Li de la Sierra-Gallay, I. | bioRxiv Science

bioRxiv2022

Polymerization cycle of actin homolog MreB from a Gram-positive bacterium

In most rod-shaped bacteria, the actin homologue MreB is an essential component of the protein complex effecting cell wall elongation. The polymerization cycle and filament properties of eukaryotic actin have studied for decades and are well characterized. However, purification and in vitro work on MreB proteins have proven very difficult. Current knowledge of MreB biochemical and polymerization properties remains limited and is based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized filaments and the first 3D-structure of MreB from a Gram-positive bacterium. We have purified MreB from the thermophilic Geobacillus stearothermophilus and shown that it forms straight pairs of protofilaments in vitro, and that polymerization depends on the presence of both lipids and nucleotide triphosphate. Two spatially close short hydrophobic sequences mediate membrane anchoring. Importantly, we demonstrate that unlike eukaryotic actin, nucleotide hydrolysis is a prerequisite for MreB interaction with the membrane, and that binding to lipids then triggers polymerization. Based on our results, we propose a molecular model for the mechanism of MreB polymerization.

biochemistry↗

bioRxiv2022

The LH-DH module of the bacterial replicative helicases is the common binding site for DciA and other helicase loaders

During the initiation step of bacterial genome replication, replicative helicases depend on specialized proteins for their loading onto oriC. DnaC and DnaI were the first loaders characterized. However, most bacteria do not contain any of these genes, which are domesticated phage elements that replaced the ancestral and unrelated loader gene dciA several times during evolution. To understand how DciA assists the loading of DnaB, we determined the crystal structure of the complex from Vibrio cholerae, in which two VcDciAs interact with a dimer of VcDnaB, without changing its canonical structure. Our data showed that the VcDciA binding site on VcDnaB is the conserved module formed by the linker helix LH of one monomer and the determinant helix DH of the second one. Interestingly, DnaC from Escherichia coli also targets this module onto EcDnaB. Thanks to their common target site, we showed that VcDciA and EcDnaC could be functionally interchanged in vitro, despite sharing no structural similarities. This is a milestone in understanding the mechanism employed by phage helicase loaders to hijack bacterial replicative helicases during evolution.

molecular 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↗