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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - IMMUNO-PEPTALK (Regulation of plant receptor kinase-mediated immunity by endogenous peptides and their receptors)

Teaser

Summary

Multicellular organisms employ cell-surface receptors to survey the extracellular environment and adjust to changing conditions. Our work aims at deciphering the molecular mechanisms that control the function of these important sensors. In addition to providing important fundamental knowledge, this research ultimately has also the potential to inform strategies to develop better crops with increased yield and resilience to an ever-changing environment affected by global climate change. We are particularly interested to understand the regulatory function of some receptor kinases on others, and the role played by endogenous peptides in controlling such processes. As these receptors are embedded into the plasma membrane, we are interested to understand the role that the dynamic nanoscale organization of the plasma membrane plays in the regulation of receptor kinase complex assembly. Similarly, as plant cells are surrounded by a cell wall, we aim to understand the role played by the cell wall itself in regulating the perception of the external environment, as well as the mechanisms underlying the perception of cell wall properties. Our overall working model is that receptor kinases are an essential part of the continuum constituted by the external environment, the cell wall and the plasma membrane (and ultimately the interior of the cell), and that this continuum is shaped by the perception of endogenous peptides as part of a highly dynamic and interconnected biological system.

Work performed

Objective 1:
Our results ruled our different hypothesis to explain the mechanisms of S1P regulation. We are therefore testing via proteomics If PAMP-induced S1P-mediated cleavage of PRORALF23 is linked to phosphorylation.

Using synthetic peptides for 32 out of the 35 RALFs, we identified 23 RALF peptides showing some level of bioactivity in different assays.
We will soon start to test the sensitivity of single and higher-order mlrk mutants to RALF peptides to begin matching RALFs with cognate MLRKs. To complement this genetic approach, we are generating a RALF peptide-array that will be screened with purified recombinant MLRK ectodomains for specific binding.
RALFs with strong bioactivity and their binding MLRKs will be prioritized for future detailed biochemical, genetic and physiological characterization.
In the meantime, we have started to characterize the genetic role of MAPLE and related MLRKs in immunity.

In collaboration with the group of Jijie Chai, we have recently determined the molecular and structural basis of RALF23 perception by a heterotypic complex involving the GPI-anchored protein LLG1 and FER (Xiao, Stegmann, Han et al., Nature 2019).
Cell wall-associated LRX proteins have also recently been shown to bind RALF peptides. We identified LRXs that are also genetically required for RALF23 responses. We are currently assessing the molecular mechanisms underlying RALF23 binding to LRXs, and testing if LRXs associate with the LLG-FER complex.

We have identified that the S1P-RALF23-FER module controls the function of additional LRR-RKs involved in growth or development, such as BRI1 and PSKR1/2 (Gronnier et al., in preparation).

Objective 2

We could now determine that several other receptor kinases, including FER, BRI1, ERECTA and PSKR1, also localize to PM nanoclusters (Gronnier et al., in preparation).
We have recently completed the molecular cloning of chimeras consisting of distinct domains of FLS2 and BRI1 to determine which protein domain(s) contribute to the specific nanocluster localization of these proteins.

We observed that FER is not required per se for the localization of FLS2 into PM nanoclusters. Instead, we determined that FER regulates the lateral mobility of FLS2, and similar observations were made upon RALF23 treatment (Gronnier et al., in preparation) â€“ consistent with the inhibitory role of this peptide on FER function. We are therefore proposing that the reduced flg22-induced FLS2-BAK1 complex formation in fer mutants could be a consequence of this increased lateral mobility â€“ a model that we are currently testing further.

We are currently generating single and higher-order CRISPR mutants, as well as over-expression lines, for the different subgroups of REMs, and for all HIRs and FLOTs.

The functional characterization of REM, FLOT and HIR mutant and over-expressing lines will start as soon as we will have genetically characterized the required material.

To help setting up the stage for the reconstitution of the FLS2 complex in nanodiscs, we have successfully implemented a HEK293T cell expression system, towards initially testing if (co-)expression of different proteins in a living heterologous system can lead to measurable biochemical and physiological outputs. Once this series of experiments will be completed, we will move forward with the proposed experiments in nanodiscs.

Objective 3

We identified a novel family of plant peptides that induced immune responses in a MIK2-dependent manner. We also obtained biochemical data showing that treatment with these peptides induces MIK2-BAK1 association in planta. Furthermore, in collaboration with Julia Santiago (Lausanne), we could show that these peptides directly bind to the extracellular domain of MIK2 with high nanomolar affinity. This family of peptides therefore represents the long sought-after ligands for MIK2. We are currently generating single and higher-order CRISPR mutants for the corresponding peptide-encoding genes to

Final results

A number of results have been achieved with some of them being already published or on their way to publication. Notably, we have defined the molecular mechanisms of RALF perception, and have also recently identified the long sought-after MIK2 ligands.
We have developed a number of novel genetic materials for most objectives, which will also be highly beneficial to the wider community. We have also successfully implemented novel super-resolution techniques to study the nanoscale dynamics of receptor kinases at the plasma membrane, have implemented the use of human cells to study the function and organization of plant receptor kinase complexes.