Biology subjects

Landeo Villanueva, S.

Publications and source records attributed to Landeo Villanueva, S..

5 recordsLinked to original sources

bioRxiv2026

TurboID-based proximity-dependent labeling using SOBIR1 as a bait in potato leads to the identification of novel defense-related signaling partners

The plant immune system comprises a complex signaling network that is activated upon perceiving molecules derived from invading pathogens. The first line of defense at the plant cell surface is mediated by receptor-like proteins (RLPs) and receptor-like kinases (RLKs). RLPs, which lack a cytoplasmic signalling domain themselves, constitutively interact with the RLK SUPRESSOR OF BIR1-1 (SOBIR1), which is a key component initiating immune signal transduction upon pathogen perception. Therefore, elucidating the composition of the SOBIR1 protein complex will contribute to understanding the basic molecular mechanisms of plant disease resistance. Most of the studies focused on the identification of SOBIR1-interacting proteins are limited to model plants, due to technical challenges and lack of reliable genome and proteome databases in crop plants. Here, we evaluate the application of the biotin ligase TurboID (TbID)-based proximity-dependent labeling (PL) approach by transiently expressing SOBIR1 from Nicotiana benthamiana (NbSOBIR1), fused to TbID in leaves of the wild potato Solanum microdontum. We show that NbSOBIR1-YFP-TbID properly accumulates in potato and that proximal proteins are biotinylated. Quantitative proteomic analysis yielded over 130 candidate proteins to be in the proximity of the cytoplasmic kinase domain of NbSOBIR1, of which some could be linked to disease resistance by KEGG pathway and gene ontology (GO) molecular function analysis. We also studied the dynamics of the proteome in proximity of NbSOBIR1 upon perception of the INF1 elicitin of Phytophthora infestans that was co-expressed in potato with the elicitin receptor ELR, which is an RLP that constitutively interacts with SOBIR1. We found more than 80 proteins, including the NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH 1 (NRC1), putatively interacting with NbSOBIR1. In conclusion, we were able to successfully apply PL in potato and a future roadmap for further research on deciphering the composition of protein complexes involved in immune signaling has been established.

plant biology↗

bioRxiv2026

Effector-centred proximity-dependent labelling enables the discovery of cell-surface immune receptors in plants

Identifying plant disease resistance proteins remains challenging, particularly for cell-surface receptors that perceive apoplastic pathogen effectors through transient or indirect interactions. Here, we establish effector-centred proximity-dependent labelling using TurboID as a protein-level approach to identify immune receptors and guarded host targets in planta. We fused three apoplastic effectors, Avr2 and Avr4 from Fulvia fulva and XEG1 from Phytophthora sojae, to TurboID and transiently expressed these fusion proteins in leaves of Nicotiana benthamiana and tomato (Solanum lycopersicum). The fusions significantly biotinylate their matching receptors, including Cf-4 by Avr4-TurboID and Cf-2 by Avr2-TurboID, as well as the guarded virulence target of Avr2, the Rcr3 protease, demonstrating that both direct and indirect recognition systems can be captured. Application of this approach to XEG1 revealed SlEix1 as the functional ortholog of NbRXEG1. Functional assays and structural modelling support SlEix1 as a signalling-competent receptor contributing to XEG1-triggered immunity. Together with previous studies, these findings position the tomato EIX locus as a multi-effector immune hub encoding closely related receptor-like proteins with distinct ligand specificities. Collectively, this study establishes effector-TurboID-mediated proximity-dependent labelling as a versatile approach for identifying cell surface plant immune receptors and apoplastic virulence targets, providing a scalable route to accelerate resistance gene discovery in crop species.

plant biology↗

bioRxiv2023

Receptor-like cytoplasmic kinases belonging to different subfamilies mediate immune responses downstream of the Cf-4 resistance protein in Nicotiana benthamiana

Cell-surface receptors, which are either receptor-like proteins (RLPs) or receptor-like kinases (RLKs), form the first layer of the plant innate immune system. The LRR-RLKs SUPPRESSOR OF BIR1-1 (SOBIR1) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) are the common regulatory co-receptors of LRR-RLPs. The tomato LRR-RLP Cf-4, which specifically detects the apoplastic effector Avr4 that is secreted by the pathogenic intercellular fungus Cladosporium fulvum, requires SOBIR1 and BAK1 to mediate resistance against this fungus. It is proposed that phosphorylation events take place between the cytoplasmic kinase domains of SOBIR1 and BAK1, which lead to the initiation of various downstream signaling outputs such as the rapid phosphorylation of receptor-like cytoplasmic kinases (RLCKs), the accumulation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) cascades, and in some cases, the hypersensitive response (HR). Here, by performing in vitro phosphorylation assays, we show that SOBIR1 directly phosphorylates BAK1, whereas BAK1, in its turn, directly phosphorylates SOBIR1, which results in the full activation of the Cf-4-containing immune complex. We found that threonine 522, present in the activation segment of the SOBIR1 kinase domain of Nicotiana benthamiana (Nb), is required for its intrinsic kinase activity, and is essential for the Avr4/Cf-4/SOBIR1/BAK1-triggered ROS burst, MAPK activation and the HR. Tyrosine residue Y469 was found to be crucial for the Avr4/Cf-4- triggered activation of MAPKs and the HR, but not for ROS production and NbSOBIR1 intrinsic kinase activity. RLCKs are well-recognized to act as the initial cytoplasmic transducers, bridging cell-surface receptor complexes with their downstream signaling partners. By knocking out multiple genes belonging to different RLCK-VII subfamilies in N. benthamiana plants stably expressing Cf-4, we show that members of the RLCK-VII subfamilies 6, 7 and 8 are required for the Avr4/Cf-4-triggered ROS production. Typically, the Avr4 protein triggers a biphasic ROS burst in leaf discs obtained from N. benthamiana:Cf-4 plants, in which the first transitory response is followed by a second, sustained ROS burst. Interestingly, the intensities of the first and second phase of the ROS burst are affected in different ways in the various rlck-vii knock-out plants, indicating that there are different downstream ROS regulatory mechanisms, involving different RLCKs in N. benthamiana. Further studies show that members from RLCK-VII-6, 7 and 8 also play an essential role in regulating the ROS production triggered by flg22, chitin, and the nlp20/RLP23 and the pg13/RLP42 combinations.

plant biology↗

bioRxiv2020

Knocking out SOBIR1 in Nicotiana benthamiana abolishes functionality of transgenic receptor-like protein Cf-4

The first layer of plant immunity is formed by pattern recognition receptors (PRRs) that are present at the cell surface and perceive extracellular immunogenic patterns. Receptor-like proteins (RLPs), such as the tomato (Solanum lycopersicum) PRR Cf-4 that provides resistance to the fungus Cladosporium fulvum secreting the matching avirulence factor Avr4, have an extracellular receptor domain consisting of leucine-rich repeats, but lack a cytoplasmic kinase domain for downstream signaling. RLPs constitutively interact with the receptor-like kinase SUPPRESSOR OF BIR1-1 (SOBIR1), thereby providing the receptor with a kinase domain, and recruit the co-receptor BRI-ASSOCIATED KINASE 1 (BAK1) upon their activation by a matching ligand. Trans-phosphorylation events, which can take place between the kinase domains of SOBIR1 and BAK1 after their association with the RLP, are thought to initiate downstream defense signaling. Currently, our knowledge on RLP/SOBIR1/BAK1-mediated defence initiation is limited and to understand the role of SOBIR1 in RLP function, we knocked out SOBIR1 and its close homolog SOBIR1-like in the model plant Nicotiana benthamiana, as well as in transgenic N. benthamiana stably expressing Cf-4. We observed that Cf-4 function is completely abolished in the knock-out mutants, and we show that these plants can be used to perform transient complementation studies with SOBIR1 mutants. Thereby, these mutants are an important tool to study the fundamentals of plant immunity mediated by RLPs.

plant biology↗

bioRxiv2020

Red-light imaging for programmed cell death visualization and quantification in plant-pathogen interactions

Studies on plant-pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility and the ability to quantify the results. In this study we show that red light detection in a multi-purpose fluorescence imaging system that is probably available in many labs can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence upon thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur either during a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables to evaluate the magnitude of programmed cell death and provides a non-invasive readout of the plant immune response in a faster and safer manner as compared to chemical staining methodologies previously developed. This application can be implemented to screen for differences in symptom severity in plant-pathogen interactions, and to visualize and quantify in a sensitive and objective manner the intensity of a plant response upon perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.

plant biology↗