Expanding the repertoire of STructurally conserved AviRulence proteins in plant pathogenic fungi to propose knowledge-driven plant resistance management – STARlep

The STARlep project is organized in four work-packages: structural characterisation of AvrLm effectors and classification into structural families by looking for structural analogues among L. maculans effectors (WP1), in-depth analysis of the interactions between AVR proteins and their cognate R proteins (WP2), determination of the plant proteins and cellular processes targeted by the AvrLm effector families, and physical understanding of effector-target interactions (WP3), identification of resistances recognizing effectors with contrasted structural patterns and functional mechanisms (WP4). The originality and strength of the STARlep project lie in its interdisciplinarity, involving three teams with internationally acknowledged expertise in the complementary fields of fungal genomics / fungal effector biology (Partner 1) and structural biology (Partners 2 and 3) and a private company developing screening of Brassica material to identify new resistance sources (Partner 4).

- Production and determination of the 3D structure of an effector of L. maculans alone or in interaction with its plant target.
- Identification of effectors from Fusarium oxysporum and Cladosporium fulvum that can, like the L. maculans effector AvrLm3, be recognized by the rapeseed resistance protein Rlm3. Identification of amino acids and protein regions in AvrLm3 and its C. fulvum homologue, Ecp11-1, that are required for recognition by Rlm3. These results will soon be published.
- Identification of L. maculans isolates displaying a presence/absence polymorphism for new effectors not yet characterized as avirulence proteins and which can be used to screen new resistances.
- Identification and start of characterization of several new potential sources of resistance to L. maculans. Definition of strategies (identification of virulent strains, crosses between strains showing a differential phenotype on these resistances, or sequencing and annotation of an avirulent strain) to identify the corresponding avirulence genes.

The STARlep project will generate knowledge on structurally conserved families of effectors, and interaction with cognate R genes and plant targets. STARlep has the potential to impulse the development of resistant crops and of novel strategies to control fungal diseases. A major aim of STARlep is to identify new resistance sources allowing recognition of new effector structural families. The knowledge acquired on structural families of AvrLm effectors will also help us to propose strategies aiming at alternating or pyramiding resistance genes recognizing different classes of effectors, with different types of protein structures. Finally, we will also be able to identify plant targets altered by effectors that could be new targets to control plant diseases.

Results from the project were presented in one national and one interantional conferences:
- Talbi N., Lazar N., Mesarich CH, Pakzad S, Petit-Houdenot Y, Li de la Sierra-Gallay I, Zélie E, Blondeau K, Gracy J, Ollivier B, Blaise F, Rouxel T, Balesdent MH, Idnurm A, van Tilbeurgh H and Fudal I. Characterization of epistatic interactions within a family of structurally conserved fungal effectors and of their broad-spectrum recognition by a plant resistance protein. Integrated Control in Oilseed Crops meeting. 17-18 mai 2022, virtual conference.
- Gallay I., Blondeau K., Lazar N, Sizun C, Fudal I., van Tilbeurgh H. Structural studies of the complex between the effector protein AvrLm1 and the Brassica napus MAP kinase Mpk9. MoDip Network Meeting. Septembre 2021, Lauret, France
A publication will be submitted soon:
- Talbi N., Pakzad S., Blaise F., Ollivier B., Rouxel T., Balesdent MH, Mesarich C, Blondeau K., Lazar N., van Tilbeurgh H., Fudal I. (in prep.). Molecular investigation of the broad-spectrum resistance Rlm3 from oilseed rape against fungal pathogens

Fungal pathogens represent a recurrent threat to human health and agriculture and possess extreme adaptive abilities. A major environment-friendly strategy to control fungal diseases is genetic control using naturally resistant crop cultivars. Crop resistance mainly relies on recognition of fungal effectors, key elements of pathogenesis having a dual role in fungal-plant interactions, both targeting plant components and being targeted by resistance (R) proteins and then termed avirulence (AVR) proteins. Breeding cultivars carrying major R genes against pathogens is a common and powerful tool to control diseases. However, the massive deployment of a single source of resistance in the fields exerts a strong selection pressure against avirulent pathogens and the resistance source can be rapidly overcome. There is then an urgent need to improve R gene management to increase their efficiency and durability.
The fungus Leptosphaeria maculans causes stem canker of oilseed rape (Brassica napus). The main strategy to control L. maculans is genetic control. However, sources of specific resistance are rare in oilseed rape and studies aim either at identifying new resistance sources to diversify available resources or to optimize the sustainability of available resistances by alternating and/or pyramiding resistances. Partner 1 and 2 of the project have recently determined the 3D structure of four L. maculans AVR effectors (called AvrLm). Surprisingly, three of them are part of a structural family including 13 effectors from L. maculans but also proteins from other plant pathogenic fungi. Our data raise crucial questions, whose clarification could improve resistance gene management: (i) Do secreted effectors, despite their lack of sequence similarity, belong to a limited set of structurally conserved protein families? (ii) Are members of a same effector family targeting the same or similar plant proteins and cellular processes? (iii) Could members of a same effector family be recognized by the same R proteins?
The STARlep project aims at exploring structural and functional diversity among L. maculans avirulence proteins to propose knowledge-driven plant resistance management. It is organized in four work-packages: structural characterisation of AvrLm effectors and classification into structural families by looking for structural analogues among L. maculans effectors (WP1), in-depth analysis of the interactions between AVR proteins and their cognate R proteins (WP2), determination of the plant proteins and cellular processes targeted by the AvrLm effector families, and physical understanding of effector-target interactions (WP3), identification of resistances recognizing effectors with contrasted structural patterns and functional mechanisms (WP4). The originality and strength of the STARlep project lie in its interdisciplinarity, involving three teams with internationally acknowledged expertise in the complementary fields of fungal genomics / fungal effector biology (Partner 1) and structural biology (Partners 2 and 3) and a private company developing screening of Brassica material to identify new resistance sources (Partner 4).
The STARlep project will generate knowledge on structurally conserved families of effectors, and interaction with cognate R genes and plant targets. STARlep has the potential to impulse the development of resistant crops and of novel strategies to control fungal diseases. A major aim of STARlep is to identify new resistance sources allowing recognition of new effector structural families. The knowledge acquired on structural families of AvrLm effectors will also help us to propose strategies aiming at alternating or pyramiding resistance genes recognizing different classes of effectors, with different types of protein structures. Finally, we will also be able to identify plant targets altered by effectors that could be new targets to control plant diseases.