</td><td>Euscelidius variegatus</td><td>148</td><td>94</td></tr><tr><td>EvClatH1_R1<br>AP2B_F5</td><td>GAAAGAGTTCCAGGGTGCAG<br>CAGAGGTTCCATCCTTTGAAC</td><td>Clathrin heavy chain</td><td>Euscelidius variegatus</td><td>120</td><td>97</td></tr><tr><td>AP2B_R1<br>LRR_15247_qF</td><td>TGGTTTGACCCCTGCTAGAC<br>CTTAGCAGCCCAAGAGAACG</td><td>AP2b adaptor complex</td><td>Euscelidius variegatus</td><td>115</td><td>100</td></tr><tr><td>LRR_15247_cR<br>3Fbl<sup>b</sup></td><td>GCTGTCATTTACGATGTTTCACC<br>TGAAGATCCAGTACGTGATTTAGAC</td><td>uK1_LRR</td><td>Euscelidius variegatus</td><td>265</td><td>94</td></tr><tr><td>3R1<sup>b</sup></td><td>TTTTAGTTTCTTTAATACCTATGATTTTC</td><td>Elongation factor Tu (EFTu)</td><td>FD92 phytoplasma</td><td>158</td><td>96</td></tr></tbody></table>

T7 promoter sequence is underlined

## 2.4. RNAi on *Euva* cells and *E. variegatus* insects

Sequences of uK1_LRR, clathrin heavy chain (chc) and AP2b adaptor complex (Ap2b) were amplified by PCR from *E. variegatus* cDNA using sequence-specific primers listed in Table 1, and cloned into the pGEM-T Easy Vector (Promega, Charbonnières-les-Bains, France). Sequence of GFP (control) was amplified from the plasmid pA-GFP-W (Carnegie, Washington, United States). The sequence of the cloned fragments was verified by sequencing (Azenta, Genewiz, Takeley, United Kingdom). PCR was then performed using primers conjugated to 21 bp of the T7 RNA polymerase promotor (Table 1). PCR products were used as templates for dsRNA synthesis using the HiScribe T7 High Yield RNA synthesis Kit (New England Biolabs, Evry, France). Precipitated dsRNAs were resuspended in 20 µL of RNAse-free water. The purity and integrity of dsRNAs were determined using the NanoVue Plus spectrophotometer (GE Healthcare, London, United Kingdom) and migration in agarose gel electrophoresis.

Euva cells were cultivated in 24-well plates (Falcon) and were transfected with 2 µg dsRNA using FuGENE HD Transfection Reagent (Promega) according to the manufacturer's instructions. After three days, total RNA was extracted (see below).

For ingestion assays of dsRNAs, phytoplasma-free young and synchronized *E. variegatus* adults were fed through a parafilm membrane for 2 days. To minimize the volume contained in the cap of the 1.5 mL microtubes, a 2 mm glass bead was placed and 18 µL of a solution composed of 8 mM HEPES, 280 mM sucrose, pH 7.4 plus 2 µg/µL dsRNA between two parafilm membranes was used. For injection assays, between 0.04 ng and 400 ng of chc dsRNA or approximately 200 ng of

<footnote><sup>a</sup> Primers were described in Galetto et al. (Galetto, L., Abbà, S., Rossi, M., Vallino, M., Pesando, M., Arricau-Bouvery, N., Dubrana, M-P., Chitarra, W., Pegoraro, M., Bosco, D., Marzachi, C., Bäumler, A. J., 2018. Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum. Infect. Immun. 86, e00042–18. https://journals.asm.org/doi/10.1128/IAI.00042-18)</footnote>

<footnote><sup>b</sup> Primers were described in Eveillard et al. (Eveillard, S., Jollard, C., Labroussaa, F., Khalil, D., Perrin, M., Desqué, D., Salar, P., Razan, F., Hévin, C., Bordenave, L., Foissac, X., Masson, J.E., Malembic-Maher, S., 2016. Contrasting Susceptibilities to Flavescence Dorée in Vitis vinifera, Rootstocks and Wild Vitis Species. Front. Plant Sci. 7, 1762. http://journal.frontiersin.org/article/10.3389/fpls.2016.01762/full)</footnote>
uk1_LRR dsRNAs was injected intra-abdominally and *E. variegatus* was engaged on broad bean for one day in the greenhouse.

## 2.5. Transmission assay

After the insects *E. variegatus* ingested dsRNA for two days in microtubes, or have been injected with dsRNA and caged on phytoplasma-free broad beans for one day, they were caged on FD phytoplasma-infected broad beans for 5 days (100 insects per plant) (Fig. 1). Broad beans were infected five weeks before with 8 *E. variegatus* containing from 8.9 × 10⁶ (± 5.4 × 10⁶) to 2.1 × 10⁷ (± 1.4 × 10⁷) FD phytoplasmas per insect. Then, insects were removed from infected plants and caged with uninfected broad beans for 18 days for the latent period. Insects were randomly transferred by groups of 4 onto two-leaf broad beans during a 5-day inoculation period, then removed and analyzed for phytoplasma presence verification (see below). Broad beans were incubated for 15 days, then 1 g petioles and 2 g stems were sampled.

Two independent experiments were conducted when dsRNA targeted uk1_LRR mRNA, and three independent experiments were conducted when dsRNA targeted chc mRNA, with GFP dsRNA as control in each experiment. All results were pooled for analysis.

## 2.6. Nucleic acid extraction and phytoplasma quantification

At the end of the 5-day inoculation, the bodies and heads of *E. variegatus* were separated after the insects had been anaesthetized with CO₂. Total nucleic acids were extracted from a single body and head separately with the TNES protocol (Sambrook and Russell, 2001). Briefly, each sample was incubated at 55°C for an overnight digestion in 190 µL TNES [Tris-Hcl 50 mM, NaCl 400 mM, EDTA 20 mM and SDS 0, 5 % w/v] plus 5 µL proteinase K at 10 mg.mL⁻¹. After adding 56 µL of 5 M NaCl, insect remains, cellular debris and proteins were roughly separated from nucleic acids by centrifugation for 20 min at 20.000 g and 4°C. The supernatant (150 µL) was transferred to a new tube and nucleic acids precipitated by adding 400 µL of cold absolute ethanol, followed by incubation for 1 h at -20°C. Nucleic acids were centrifuged at 20.000 g and 4°C for 30 min, rinsed with 70% ethanol and resuspended in 80 µL of nuclease free water for bodies and 30 µL of nuclease free water for head. For plants, total nucleic acids were extracted from the 1 g petioles plus 2 g stems using 3 mL cetyltrimethylammonium bromide (CTAB) buffer as described (Maixner, 2005). For the extraction procedure, 1 mL of the ground mixture was used and nucleic acids were resuspended in 200 µL of 1 × TE [10 mM Tris, 1 mM ethyl-enediaminetetraacetic acid (EDTA) pH 8]. Concentrations were determined with a NanoVue™ Plus and reduced to 100 ng.mL⁻¹ for qPCR analysis. Purity was assessed by calculating the ratio of the absorbance at 260 nm to absorbance at 280 nm and 230 nm. Nucleic acids' extracts were stored at -20°C before used.

FD phytoplasmas were quantified in plants and insect parts by absolute quantitative PCR of the *tuf* gene (Eveillard et al., 2016) performed on the LightCycler 480 Real-Time PCR system (Roche Diagnostics Corp, Indianapolis, United States) using the sequence-specific primers 3Fbl and 3R1 detailed in Table 1 and N',N'-dimethyl-N-[4-[(E)-(3-methyl-1, 3-benzothiazol-2-ylidene)methyl]-1--phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine (SYBR) Green (Roche, Bâle, Switzerland) according to the manufacturer's instructions. Two technical replicates were included. The cycling program for all amplifications was as followed: 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 sec and 72°C for 30 s.

The relative number of phytoplasmas per insect part and plant fresh weight in the different conditions (uk1_LRR dsRNA and chc dsRNA) was calculated by dividing the number of phytoplasmas measured in each condition by the mean of phytoplasma numbers in the control condition (GFP dsRNA).

<figure><img src="image_2.png" /><figcaption>Fig. 1. Experimental procedure used to assess the silencing effect on the ability of dsRNA treated *E. variegatus* to transmit phytoplasmas to broad beans.</figcaption></figure>

## 2.7. mRNA analysis

Total RNA was extracted from Euva cells transfected with dsRNA using TRIzol reagent (Life Technologies, Carlsbad, United States) according to the manufacturer's instructions. The insects used for mRNA
analysis were different from those used for total DNA extraction and phytoplasma quantification. Heads and bodies were separated from *E. variegatus* and grinded using micropestle with TRIzol reagent for total RNA extractions. DNA was removed by treating RNA extractions with RNase-free DNase (QIAGEN, Hilden, Germany). First-strand cDNA were synthetized using SuperScript III Reverse Transcripase (Thermo Fisher

Scientific) and 2.5 µM Random hexamer primers (Invitrogen). Subsequently, quantitative PCR was performed on the LightCycler 480 Real-Time PCR system using sequence-specific primers listed in Table 1 and SYBR Green according to the manufacturer's instructions. Two technical replicates were included for each cDNA-primer combination. The cycling program for all amplifications was as follows: 95°C for 15 min,

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Effect of RNAi and drugs on the internalization of VmpA-His<sub>6</sub>-coated beads into S2 cells and Euva cells. (A) Observation by fluorescent microscopy of Euva cells incubated with VmpA-His<sub>6</sub>-coated fluorescent beads. One Z-projection of one field is shown. Cell nuclei were stained with DAPI (grey) and VmpA of extracellular VmpA-His<sub>6</sub>-coated beads with anti-VmpA antibodies and Alexa 568-secondary antibodies (red). The beads used were yellow-green fluorescent microsphere of 1 µm diameter (green). (B) RNAi efficiency into Euva cells transfected at the same time as cells incubated with beads in (C). The expression of the chc (clathrin heavy chain), AP2b (adaptor protein 2b) and uk1_LRR genes were measured in Euva cells with regard to the reference genes glutathione S-transferase and tubulin beta (C) Internalization of VmpA-His<sub>6</sub>-coated beads into Euva cells 3 days after the cells were transfected with GFP dsRNA (white), chc dsRNA, AP2b dsRNA or uk1_LRR dsRNA (gray). Expression of the genes chc (D) and uk1_LRR (E) in presence of GFP dsRNA, chc dsRNA, AP2b dsRNA or uk1_LRR in Euva cells. Internalization of VmpA-His<sub>6</sub>-coated beads into S2 cells (F) and into Euva cells (G) in presence of nystatin (Nyst), chlorpromazine (CPZ) and cytochalasin D (CytoD). The concentrations used for each inhibitor were indicated above the inhibitors. * indicates a significant difference with p < 0.05, ** with p < 0.01 and *** with p < 0.001 compared to the GFP condition under the Kruskal-Wallis rank sum test of the R commander package of R software version 4.0.3.</figcaption></figure>
followed by 40 cycles at 95°C for 30 s, 60°C for 30 sec and 72°C for 30 s. Glutathione S-transferase (GST) and tubulin beta (Tub beta) were used as reference genes. The pGEM-T Easy Vector containing the open reading frames of chc, AP2b, uk1_LRR, GST and Tub beta was used to verify that qPCR efficiency was similar for all genes (Table 1). The primers used for qPCR were chosen outside the sequence used to synthesize dsRNA and to amplify DNA fragments of 100–270 bp, with primers rich in G/C nucleotides at their 3' end. The relative fold change in gene expression was calculated using the 2^(-ΔΔCt) method (Livak and Schmittgen, 2001) and by integrating the two reference genes (Vandesompele et al., 2002).

## 2.8. Statistical analysis

The Kruskal-Wallis rank sum test of the R commander package of R software version 4.0.3 was used to compare the values of the experimental conditions with those of the control condition in each experiment.

# 3. Results

## 3.1. *Uk1_LRR and clathrin are required for internalization of VmpA_His<sub>6</sub>-coated beads in Euva cells*

Uk1_LRR is a predictive surface protein of *E. variegatus* that is predicted to interact with the adhesin VmpA (Canuto et al., 2023). It was also demonstrated that FD phytoplasma enters *Drosophila* S2 cells using clathrin (Arricau-Bouvery et al., 2023). These results suggest that the VmpA adhesin might be involved in phytoplasma internalization via Uk1_LRR and clathrin-mediated endocytosis. S2 and Euva cells were incubated with VmpA-His<sub>6</sub>-coated fluorescent beads and assessed the percentage of intracellular beads. Twenty fields were imaged per condition (Fig. 2 A and S1), and three independent experiments were performed and pooled. When S2 cells were incubated with VmpA-His<sub>6</sub>-coated beads, averages of 49 (± 9) S2 cells and 194 (± 44) beads were observed per field. For Euva cells, averages of 28 (± 6) cells and 179 (± 39) beads were observed per field. Both S2 and Euva cells internalized VmpA-His<sub>6</sub>-coated beads with high efficiency, with 80% (±8) and 82% (±7) of beads localized in the cytoplasm, respectively. Thus, VmpA appears to enable fluorescent beads to enter Euva cells.

To assess whether Uk1_LRR and clathrin are implicated in endocytosis of VmpA, the internalization of VmpA-His<sub>6</sub>-coated beads was measured in Euva cells in which expressions of uk1_LRR gene, clathrin heavy chain (chc) gene and AP2b adaptor complex (AP2b) gene, which codes a protein that binds clathrin during endocytosis process, were inhibited by RNAi. As expected, strong inhibition of uk1_LRR, chc and AP2b mRNAs was observed in Euva cells after transfection with uk1_LRR dsRNA, chc dsRNAs and AP2b dsRNA (Fig. 2B). Incubation of the Euva cells with VmpA-His<sub>6</sub>-coated beads was performed 3 days after transfection of Euva cells with dsRNAs. On average, 45 (± 11) Euva-11 cells and 169 (± 58) beads were observed per field, 20 fields were photographed per condition, and three independent experiments were performed and pooled. A significant decrease in internalization of VmpA-His<sub>6</sub>-coated beads was observed after transfection with uk1_LRR dsRNA, chc dsRNA and AP2b dsRNA compared to GFP dsRNA (Fig. 2C). These results suggest a role of Uk1_LRR and clathrin-mediated endocytosis in the entry of FD phytoplasma via VmpA into insect cells.

The off-target effect of gene inhibition by non-homologous dsRNA on chc and uk1_LRR gene expression was monitored in Euva cells. The results showed that when cells were transfected with AP2b or uk1_LRR dsRNAs, no difference was observed for chc gene expression compared with the GFP control (Fig. 2D). On the contrary, when Euva cells were transfected with chc or AP2b dsRNAs, a slight but significant decrease was observed for uk1_LRR gene expression (Fig. 2E). Comparison of the uk1_LRR mRNA sequence with the sequence of the chc and AP2b dsRNA sequences only showed very limited homologies with a maximum of 8

consecutive bases of chc dsRNA and only a maximum of 10 consecutive bases of AP2b dsRNA were found to be homologous to the uk1_LRR mRNA sequence. For chc mRNA, only a maximum of 8 consecutive bases of uk1_LRR dsRNA and only a maximum of 9 consecutive bases of AP2b dsRNA were found to be homologous to the chc mRNA sequence. In regards to these results, it seems that each RNAi inhibitory effect is specific of the targeted gene.

To confirm the role of clathrin in the internalization of VmpA-His<sub>6</sub>-coated beads in Euva cells different inhibitory drugs were tested. The inhibitor chlorpromazine (CPZ), which inhibits clathrin-mediated endocytosis, was compared to cytochalasin D (Cyto D), which inhibits actin filament polymerization and thus inhibits various endocytosis pathways, and nystatin (Nyst), which is a cholesterol sequestering agent and an inhibitor of caveolin-dependent endocytosis. Nyst had no effect on internalization of VmpA-His<sub>6</sub>-coated beads in S2 and Euva cells (Fig. 2F and G). In contrast, Cyto D had a significant effect and significantly inhibited bead internalization. Only 17% of the VmpA-His<sub>6</sub>-coated beads were internalized into S2 cells, and 31% in the Euva cells in the presence of Cyto D. The addition of CPZ inhibited bead internalization in a dose-dependent manner, and this decrease was significant as early as the 15 µM concentration in both cell types. The effect was stronger in Euva cells than in S2 cells, with only 42% VmpA-His<sub>6</sub>-coated beads internalized in Euva cells at 60 µM CPZ and 66% in S2 cells. Conversely, the effect of CytoD was stronger in S2 cells. These results are consistent with the fact that S2 cells are phagocytic cells unlike Euva cells that are epithelial-like cells.

## 3.2. Inhibition of uk1_LRR gene expression increases the colonization of *E. variegatus* body and salivary glands by FD phytoplasmas

As uk1_LRR is more highly expressed in the midgut and in the early stages of insect colonization by FD phytoplasmas, with a two-fold increase but not statistically significant (Canuto et al., 2023), first RNAi assays were carried out with *E. variegatus* that ingested uk1_LRR dsRNA followed by acquisition of FD phytoplasmas to target the midgut barrier. Next, dsRNA injection was used to confirm the results with ingested dsRNA. No difference in mortality was observed between insects that ingested or were injected with uk1_LRR dsRNA and GFP dsRNA (69 ± 0.6 and 66 ± 0.5 for ingestion of uk1_LRR dsRNA and GFP dsRNA, respectively, 56 ± 6.4 and 58 ± 7.3 for injection of uk1_LRR dsRNA and GFP dsRNA, respectively). Regardless of how the dsRNAs were administered, a significant decrease in uk1_LRR mRNA levels was observed in *E. variegatus* treated with uk1_LRR dsRNA compared with GFP dsRNA (Fig. 3A, B, E and F). It should be noted that the efficiency of RNAi varied from one insect to another. In some, RNAi was not effective, while in others it was very significant.

The decrease in uk1_LRR expression induced by ingestion or injection of dsRNA caused a significant increase in FD phytoplasmas in both insect body (1.6- and 3.5-fold, respectively) and head (2.2- and 2.7-fold, respectively) mainly in the salivary glands due to the high multiplication of phytoplasmas in this organ, compared with ingestion and injection of GFP dsRNA (Fig. 3C and G). In controls, median FD phytoplasma counts were $1.4 \times 10^7$ and $1.1 \times 10^7$ in insect bodies after GFP dsRNA ingestion, and $9.3 \times 10^6$ and $1.4 \times 10^6$ after GFP dsRNA injection. They were $1.7 \times 10^7$ and $1.4 \times 10^7$ in the heads of *E. variegatus* ingesting GFP dsRNA, and $1.6 \times 10^7$ and $1.3 \times 10^6$ after injection of GFP dsRNA.

## 3.3. *Uk1_LRR is not required for phytoplasmas to be efficiently transmitted to the plant*

After *E. variegatus* had ingested or been injected with uk1_LRR or GFP dsRNA and been infected with FD phytoplasmas, the insects were placed on uninfected broad beans for five days to inoculate the phytoplasmas. After 15 days incubation, 55 plants out of 58 (95%) were infected when insects had been treated with uk1_LRR dsRNA versus 53 plants out of 55 (96%) when insects had been treated with GFP dsRNA. No significant
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effect of uk1_LRR dsRNA on the colonization of *E. variegatus* by FD phytoplasmas and on their transmission to broad bean after the insects had ingested uk1_LRR dsRNA (A-D) or have been injected with uk1_LRR dsRNA (E-H). Expression of the uk1_LRR gene in the bodies and in the heads regards to the reference genes glutathione S-transferase and tubulin beta five (A and E), ten (F) and twelve (B) days after dsRNA ingestion (A and B) or dsRNA injection (E and F). C and G, relative number of FD phytoplasmas (FDp) in each insect part indicated under the graph. White boxes correspond to *E. variegatus* that ingested GFP dsRNA for 2 days (C) or were injected with GFP dsRNA (G) and acquired phytoplasmas on infected broad beans, and grey boxes correspond to *E. variegatus* that ingested uk1_LRR dsRNA (C) or were injected with uk1_LRR dsRNA (G) before acquisition of phytoplasmas. D and H, relative number of FD phytoplasmas per g of plant fresh weight (gFWg) in broad beans 15 days after inoculation by *E. variegatus* that were treated with GFP dsRNA (white box) or uk1_LRR dsRNA (grey box). dsGFP, GFP dsRNA ingested or injected; dsLRR, uk1_LRR dsRNA ingested or injected; n, number of *E. variegatus* or plants treated, ** indicates a significant difference with p < 0.01 and *** with p < 0.001 under the Kruskal-Wallis rank sum test of the R commander package of R software version 4.0.3.</figcaption></figure>

difference was observed between the number of FD phytoplasmas in plants infected with GFP *E. variegatus* and uk1_LRR *E. variegatus* (Fig. 3D and H). The median values for the number of FD phytoplasmas per g of plant fresh weight were 2.10⁹ and 2.10⁸, and 6.10⁸ and 1.10⁹ for the GFP condition of the four experiments.

**3.4. Clathrin is involved in the infection of *E. variegatus* body and salivary glands by phytoplasmas**

As FD phytoplasma use clathrin to enter insect cells and that ingestion of chc dsRNA by *E. variegatus* decreased the quantity of phytoplasma tuf mRNA in the body and the head of insects (Arricau-Bouvery et al., 2023), RNAi using chc dsRNA should be accompanied by a reduction of phytoplasma cells' number in insects and by a lower transmission efficiency to the plant. Young *E. variegatus* adults ingested chc dsRNA and

GFP dsRNA, and were fed on broad bean infected with FD phytoplasmas. Results show that chc dsRNA ingestion significantly decreased chc mRNA with a 3.7 and 3.5-fold change of mean values in bodies and heads, respectively, five days after dsRNA ingestion (Fig. 4A). As for RNAi targeting uk1_LRR gene expression, RNAi varied from one insect to another with some where RNAi was not effective, while in others it was very significant.

Median values for the number of FD phytoplasmas were 2.10⁴, 8.10³ and 7.10⁴ in the body of GFP insects, and were 4.10³, 4.10⁴ and 7.10⁴ in the heads of GFP insects for all three experiments. Ingestion of chc dsRNA significantly decreased the amount of phytoplasmas in the bodies (9-fold on medians) and in the heads, (120-fold on medians) compared to ingestion of GFP dsRNA (Fig. 4B). The high variability between insects ingesting chc dsRNA was probably due to heterogeneity in dsRNA ingestion and thus RNAi efficiency. The reduction in phytoplasma
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Effects of chc dsRNA on the colonization of *E. variegatus* by FD phytoplasmas and on their transmission to broad bean. A, expression of the clathrin heavy chain (chc) gene in the bodies and in the heads regards to the reference genes glutathione S-transferase and tubulin beta five days after dsRNA ingestion. B, relative number of FD phytoplasmas in each insect part indicated under the graph. White boxes correspond to *E. variegatus* that ingested GFP dsRNA for 2 days and acquired FD phytoplasmas on infected broad beans, and grey boxes correspond to *E. variegatus* that ingested chc dsRNA before acquisition of FD phytoplasmas. C, relative number of FD phytoplasmas per g of plant fresh weight (gFWg) in broad beans 15 days after inoculation by *E. variegatus* that had ingested GFP dsRNA (white box) or chc dsRNA (grey box). dsGFP, GFP dsRNA ingested; dschc, chc dsRNA ingested; n, number of *E. variegatus* or broad beans treated, ** indicates a significant difference with *p* < 0.01 and *** with *p* < 0.001 under the Kruskal-Wallis rank sum test of the R commander package of R software version 4.0.3.</figcaption></figure>

numbers was greater in the salivary glands than in the bodies, but this could be a consequence of RNAi efficiency in the midgut, which reduced the number of phytoplasmas reaching the hemolymph and then the salivary glands. Thus, the injection of dsRNAs and phytoplasmas was intended to be evaluated to bypass the midgut and directly target salivary gland cells to verify that clathrin-mediated endocytosis is necessary for phytoplasmas to cross this barrier.

Injection of chc dsRNA into the body of *E. variegatus* was first checked and showed higher efficiency and very little variation in RNAi response between insects injected (Table S1). However, chc gene is involved in endocytosis and lysosome pathways, and its expression is critical in most *E. variegatus* tissues and organs. High mortalities were observed when chc dsRNAs were injected into insects. The efficiency of RNAi decreased with decreasing dsRNA concentration. When insects were injected with the highest concentrations of dsRNA, fewer than 1 in 10 survived compared with *E. variegatus* injected with GFP dsRNA. At concentrations of 1 and 0.4 µg/mL, only half of *E. variegatus* survived treatment with chc dsRNA compared with those injected with GFP dsRNA. As a result, due to high insect mortality or response heterogeneity depending on dsRNA concentrations, experiments to test salivary gland infection and phytoplasma transmission to the plant after a double injection of dsRNA and phytoplasmas were not performed.

### 3.5. Clathrin is involved in the transmission of FD phytoplasmas to the plant

Broad bean inoculation was performed as previously described using *E. variegatus* treated with GFP and chc dsRNAs. After 15 days of incubation, 12 out of 21 plants (57 %) were infected when the insects had ingested chc dsRNA compared to 23 out of 28 (82 %) when the plants were infected by insects that had ingested GFP dsRNA. The number of FD phytoplasmas in plants infected with *E. variegatus* ingesting chc dsRNA was significantly lower (2.10³-fold) than the number of FD phytoplasmas in GFP plants (Fig. 4C). The median values for the number

of FD phytoplasmas per g of plant fresh weight were 3.10⁵, 1.10⁸ and 2.10⁴ for the GFP condition of the three experiments. Thus, the decrease of phytoplasma load in insects, particularly in heads, was correlated with a reduction in the number of infected plants.

### 3.6. FD phytoplasmas localize mainly outside the salivary gland cells of *E. variegatus* treated with dsRNA chc whereas they are mainly inside when treated with dsRNA uk1_LRR

To visualize the effect of RNAi on phytoplasma infection of salivary glands, *E. variegatus* ingested uk1_LRR or chc dsRNAs and were infected with FD phytoplasmas using infected broad beans. Phytoplasmas were detected on the surface and in the salivary gland cells of insects treated with GFP, uk1_LRR or chc dsRNA (Fig. 5; labeling by the different fluorochromes is shown in Figure S2). Phytoplasmas appeared spherical and some had protruding filaments (arrows in panels labeled Surface). Phytoplasmas were not counted in images taken by laser scanning microscopy, but it was repeatedly found that *E. variegatus* salivary gland cells treated with uk1_LRR dsRNA and infected with FD phytoplasmas showed numerous phytoplasmas inside the cells (arrowheads in Fig. 5). These results suggest that silencing of uk1_LRR expression can enhance phytoplasma accumulation in salivary glands of *E. variegatus*. On the contrary, fewer phytoplasmas were detected inside salivary gland cells when insects ingested chc dsRNA than when insects ingested GFP or uk1_LRR dsRNAs (arrowheads in Fig. 5). This suggests that phytoplasmas took longer to enter these cells when RNAi targeted the clathrin heavy chain gene, or that fewer phytoplasmas had access to the salivary glands.

## 4. Discussion

Phytoplasmas express a variety of surface-exposed membrane proteins (Kakizawa et al., 2006; Konnerth et al., 2016; Rossi et al., 2019). The proteins Amp of 'Ca. P. asteris' and Imp of 'Ca. P. mali' and
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Observation by laser scanning microscopy with an Airyscan detector of salivary gland cells of FD phytoplasma infected *E. variegatus* that have ingested GFP, uk1_LRR or chc dsRNA. Cell nuclei were stained with DAPI (cyan), actin filaments with Alexa 568-phalloidin (red) and phytoplasmas with anti-VmpA antibodies and Alexa 488-secondary antibodies (green). "Surface" indicates the view of the infected salivary gland cell surface; arrows indicate protruding filaments. "Inside" indicates the view of the inside of the same infected salivary gland cell; arrows indicate FD phytoplasmas at the surface of salivary gland cells and arrowheads FD phytoplasmas inside these cells. Scale bar 10 µm.</figcaption></figure>

*Crotalaria* witches' broom phytoplasma, homologous to the Imp protein of FD phytoplasma, bind to host cell actin and are supposed to be involved in the intracellular trafficking of phytoplasmas in both insect and plant hosts (Boonrod et al., 2023, 2012; Galetto et al., 2011; Liu et al., 2024; Suzuki et al., 2006). No direct involvement of these two proteins in the endocytosis process has been demonstrated to date, although Amp's binding to ATP synthase may indicate a role in host energy exploitation during phytoplasma endocytosis. The Imp protein should have the same role as the Amp protein in FD phytoplasma, which does not possess an Amp gene (Debonneville et al., 2022). A recent study has shown that the interaction between Imp and the natterin and legu- main proteins, expressed in the midgut of the insect vector, is involved in the infection of the insect vector by FD phytoplasmas (Galetto et al.,

2025) but the precise role of these two insect proteins reminds to be determined. VmpA is another protein exposed on the surface of FD phytoplasma and acts as an adhesin. In this study, VmpA was shown to be able to induce VmpA-coated bead internalization in *E. variegatus* cells in culture, and would therefore most likely be involved in FD phyto- plasma endocytosis in insect cells. Many adhesins of pathogenic bacteria have invasive properties, enabling the internalization of bacteria into the host cell. They are therefore often called invasins. The first to be studied were *Yersinia* and *Listeria* invasins, which bind integrins with sufficient affinity to induce internalization (Isberg and Leong, 1990; Mengaud et al., 1996). Moreover, VmpA proteins are able to stick together in the presence of mannose (Arricau-Bouvery et al., 2021). The example of *Neisseria meningitidis* NadA (*Neisseria* adhesin A) and
Haemophilus influenzae HadA adhesins belonging to the ‘oligomeric coiled-coil adhesin’, which mediate adhesion and entry into host cells of these bacteria (Capecchi et al., 2005; Serruto et al., 2009), suggests that this may be the case for FD phytoplasma via VmpA.

The fibronectin-binding protein FnBP of *Staphylococcus aureus* binds directly to the heat shock protein 60 exposed on the surface of mammary epithelial cells, but the interaction of FnBP with fibronectin of the host extracellular matrix, which binds to the β integrin present on the surface of eukaryotic cells, is also necessary for maximal uptake of *S. aureus* (Dziewanowska et al., 2000). VmpA interacts with several glycoproteins of *E. variegatus* cells and a likely receptor, Uk1_LRR, has been identified (Arricau-Bouvery et al., 2021; Canuto et al., 2023). Here, internalization of VmpA-His<sub>6</sub>-coated beads was shown to be partially dependent on this Uk1_LRR protein. The interaction between Uk1_LRR and VmpA is probably required for the entry of FD phytoplasmas into insect cells, although this receptor does not appear to be the only VmpA receptor enabling internalization of VmpA-coated beads. Indeed, many *E. variegatus* proteins that potentially interact with VmpA have been identified by mass spectrometry proteins after purification on affinity column and far-western blot assays (Canuto et al., 2023). For example, integrin β has been identified, and a decrease in its gene expression in Euva cells leads to a decrease in the adhesion of VmpA-His<sub>6</sub>-coated beads with a p-value of 0.067. As mentioned above, integrin β is a eucaryotic surface protein targeted by bacterial adhesins to enable the entry of pathogenic bacteria into cells. As for the FnBP, interaction of VmpA with several insect cell proteins could enhance phytoplasma entry into host cells.

FD phytoplasmas are pathogenic and detrimental for *E. variegatus* as deleterious effects on the longevity and fecundity of *E. variegatus* have been observed (Bressan et al., 2005). When uk1_LRR gene expression was inhibited by RNAi, an increase in phytoplasma number was observed in *E. variegatus*. Furthermore, uk1_LRR expression had increased, but not significantly, in the midgut of *E. variegatus* shortly after the insects had been infected with FD phytoplasmas (Canuto et al., 2023). These results indicate that Uk1_LRR may be a pattern recognition receptor (PRR) involved in the immune response of *E. variegatus* to decrease FD phytoplasma infection. Indeed, Toll-like receptors (TLRs) are well-known PRRs containing LRR domain and play an indispensable role in the activation of innate immunity. When the expression level of LsToll-13 mRNA was decreased by RNAi in *Laodelphax striatellus*, the expression of virus mRNA was increased (Zhou et al., 2020). Uk1_LRR shares sequence and structural homologies with Toll-like receptors from several aphids (*Daktulosphaira vitifoliae*, *Rhopalosiphum maidis*, *Aphis craccivora*). However, no Toll/interleukin-1 receptor/resistance protein (TIR) domain homology has been predicted in the C-terminal part of the Uk1_LRR protein. Recently, the CfLRRop-1 protein from *Chlamys farreri*, which solely contains LRR motifs, was shown to be part of the PRR (Wang et al., 2016). Moreover, in the early phase of infection, the uk1_LRR gene was more highly expressed in *E. variegatus* as it was the case for the phenoloxidase gene, coding a protein implicated in melanization during immune response of insects, after infection of *E. variegatus* by FD phytoplasma (Gonella et al., 2019) or after infection of *Recilia dorsalis* by rice orange leaf phytoplasma (Zhang et al., 2025). Expression of the gene serine/arginine repetitive matrix protein 1-like also implicated in melanization was found to be upregulated in *Cacopsylla pyricola* infected by *Ca. P. pyri* (Easterling et al., 2024). Thus, the Uk1_LRR protein could be involved in protein-protein interactions occurring during the *E. variegatus* immune response by enabling recognition of FD phytoplasmas and inducing a protective response. The insect vectors *S. titanus* and *E. variegatus* have another way to detect phytoplasmas via the natterin protein that interacts with the surface protein Imp of the FD phytoplasma (Galetto et al., 2025). The natterin proteins are other important constituents of the innate immune defense system (Lima et al., 2021). Thus, despite phytoplasmas do not possess peptidoglycan that is usually the major target of immune receptors, these bacteria are recognized by insect receptors and induce insect immune responses including activation of the prophenoloxidase (Galetto et al., 2018).

Clathrin-mediated endocytosis is frequently used by bacteria to enter their host cells (Rasschaert et al., 2010; Veiga and Cossart, 2005) and by plant pathogens to infect their insect vectors. It occurs for viruses (Pan et al., 2017) and the bacterium *Ca. Liberibacter asiaticus* (Xie et al., 2024). FD phytoplasmas enter insect cells using clathrin-mediated endocytosis (Arricau-Bouvery et al., 2023). The involvement of clathrin-mediated endocytosis in insect vector colonization by FD phytoplasma is reinforced by the fact that inhibition of clathrin expression in *E. variegatus* using RNAi induced a significant decrease in insect colonization and plant infection by FD phytoplasmas as is the case for *Ca. L. asiaticus*. Here VmpA-coated fluorescent beads were shown to penetrate insect cells, including cells of FD phytoplasma insect vector, using clathrin. Several bacterial adhesins enable clathrin-mediated internalization of bacteria during the process of internalization into eukaryotic cells. For example, *H. influenzae* HadA adhesin and *Staphylococcus autolysin* Atl, which interacts with its receptor heat shock cognate protein 70, promote bacterial internalization involving cytoskeletal rearrangement and clathrin-mediated endocytosis (Schlesier et al., 2020; Serruto et al., 2009). It remains to be defined whether the interaction between VmpA and the membrane protein Uk1_LRR is involved in clathrin-mediated endocytosis of phytoplasmas by determining if Uk1_LRR interacts with AP2b.

## 5. Conclusion

In conclusion, phytoplasmas establish various types of molecular interactions to complete their life cycle in their insect vector, invading insect cells while trying to evade the host response. The VmpA surface protein appears to be involved in insect cell invasion and to be recognized by the *E. variegatus* defense system.

### CRediT authorship contribution statement

**Sybille Duret:** Validation, Methodology, Investigation. **Marie-Pierre Dubrana:** Visualization, Validation, Methodology, Investigation, Data curation. **Xavier Foissac:** Writing – review & editing, Funding acquisition, Conceptualization. **Nathalie Arricau-Bouvery:** Writing – original draft, Visualization, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Sylvie Malembic-Maher:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

### Acknowledgment

This study was possible thanks to the continuous management of the confined greenhouses and laboratories in the INRAE EMERGREEN platform (Greenhouses Platform for Studying Emerging Plant Diseases) by Adelaïde Salvado with the help of Denis Lacaze, Marielle Levillain and Thierry Lusseau. We acknowledge the “Pole Imagerie du Végétal” of Bordeaux Imaging Center (BIC, UAR 3420, France-BioImaging infrastructure) for the use of the platform facilities and the invaluable support accorded to us during microscopic imaging by Lysiane Brocard and Sandra Pedemay. This work was supported by the Plan National contre le Dépérissement du Vignoble (Project RISCA II coordinated by Audrey Petit of French Wine and Vine Institute).

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.micres.2025.128416.

### Data availability

Data will be made available on request.
## References

Angelini, E., Clair, D., Borgo, M., Bertaccini, A., Boudon-Padieu, E., 2001. Flavescence dorée in France and Italy - Occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma. VITIS J. Grapevine Res. 40, 79-86. https://doi.org/10.5073/vitis.2001.40.79-86.

Arricau-Bouvery, N., Dubrana, M.-P., Canuto, F., Duret, S., Brocard, L., Claverol, S., Malembic-Maher, S., Foissac, X., 2023. Flavescence dorée phytoplasma enters insect cells by a clathrin-mediated endocytosis allowing infection of its insect vector. Sci. Rep. 13, 2211. https://doi.org/10.1038/s41598-023-29341-1.

Arricau-Bouvery, N., Duret, S., Dubrana, M.-P., Batailler, B., Desqué, D., Béven, L., Danet, J.-L., Monticone, M., Bosco, D., Malembic-Maher, S., Foissac, X., 2018. Variable membrane protein A of flavescence dorée phytoplasma binds the midgut perimicrovillar membrane of *Eucelidius variegatus* and promotes adhesion to its epithelial cells. Appl. Environ. Microbiol. 84, e02487-17. https://doi.org/10.1128/AEM.02487-17.

Arricau-Bouvery, N., Duret, S., Dubrana, M.-P., Desqué, D., Eveillard, S., Brocard, L., Malembic-Maher, S., Foissac, X., 2021. Interactions between the flavescence dorée phytoplasma and its insect vector indicate lectin-type adhesion mediated by the adhesin VmpA. Sci. Rep. 11, 11222. https://doi.org/10.1038/s41598-021-90809-z.

Bertaccini, A., 2022. Plants and phytoplasmas: when bacteria modify plants. Plants 11, 1425. https://doi.org/10.3390/plants1111425.

Boonrod, K., Kuaguil, M., Braun, M., Müller-Renno, C., Ziegler, C., Krczal, G., 2023. Identification of the actin-binding region and binding to host plant apple actin of immunodominant transmembrane protein of *Candidatus* Phytoplasma mali. Int. J. Mol. Sci. 24, 968. https://doi.org/10.3390/ijms24020968.

Boonrod, K., Munteanu, B., Jarausch, B., Jarausch, W., Krczal, G., 2012. An immunodominant membrane protein (Imp) of *Candidatus* Phytoplasma mali* binds to plant actin. Mol. PlantMicrobe Interact. ® 25, 889-895. https://doi.org/10.1094/MPMI-11-11-0303.

Bosco, D., D'Amelio, R., 2009. Transmission specificity and competition of multiple phytoplasmas in the insect vector. In: Weintraub, P.G., Jones, P. (Eds.), Phytoplasmas: Genomes, Plant Hosts and Vectors. CABI, UK, pp. 293-308. https://doi.org/10.1079/9781845935306.0293.

Bressan, A., Clair, D., Sémétey, O., Boudon-Padieu, É., 2005. Effect of two strains of Flavescence dorée phytoplasma on the survival and fecundity of the experimental leafhopper vector *Eucelidius variegatus* Kirschbaum. J. Invertebr. Pathol. 89, 144-149. https://doi.org/10.1016/j.jip.2005.03.001.

Canuto, F., Duret, S., Dubrana, M.-P., Claverol, S., Malembic-Maher, S., Foissac, X., Arricau-Bouvery, N., 2023. A knockdown gene approach identifies an insect vector membrane protein with leucin-rich repeats as one of the receptors for the VmpA adhesin of flavescence dorée phytoplasma. Front. Cell. Infect. Microbiol. 13, 1289100. https://doi.org/10.3389/fcimb.2023.1289100.

Capecchi, B., Adu-Bobie, J., Di Marcello, F., Ciucchi, L., Masignani, V., Taddei, A., Rappuoli, R., Pizza, M., Aricò, B., 2005. *Neisseria meningitidis* NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells. Mol. Microbiol. 55, 687-698. https://doi.org/10.1111/j.1365-2958.2004.04423.x.

Caudwell, A., Kuszala, C., Bachelier, J.C., Larrue, J., 1970. Transmission de la flavescence dorée de la vigne aux plantes herbacees par l'allongement du temps d'utilisation de la cicadelle *Scaphoideus littoralis* Ball et l'étude de sa survie sur en grand nombre d'espetes vegetales. Ann. Phytopathol. 1572, 415-428.

Caudwell, A., Kuszala, C., Larrue, J., Bachelier, J.C., 1972. Transmission de la flavescence dorée de la feve a la feve par des cicadelles des genres *Eucelis et Eucelidius*: intervention possible de ces insectes dans l'épidémiologie du bois noir en Bourgogne. Ann. Phytopathol. 84, e02487.

Chuche, J., Thiéry, D., 2014. Biology and ecology of the flavescence dorée vector *Scaphoideus titanus*: a review. Agron. Sustain. Dev. 34, 381-403. https://doi.org/10.1007/s13593-014-0208-7.

Debonneville, C., Mandelli, L., Brodard, J., Groux, R., Roquis, D., Schumpp, O., 2022. The complete genome of the "Flavescence Dorée" phytoplasma reveals characteristics of low genome plasticity. Biology 11, 953. https://doi.org/10.3390/biology11070953.

Ding, L., Lu, W., Yang, Y., Zhong, Q., Zhou, T., Wang, G., Song, C., Ma, W., Chen, W., Wu, Y., 2022. Immunodominant membrane protein (Imp) promotes the transmission of wheat blue dwarf (WBD) phytoplasma by directly interacting with α-tubulin in leafhoppers. Eur. J. Plant Pathol. 162, 357-367. https://doi.org/10.1007/s10658-021-02408-3.

Duret, S., Batailler, B., Danet, J.-L., Béven, L., Renaudin, J., Arricau-Bouvery, N., 2010. Infection of the *Circulifer haematoceps* cell line Ciha-1 by *Spiroplasma citri*: the non-insect-transmissible strain 44 is impaired in invasion. Microbiology 156, 1097-1107. https://doi.org/10.1099/mic.0.035063-0.

Duret, S., Batailler, B., Dubrana, M.-P., Saillard, C., Renaudin, J., Béven, L., Arricau-Bouvery, N., 2014. Invasion of insect cells by *Spiroplasma citri* involves spiralin relocalization and lectin/glyco conjugate-type interactions: spiralin relocalization and insect cell adhesion. Cell. Microbiol. 16, 1119-1132. https://doi.org/10.1111/cmi.12265.

Dziewanowska, K., Carson, A.R., Patti, J.M., Deobald, C.F., Bayles, K.W., Bohach, G.A., 2000. Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells. Infect. Immun. 68.

Easterling, K.A., Marshall, A.T., Pitino, M., Walker, W.B., Cooper, W.R., 2024. Gene expression profiling of *Cacopylla pyricola* (Hemiptera: Psyllidae) infected with *Ca. Phytoplasma pyri* (*Acholeplasmatales: Acholeplasmataceae*) reveals candidate effectors and mechanisms of infection. Environ. Entomol. 53, 771-781. https://doi.org/10.1093/ee/nvae074.

Eveillard, S., Jollard, C., Labroussaa, F., Khalil, D., Perrin, M., Desqué, D., Salar, P., Razan, F., Févin, C., Bordenave, L., Foissac, X., Masson, J.E., Malembic-Maher, S., 2016. Contrasting susceptibilities to flavescence dorée in *Vitis vinifera*, rootstocks and wild *Vitis* species. Plant Biot. Inter. 1762. https://doi.org/10.3389/fpls.2016.01762.

Galeto, L., Abbà, S., Rossi, M., Vallino, M., Pesando, M., Arricau-Bouvery, N., Dubrana, M.-P., Chitarra, W., Pegoraro, M., Bosco, D., Marzachi, C., 2018. Two phytoplasmas elicit different responses in the insect vector *Eucelidius variegatus* Kirschbaum. Infect. Immun. 86, e00042-18. https://doi.org/10.1128/IAI.00042-18.

Galeto, L., Bosco, D., Balestrini, R., Genre, A., Fletcher, J., Marzachi, C., 2011. The major antigenic membrane protein of "*Candidatus* Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors. PLoS ONE 6, e22571. https://doi.org/10.1371/journal.pone.0022571.

Galeto, L., Lucetti, G., Bucci, L., Canuto, F., Rossi, M., Abbà, S., Vallino, M., Parise, C., Palmano, S., Manfredi, M., Bosco, D., Marzachi, C., 2025. Natterin-like and legumin insect gut proteins promote the multiplication of a vector-borne bacterial plant pathogen. Microbiol. Res. 291, 127984. https://doi.org/10.1016/j.micres.2024.127984.

Glebov, O.O., 2020. Understanding SARS-CoV-2 endocytosis for COVID-19 drug repurposing. FEBS J. 287, 3664-3671. https://doi.org/10.1111/febs.15369.

Gonella, E., Benelli, G., Arricau-Bouvery, N., Bosco, D., Duso, C., Dietrich, C.H., Galeto, L., Rizzoli, A., Jović, J., Mazzoni, V., Mori, N., Nieri, R., Roversi, P.F., Strauss, G., Thiéry, D., Trivellone, V., Virant-Doberlet, M., Lucchi, A., Alma, A., 2024a. *Scaphoideus titanus* forecasting and management: *quo vadis*? Entomol. Gen. 44, 497-510. https://doi.org/10.1127/entomologia/2023/2598.

Gonella, E., Benelli, G., Arricau-Bouvery, N., Bosco, D., Duso, C., Dietrich, C.H., Galeto, L., Rizzoli, A., Jović, J., Mazzoni, V., Mori, N., Nieri, R., Roversi, P.F., Strauss, G., Thiéry, D., Trivellone, V., Virant-Doberlet, M., Lucchi, A., Alma, A., 2024b. *Scaphoideus titanus* up-to-the-minute: biology, ecology, and role as a vector. Entomol. Gen. 44, 481-496. https://doi.org/10.1127/entomologia/2023/2597.

Gonella, E., Mandrioli, M., Tedeschi, R., Crotti, E., Pontini, M., Alma, A., 2019. Activation of immune genes in leafhoppers by phytoplasmas and symbiotic bacteria. Front. Physiol. 10, 795. https://doi.org/10.3389/fphys.2019.00795.

Hogenhout, S.A., Oshima, K., Ammar, E., Kakizawa, S., Kingdom, H.N., Namba, S., 2008. Phytoplasmas: bacteria that manipulate plants and insects. Mol. Plant Pathol. 9, 403-423. https://doi.org/10.1111/j.1364-3703.2008.00472.x.

Isberg, R.R., Leong, J.M., 1990. Multiple /1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60, 861-871. https://doi.org/10.1016/0092-8674(90)90099-z.

Jeger, M., Bragard, C., Caffier, D., Candresse, T., Chatzivassiliou, E., Dehnen-Schmutz, K., Gilioli, G., Jaques Miret, J.A., MacLeod, A., Navajas Navarro, M., Niere, B., Parnell, S., Potting, R., Rafoss, T., Rossi, V., Urek, G., Van Bruggen, A., Van Der Werf, W., West, J., Winter, S., Bosco, D., Foissac, X., Strauss, G., Hollo, G., Mosbach-Schulz, O., Grégoire, J.-C., 2016. Risk to plant health of flavescence dorée for the EU territory. EFSA J. 14, e04603. https://doi.org/10.2903/j.efsa.2016.4603.

Johnson, A., 2024. Mechanistic divergences of endocytic clathrin-coated vesicle formation in mammals, yeasts and plants. J. Cell Sci. 137, jcs261847. https://doi.org/10.1012/jcs.261847.

Kakizawa, S., Oshima, K., Namba, S., 2006. Diversity and functional importance of phytoplasma membrane proteins.

Koinuma, H., Maejima, K., Tokuda, R., Kitazawa, Y., Nijo, T., Wei, W., Kumita, K., Miyazaki, A., Namba, S., Yamaji, Y., 2020. Spatiotemporal dynamics and quantitative analysis of phytoplasmas in insect vectors. Sci. Rep. 10, 4291. https://doi.org/10.1038/s41598-020-61042-x.

Konnerth, A., Krczal, G., Boonrod, K., 2016. Immunodominant membrane proteins of phytoplasmas. Microbiology 162, 1267-1273. https://doi.org/10.1099/mic.000331.

Lima, C., Disner, G.R., Falcão, M.A.P., Seni-Silva, A.C., Maleski, A.L.A., Souza, M.M., Reis Tonello, M.C., Lopes-Ferreira, M., 2021. The natterin proteins diversity: a review on phylogeny, structure, and immune function. Toxins 13, 538. https://doi.org/10.3390/toxins13080538.

Liu, C.-Y., Cheng, H.-P., Lin, C.-P., Liao, Y.-T., Ko, T.-P., Lin, S.-J., J., Lin, S.-S., Wang, H.-C., 2024. Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein. IUCrJ 11, 384-394. https://doi.org/10.1107/S205225524003075.

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻ΔΔCT method. Methods 25, 402-408. https://doi.org/10.1006/meth.2001.1262.

Maixner, M., 2005. Risks posed by the spread and dissemination of grapevine pathogens and their vectors., in: Proceedings of the Symposium Held at Humboldt University: Introduction and Spread of Invasive Species. Berlin, pp. 141-146.

Malembic-Maher, S., Desqué, D., Khalil, D., Salar, P., Bergey, B., Danet, J.-L., Duret, S., Dubrana-Ourabah, M.-P., Beven, L., Ember, I., Acs, Z., Della Bartola, M., Materazzi, A., Filippin, L., Krnjajic, S., Krstić, O., Toševski, I., Lang, F., Jarausch, B., Kölber, M., Jović, J., Angelini, E., Arricau-Bouvery, N., Maixner, M., Foissac, X., 2020. When a Palearctic bacterium meets a Nearctic insect vector: genetic and ecological insights into the emergence of the grapevine flavescence dorée epidemics in Europe. PLOS Pathog. 16, e1007967. https://doi.org/10.1371/journal.ppat.1007967.

Mengaud, J., Ohayon, H., Gounon, P., Mêge, R.-M., Cossart, P., 1996. E-cadherin is the receptor for internalin, a surface protein required for entry of *L. monocytogenes* into epithelial cells. Cell 84, 923-932. https://doi.org/10.1016/S0092-8674(00)81070-3.

Namba, S., 2019. Molecular and biological properties of phytoplasmas. Proc. Jpn. Acad. Ser. B 95, 401-418. https://doi.org/10.2183/pjab.95.028.

Oliveira, M.J.R.A., Roriz, M., Vasconcelos, M.W., Bertaccini, A., Carvalho, S.M.P., 2019. Conventional and novel approaches for managing "flavescence dorée" in grapevine:
knowledge gaps and future prospects. Plant Pathol. 68, 3–17. https://doi.org/10.1111/ppa.12938.

Pan, L.-L., Chen, Q.-F., Zhao, J.-J., Guo, T., Wang, X.-W., Hariton-Shalev, A., Czosnek, H., Liu, S.-S., 2017. Clathrin-mediated endocytosis is involved in Tomato yellow leaf curl virus transport across the midgut barrier of its whitefly vector. Virology 502, 152–159. https://doi.org/10.1016/j.virol.2016.12.029.

Rashidi, M., Galetto, L., Bosco, D., Bulgarelli, A., Vallino, M., Veratti, F., Marzachì, C., 2015. Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species. BMC Microbiol 15, 193. https://doi.org/10.1186/s12866-015-0522-5.

Rasschaert, K., Devriendt, B., Favoreel, H., Goddeeris, B.M., Cox, E., 2010. Clathrin-mediated endocytosis and transcytosis of enterotoxigenic *Escherichia coli* F4 fimbriae in porcine intestinal epithelial cells. Vet. Immunol. Immunopathol. 137, 243–250. https://doi.org/10.1016/j.vetimm.2010.05.016.

Renaudin, J., Béven, L., Batailler, B., Duret, S., Desqué, D., Arricau-Bouvery, N., Malembic-Maher, S., Foissac, X., 2015. Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in *Spiroplasma citri*. BMC Microbiol 15, 82. https://doi.org/10.1186/s12866-015-0417-5.

Rossi, M., Samarzija, I., Seruga-Musić, M., Galetto, L., 2019. Diversity and functional importance of phytoplasma membrane proteins. In: Bertaccini, A., Oshima, K., Kube, M., Rao, G.P. (Eds.), Phytoplasmas: Plant Pathogenic Bacteria - III. Springer Singapore, Singapore, pp. 69–88. https://doi.org/10.1007/978-981-13-9632-8_5.

Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual. CSHL Press.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682. https://doi.org/10.1038/nmeth.2019.

Schlesier, T., Siegmund, A., Rescher, U., Heilmann, C., 2020. Characterization of the AtI-mediated staphylococcal internalization mechanism. Int. J. Med. Microbiol 310, 151463. https://doi.org/10.1016/j.ijmm.2020.151463.

Serruto, D., Spadafina, T., Scarselli, M., Bambini, S., Comanducci, M., Höhle, S., Kilian, M., Veiga, E., Cossart, P., Oggioni, M.R., Savino, S., Ferlenghi, I., Taddei, A. R., Rappuoli, R., Pizza, M., Massignani, V., Aricò, B., 2009. HadA is an atypical new multifunctional trimeric coiled-coil adhesin of *Haemophilus influenzae* biogroup *aegyptius*, which promotes entry into host cells. Cell. Microbiol 11, 1044–1063. https://doi.org/10.1111/j.1462-5822.2009.01306.x.

Suzuki, S., Oshima, K., Kakizawa, S., Arashida, R., Jung, H.-Y., Yamaji, Y., Nishigawa, H., Ugaki, M., Namba, S., 2006. Interaction between the membrane protein of a pathogen and insect microfilament complex determines insect-vector specificity. Proc. Natl. Acad. Sci. 103, 4252–4257. https://doi.org/10.1073/pnas.0508668103.

Tomkins, M., Kliot, A., Marée, A.F., Hogenhout, S.A., 2018. A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment. Curr. Opin. Plant Biol. 44, 39–48. https://doi.org/10.1016/j.pbi.2018.02.002.

Trivellone, V., Ripamonti, M., Angelini, E., Filippin, L., Rossi, M., Marzachì, C., Galetto, L., 2019. Evidence suggesting interactions between immunodominant membrane protein Imp of flavescence dorée phytoplasma and protein extracts from distantly related insect species. J. Appl. Microbiol 127, 1801–1813. https://doi.org/10.1111/jam.14445.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034.1. https://doi.org/10.1186/gb-2002-3-7-research0034.

Veiga, E., Cossart, P., 2005. *Listeria* hijacks the clathrin-dependent endocytic machinery to invade mammalian cells. Nat. Cell Biol. 7, 894–900. https://doi.org/10.1038/ncb1292.

Wang, M., Wang, L., Guo, Y., Yi, Q., Song, L., 2016. An LRR-only protein representing a new type of pattern recognition receptor in *Chlamys farreri*. Dev. Comp. Immunol. 54, 145–155. https://doi.org/10.1016/j.dci.2015.09.006.

Xie, L., Zeng, X., Amir, M.B., Zou, X., Yuan, J., Yu, H., Lu, Z., Chen, W., 2024. Clathrin heavy chain is involved in infection of 'Candidatus Liberibacter asiaticus' in the host vector *Diaphorina citri*. Insect Sci. 31, 1326–1332. https://doi.org/10.1111/1744-7917.13305.

Zhang, X.-F., Li, Z., Qiu, J., Zhang, R., Jiang, Z., Wang, T., Chen, H., Wei, T., 2025. A phytoplasma effector suppresses insect melanization immune response to promote pathogen persistent transmission. Sci. Adv. 11, eads9781. https://doi.org/10.1126/sciadv.ads9781.

Zhou, X., Hu, J., Fu, M., Jin, P., Zhang, Y., Xiang, Y., Li, Y., Ma, F., 2020. Identification and characterization of a TLR13 gene homologue from *Laodelphax striatellus* involved in the immune response induced by rice stripe virus. J. Integr. Agric. 19, 183–192. https://doi.org/10.1016/S2095-3119(19)62795-4.