Blondeau, K. | bioRxiv Science

bioRxiv2025

Molecular investigation of Rlm3 from rapeseed as a broad-spectrum resistance gene against fungal pathogens producing structurally conserved effectors

Recognition of a pathogen avirulence (AVR) effector protein by its cognate plant resistance (R) protein triggers immune responses that render the plant resistant, representing an efficient disease control strategy. While (AVR) effectors have long been considered species- or genotype-specific, based on a lack of homology in sequence databases, a growing number of studies have now shown that these proteins belong to a limited set of structural families. This is an important finding because it paves the way for the identification or engineering of broad- spectrum R proteins capable of recognizing several members of the same structural family. In the Leptosphaeria maculans / rapeseed (Brassica napus) pathosystem, 13 AVR genes have been cloned, of which four encode effectors belonging to the LARS (Leptosphaeria AviRulence and Supressing) structural family that has also been found in thirteen other phytopathogenic fungi. Homologues of the L. maculans AvrLm3 AVR protein, a LARS family member, have been identified in other fungal species, including an AVR protein from Fulvia fulva called Ecp11-1. We have previously shown that Ecp11-1 is recognized by rapeseed varieties carrying the Rlm3 R gene, and that this recognition is masked by the presence of another LARS AVR gene, AvrLm4-7. In this study, we expanded our characterization of the Rlm3 resistance spectrum to putative effectors from Fusarium oxysporum and Zymoseptoria ardabiliae and showed that one effector from F. oxysporum f. sp. narcissi behaves like Ecp11-1, being recognized by Rlm3, and this recognition also being masked by the presence of AvrLm4-7. We finally investigated which protein regions and amino acids were necessary for the recognition of AvrLm3 and Ecp11-1 by Rlm3. This analysis is a first step towards the identification or engineering of broad- spectrum R proteins that confer protection against multiple phytopathogens through the recognition of structural effector families.

plant biology↗

bioRxiv2020

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9- mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3. Author summaryAn efficient strategy to control fungal diseases in the field is genetic control using resistant crop cultivars. Crop resistance mainly relies on gene-for-gene relationships between plant resistance (R) genes and pathogen avirulence (AVR) genes, as defined by Flor in the 1940s. However, such gene-for-gene relationships can increase in complexity over the course of plant-pathogen co-evolution. Resistance against the plant-pathogenic fungus Leptosphaeria maculans by Brassica napus and other Brassica species relies on the recognition of effector (AVR) proteins by R proteins; however, L. maculans produces an effector that suppresses a subset of these specific resistances. Using a protein structure approach, we revealed structural analogy between several of the resistance-triggering effectors, the resistance-suppressing effector, and effectors from other plant-pathogenic species in the Dothideomycetes and Sordariomycetes classes, defining a new family of effectors called LARS. Notably, cross-species expression of one LARS effector from Fulvia fulva, a pathogen of tomato, in L. maculans resulted in recognition by several resistant cultivars of oilseed rape. These results highlight the need to integrate knowledge on effector structures to improve resistance management and to develop broad-spectrum resistances for multi-pathogen control of diseases.

plant biology↗