Periodic Reporting for period 1 - ENDOLOGISTIC (Endocytic logistics of defense signaling in plants)
Periodo di rendicontazione: 2022-02-01 al 2024-01-31
Being sessile, plants do not have an immune system as advanced as animals; they rely on innate immunity to respond rapidly and accurately to changing environment. In the event of pathogen infection, plants recognize molecules produced by microorganisms or derived from insect predation named microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs), respectively, via plasma membrane (PM)-bound pattern recognition receptors (PRRs). Concomitant with receptor activation, ligand binding also triggers internalization of the signaling receptors from the PM into early endosomes, where receptors are sorted for degradation to late endosomes or recycled back to the PM to be reused. Receptor-mediated endocytosis (RME) plays an essential role in cell signaling as it protect plants from over-activation of signaling by way of receptor removal from the PM. In addition to signaling attenuation, RME might also be required for signaling as endosomes can carry active receptor complexes or signaling components and they can be used as signaling platforms in the cytoplasm. In mammals, endosomal signaling was demonstrated for many receptor families including receptor tyrosine kinases, G-protein‐ coupled receptors and toll‐like receptors. Unlike mammals, the mechanisms of endosomal signaling in plants are unknown.
In plants, most of PM receptor kinases (RKs) undergo Clathrin mediated endocytosis (CME). In addition to maintaining basic cellular functions, CME has a role in growth and development, hormone signaling and interaction with the environment, nutrient uptake, restrain toxin and pathogen defense. The very well -known immune receptors FLAGELLIN SENSING2 (FLS2, receptor for flg22) or PEPR1 (a receptor for AtPep1) both requires clathrin for their downstream responses. Interference with clathrin function in Arabidopsis completely blocked the endocytosis of PEPR1 and impaired MAPK activation. While, slight decrease in FLS2 endocytosis only affected the activation of a subset of flg22-triggered signaling.
Recent study of the immune receptor PEPR1 that perceives the endogenous peptide AtPep1, showed that endocytosis is required for mitogen-activated protein kinase (MAPK) activation after elicitation with AtPep1. However, because MAPK activation occurs faster than the endocytosis of the main receptor it raised the question if endocytosis of so far unknown signaling components is required for AtPep1-elicited immune responses.
The overall objective of ENDOLOGISTIC is to decode signaling components involved in early MAPK activation and late endocytosis of PEPR1 upon AtPep1 perception using Arabidopsis thaliana as a model plant. The goal of the study was addressed by combining mass spectrometry analysis on clathrin coated vesicles (CCVs) and TurboID based proximity labelling (PL) approach using PEPR1 as bait upon AtPep1 elicitation.
The study of Identification of unknown signals and endocytic components will be major advance in understanding the regulation of immunity and will fill the gap in our understanding of how endocytosis controls immunity in plants.
In plants, most of PM receptor kinases (RKs) undergo Clathrin mediated endocytosis (CME). In addition to maintaining basic cellular functions, CME has a role in growth and development, hormone signaling and interaction with the environment, nutrient uptake, restrain toxin and pathogen defense. The very well -known immune receptors FLAGELLIN SENSING2 (FLS2, receptor for flg22) or PEPR1 (a receptor for AtPep1) both requires clathrin for their downstream responses. Interference with clathrin function in Arabidopsis completely blocked the endocytosis of PEPR1 and impaired MAPK activation. While, slight decrease in FLS2 endocytosis only affected the activation of a subset of flg22-triggered signaling.
Recent study of the immune receptor PEPR1 that perceives the endogenous peptide AtPep1, showed that endocytosis is required for mitogen-activated protein kinase (MAPK) activation after elicitation with AtPep1. However, because MAPK activation occurs faster than the endocytosis of the main receptor it raised the question if endocytosis of so far unknown signaling components is required for AtPep1-elicited immune responses.
The overall objective of ENDOLOGISTIC is to decode signaling components involved in early MAPK activation and late endocytosis of PEPR1 upon AtPep1 perception using Arabidopsis thaliana as a model plant. The goal of the study was addressed by combining mass spectrometry analysis on clathrin coated vesicles (CCVs) and TurboID based proximity labelling (PL) approach using PEPR1 as bait upon AtPep1 elicitation.
The study of Identification of unknown signals and endocytic components will be major advance in understanding the regulation of immunity and will fill the gap in our understanding of how endocytosis controls immunity in plants.
The first objective aims to identify components of the AtPep1-PEPR1 receptor complex that are rapidly dissociated and internalized by employing Mass spectrometry analysis on CCVs. Initially, I tried to isolate CCVs from Arabidopsis cell culture expressing 35S-PEPR1-GFP upon AtPep1 elicitation at different time points. Further, I confirmed purity of CCVs by immunoblot assay using antibodies against different cellular compartments. Though working with cell cultures always had some organelle contaminants in CCV samples, I preferred using Arabidopsis stable lines expressing RPS5A-PEPR1-GFP. The challenging part with the CCV isolation was 60-80mg of initial material yielded only 15-20ug of purified CCV protein which was not enough for MS analysis. After many initial failures, I succeeded to achieve purified CCV protein for MS analysis. I examined the purity of CCV samples by immunoblot and quality by scanning electron microscopy. With the limited final CCV samples, MS analysis was performed.
The second objective aims to identify the machinery responsible for AtPep1-PEPR1 receptor complex internalization by performing TurboID-based PL-MS. I have used Arabidopsis cell culture expressing p35S-gPEPR1-shortlinkerTurboID or Arabidopsis stable lines expressing pRPS5A-gPEPR1-shortlinkerTurboID in pepr1/pepr2. In the initial phase of project, I tried to optimize experimental conditions with Biotin and AtPep1 treatment. Unfortunately, no interesting proteins were identified in MS data after AtPep1 treatment at 30 min suggesting internalization of activated receptor PEPR1 is much rapid process than labelling; we ended up with no interested proteins. Alternately, the objective was achieved by using Arabidopsis cell culture expressing p35S-gPEPR1-shortlinkerTurboID followed by Biotin treatment only for TurboID-based PL-MS. The MS data was analysed and compared with big data set available at host institute, 128 candidates were identified with >100 enrichment score. Further, 37 candidates were selected based on less frequently found with other baits and predicted localization at PM.
The third objective aims to validate candidates from objective 1 and 2 which was not achieved due to experimental constraint, shift in work schedule and end of project tenure
Furthermore, I presented my work as poster at conference ‘ENPER 2023’ held at Konstanz, Germany. I also presented my work regularly internally and in cluster meetings. A manuscript related to the topic of project is under review and is supported by EU funding.
The second objective aims to identify the machinery responsible for AtPep1-PEPR1 receptor complex internalization by performing TurboID-based PL-MS. I have used Arabidopsis cell culture expressing p35S-gPEPR1-shortlinkerTurboID or Arabidopsis stable lines expressing pRPS5A-gPEPR1-shortlinkerTurboID in pepr1/pepr2. In the initial phase of project, I tried to optimize experimental conditions with Biotin and AtPep1 treatment. Unfortunately, no interesting proteins were identified in MS data after AtPep1 treatment at 30 min suggesting internalization of activated receptor PEPR1 is much rapid process than labelling; we ended up with no interested proteins. Alternately, the objective was achieved by using Arabidopsis cell culture expressing p35S-gPEPR1-shortlinkerTurboID followed by Biotin treatment only for TurboID-based PL-MS. The MS data was analysed and compared with big data set available at host institute, 128 candidates were identified with >100 enrichment score. Further, 37 candidates were selected based on less frequently found with other baits and predicted localization at PM.
The third objective aims to validate candidates from objective 1 and 2 which was not achieved due to experimental constraint, shift in work schedule and end of project tenure
Furthermore, I presented my work as poster at conference ‘ENPER 2023’ held at Konstanz, Germany. I also presented my work regularly internally and in cluster meetings. A manuscript related to the topic of project is under review and is supported by EU funding.
ENDOLOGISTIC project involved state-of-the-art techniques, which improved my technical skills and competencies. I gained valuable expertise in CCV isolation protocol and proximity labelling during my project. The data generated through this project will allow new insights into the field of endocytic regulation in plant immunity.