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Structure Assisted Investigation of Critical Protein Families Involved in Plant Immunity

Structure Assisted Investigation of Critical Protein Families Involved in Plant Immunity 2017-2019
Acronym: STRASSIST
Project director: Andrei-José Petrescu

This project aims to address a number of structural aspects related to key elements of the plant immune system and its pathogen interactors using a combined approach intricating experimental and computational steps. To this end we intend to build on our previous results in the field and further develop experimental, bioinformatics and molecular modeling methods appropriate for solving the specific problems implied by this proposal.

This project aims to address a number of structural aspects related to key elements of the plant immune system and its pathogen interactors using a combined approach intricating experimental and computational steps. To this end we intend to build on our previous results in the field and further develop experimental, bioinformatics and molecular modeling methods appropriate for solving the specific problems implied by this proposal.

Despite the massive investment in structural biology, results on plant molecular system are scarce. There are not many groups with consistent and systematic interest in structural plant biology. This makes computational techniques very useful but at the same time extremely challenging given the small number of known structures which translates into a very low level or absence of homology.

Over a decade ago we started a long standing collaboration with plant biologists form Wageningen. This led us, in time, to tackle increasingly complex problems related to protein families of the first and second layer of immunity and their pathogen interactors in studies in which we progressively started to intricate computation and experiment, mainly mutational analysis. Such combined strategies allowed us to significantly reduce the dimensionality of the tackled problems but on the other hand it allowed us to validate our computational models. In the end this strategy equates to a structure guided investigation of protein families involved in plant pathogen interaction. Gradually more groups started to show interest in our approach which led nowaday to an extended network of colaborations all over the Europe, Israel & US; and an increasing spread of this approach in the last couple of years.

The paucity of structural data in the field pose significant problems in computational modelling of these systems. This led us to improve and develop de-novo remote homology and ab-initio modelling workflows that we used to generate the first ever R-protein LRR and CC models improved NB models; and also models of some important effectors.

The present proposal intends to further build upon the experience we get in the field by addressing some new systems and new methodology challenges.

We aim here to address new aspects related to the molecular structure, interactions and functioning of two major protein classes of plant immune system: (-a) R-gene products and (-b) the constitutive tandems formed as a first layer of plant immunity in tomato by cell surface receptor-like kinases (RLKs) and receptor like proteins (RLPs), in an attempt to understanding their functioning.

Related to (-a) we intend to further build upon results obtained on the structrue of CC-, NB- and LRR- domains and their interactions, by adressing new problems, as follows:

  • -a1) Quantify the structural diversity of LRR domains of the 130 R proteins that we have recently identified with a software developed by our group and used in analyzing the potato genome in collaboration with Aska Goverse from Wageningen University, member of the Potato Genome Consortium.;
  • -a2) Predict the structure, dynamics and interactions of the CC domains in RGA4/5 R-proteins from rice by combining molecular modeling and simulation with experiment, in collaboration with the group of Thomas Kroj form INRA Montpelier.;
  • -a3) Predict the structure and interactions within the complexes formed by Arabidopsis ZAR1, ZED1 and the avr HopZ1a by combining bioinformatics, molecular modelling with lab experiments in collaboration with the group of Jennifer Lewis from Berkeley University..;

Related to (-b) we intend to combine our experience in glycobiology and biocomputing in aiming to:

  • -b1) Investigate the glycosylation of tomato Cf4/9 and model its constitutive interactions with SOBIR1 - the supressor of BIR1 RLK, in collaboration with Matthieu Joosten from the Laboratory of Phytopathology from Wageningen.;
Principal Investigator Andrei-J Petrescu - IBAR
Senior Researcher Adina Milac - IBAR
PostDoc Researcher Marius Micluta - IBAR
PostDoc Researcher Marius Surleac - IBAR
PostDoc Researcher Cristian A Munteanu - IBAR
PhD Student Eliza Martin - IBAR
PhD Student Laura Georgiana Manica - IBAR

Research on all four topics tackled here might result in high impact outcomes and a predicted publication rate similar to that of our former ID-PCE projects. Funding will allow us in the first place to tackle some sensitive issues in plant biology taking into account the interest showed by our collaborators, known figures in their fields.

Secondly we believe the interest in the computational and experimental techniques developed herein might exceed the plant biology community and reach a broader audience of structural biologists and researchers interested in model driven approach.

Finaly and more importantly a better understanding of the structures underlying plant-pathogen interactions could potentially result in improving the technologies currently in use for preventing effector-mediated pathogenicity in plants, which is of critical importance in developing strategies for durable, broad-range plant resistance.

Results 2017 - 2018

In implementing STRASSIST, during 2017 and 2018, we addressed a number of specific problems related to the overall goals of the project concerning the domain structure of resitance gene products and pattern-recognition receptors (PRR) investigated.

 

Hence, so far:

Along the first line of the proposed research: (a1) quantifying the structural diversiy of LRR domains identified at genome level - we have developed prediction tools for LRR sequence assessment and we clustered the LRR sequences in the structure propensity space.

On the second line of our proposal: (a2) predicting the structure, dynamics and interactions of the RGA-CC domains - we have generated so far the starting structural models of the coiled-coil domain of RGA protein consistent with known experimental and bioinformatic data.

Related to: (a3) predicting the structure and interactions within Arabidopsis ZAR1/ZED1/avr complexes - we have built the predictive frame models of Zar1 domains and Zed1 & avr proteins and probed the complex architecture using the determined constraints and characterised the occupancy of glycosylation sites in gf4.

And finaly related to (b1) the investigation of the first RLN-RNK layer of immunity in tomato - we have characterised the occupancy of glycosylation sites in gf4 and have built the complete predictive models of Cf4/9 & SOBIR1

This work and the methods developed herein resulted so far in two papers published in: Plant Physiol. e-pub ahead of print doi: 10.1104/pp.18.00603 (2018) and Gynecol Oncol Rep. 23: 41-44 (2018), while the manuscript "Genome-wide functional analyses of plant coiled-coil NLR-type pathogen receptors reveal essential roles of their N-terminal domain in ligomerization and immunity" was accepted on 9 October 2018 for publication in PLoS Biology.

 

Publications on issues related to the structure of R-gene products:

Slootweg EJ, Spiridon LN, Martin EC, Tameling WI, Townsend PD, Pomp R, Roosien J, Drawska O, Sukarta OCA, Schots A, Borst JW, Joosten MHAJ, Bakker J, Smant G, Cann MJ, Petrescu AJ, Goverse A. " Distinct roles of non-overlapping surface regions of the coiled-coil domain in the potato immune receptor Rx1.", Plant Physiol, 178(3) 1310-1331 (2018)
doi: 10.1104/pp.18.00603 // PMID: 30194238

Wroblewski T, Spiridon L, Martin EC, Petrescu AJ, Cavanaugh K, Jose-Truco M, Xu H, Gozdowski D, Pawlowski K, Michelmore RW, Takken FLW, " Genome-wide functional analyses of plant coiled-coil NLR-type pathogen receptors reveal essential roles of their N-terminal domain in oligomerization and immunity" PLoS Biol. 16 (12), e2005821 (2018)

Publications based on methods developped in STRASSIST:

Norris EJ, Jones WD, Surleac MD, Petrescu AJ, Destephanis D, Zhang Q, Hamadeh I, Kneisl JS, Livasy CA, Ganapathi RN, Tait DL, Ganapathi MK. " Clonal lineage of high grade serous ovarian cancer in a patient with neurofibromatosis type 1." Gynecol Oncol Rep. 23: 41-44 (2018)