| dc.description.abstractEn | INTRODUCTIONLike other bacterial pathogens, phytoplasmas produce proteins termed 'effectors' that target specific host proteins and alter host-cell structure and function. Several phytoplasma effectors have already been well documented (Wang et al., 2024). However, they are not shared by all phytoplasmas, and currently only limited data are available regarding pathogenicity factors deployed by flavescence dorée phytoplasma. In a previous study, we predicted candidate effectors from the assembled genome of flavescence dorée phytoplasma, and set out to determine and compare their expression level and subcellular localization in plant and insect cells. A major objective was to provide assumptions for each effector about the targeted host, i.e. the plant or insect host (Garcion et al., 2019).In this study, we carried on the functional analysis of these candidate effectors and looked for their putative targets in grapevine using the yeast two-hybrid system. In this technique, the candidate effector is fused to the GAL4 DNA-binding domain that binds to a specific DNA sequence, strategically engineered in the promoter of reporter genes. The putative interacting protein is fused to the GAL4 transcription activation domain. Both fusion proteins are expressed in a suitable strain of the yeast Saccharomyces cerevisiae (Fields & Song, 1989). Interaction of the candidate effector with its target leads to transcriptional activation of reporter genes and growth on defined media.MATERIALS AND METHODSThe yeast two-hybrid library was created using the kit "Make your own 'Mate & Plate' library system" (Takara Bio). Briefly, RNA was extracted from Vitis vinifera cv. 'Cabernet Sauvignon' young healthy leaves using the Monarch total RNA isolation kit. Poly-adenylated mRNAs were isolated from total RNA using the Dynabeads™ mRNA purification kit (Invitrogen). cDNAs were produced using oligo-dT primers, amplified by PCR and simultaneously transformed in yeast strain Y187 and cloned in pGADT7-Rec vector by following the protocol indicated in Yeastmaker Yeast Transformation System 2 User Manual. Yeast strain Y2H Gold was used to perform the yeast two-hybrid screen and assays, according to the instructions of the Matchmaker Gold yeast two-hybrid system user manual (Clontech). Yeast media employed in this study were from Takara Bio.RESULTS AND DISCUSSIONFive candidate effectors were selected for serving as baits in yeast two-hybrid screens. For three of them, previous subcellular localization studies showed an association with undetermined punctuate structures, or vesicle-like structures that could be linked to the secretion system in plant or insect cells (Garcion et al., 2019). Unfortunately, none of the two-hybrid screens performed for these three candidates allowed to disclose any putative target. Indeed, although the yeast two-hybrid system is a popular technique that is widely used for characterization of pathogens effectors, it may not always succeed in identifying interacting proteins, and several reasons could explain such a lack of results. For instance, the screened cDNA library may not contain the coding sequences of the targets, due to a low level of expression in our plant material. Another hypothesis is that the fusion of the candidate of interest with the GAL4 DNA binding domain, as required by the yeast two-hybrid system, prevents the interaction of the candidate effector with its targets. Another possibility would be that these three candidates retain in yeast cells their capacity to be addressed to specific cell compartments, whereas the yeast two-hybrid system employed here requires that the "hybrid" fusion proteins interact inside the nucleus of yeast cells to activate reporter genes. A fourth candidate effector showed sequence similarity to the candidate effector SAP68 of aster yellows phytoplasma strain witches' broom. Because SAP68 was demonstrated to interact with Arabidopsis TCP transcription factors (Correa Marrero et al., 2024), we reasoned that our fourth effector might similarly interact with grapevine TCP transcription factors. However, when we performed the screen of the cDNA library, we obtained a large number of positive clones (> 5000). Sequence analyses of a set of random clones indicated that they corresponded to functionally unrelated genes, and that no gene appeared more frequently than others in the selected set. No TCP transcription factor was detected in the set of sequenced clones. From this experiment, it was concluded that our candidate effector likely behaves as a "sticky" protein in yeast cells, preventing the identification of a plausible target with confidence.We obtained more satisfying results with a fifth effector. Previous characterization indicated that a GFP fusion to this effector was localized in the nucleolus of plant cells, suggesting that it recognized a nucleolus-specific component or mechanism (Garcion et al., 2019). A yeast two-hybrid screen of the grapevine cDNA library allowed to recover one clone coding for a putatively interacting gene product with no known function in grapevine. Related genes have been reported in Arabidopsis thaliana, but further experiments suggested that the function of these homologs may not be strictly identical between grapevine and Arabidopsis. Current experiments aim at confirming the interaction with other techniques than the yeast two-hybrid system (i.e. bimolecular fluorescence complementation or pull-down).The knowledge of the targets of phytoplasma effectors will help to provide a fundamental understanding of how phytoplasmas manipulate the physiology of their host plants to their own benefit, especially in the case of a phytoplasma that has recently spilled-over from its original ecological niche. Furthermore, such data will contribute to the efforts of plant pathologists and breeders to improve crop varieties with altered effector targets and increased resistance (McLellan et al., 2022).ACKNOWLEDGEMENTSWe thank Lucie Dutilh and Lina Fawaz for their help at the bench, and Denis Lacaze, Jean-Said Bey and Thierry Lusseau for assistance in growing plants. The authors acknowledge the support of the French National Research Agency (ANR) under the grant 20‐PCPA‐0010 (VITAE).REFERENCESCorrea Marrero M., Capdevielle S., Huang W., Al-Subhi A. M., Busscher M., Busscher-Lange J., van der Wal F., de Ridder D., van Dijk A. D. J., Hogenhout S. A., & Immink R. G. H. (2024). Protein interaction mapping reveals widespread targeting of development-related host transcription factors by phytoplasma effectors. The Plant Journal: For Cell and Molecular Biology, 117(4), 1281–1297. https://doi.org/10.1111/tpj.16546Fields S., & Song O. (1989). A novel genetic system to detect protein-protein interactions. Nature, 340(6230), 245–246. https://doi.org/10.1038/340245a0Garcion C., Métais F., Razan F., Foissac X., Eveillard S., Duret S., Batailler B., & Arricau-Bouvery N. (2019). Characterization of candidate effectors of “flavescence dorée” phytoplasma: Expression and subcellular localization. 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