The antifungal plant defensin AhPDF1.1b is a beneficial factor involved in adaptive response to zinc overload when it is expressed in yeast cells

Antimicrobial peptides represent an expanding family of peptides involved in innate immunity of many living organisms. They show an amazing diversity in their sequence, structure, and mechanism of action. Among them, plant defensins are renowned for their antifungal activity but various side activities have also been described. Usually, a new biological role is reported along with the discovery of a new defensin and it is thus not clear if this multifunctionality exists at the family level or at the peptide level. We previously showed that the plant defensin AhPDF1.1b exhibits an unexpected role by conferring zinc tolerance to yeast and plant cells. In this paper, we further explored this activity using different yeast genetic backgrounds: especially the zrc1 mutant and an UPRE-GFP reporter yeast strain. We showed that AhPDF1.1b interferes with adaptive cell response in the endoplasmic reticulum to confer cellular zinc tolerance. We thus highlighted that, depending on its cellular localization, AhPDF1.1b exerts quite separate activities: when it is applied exogenously, it is a toxin against fungal and also root cells, but when it is expressed in yeast cells, it is a peptide that modulates the cellular adaptive response to zinc overload

Identification du mode d'action de la défensine AhPDF1.1b dans la tolérance au zinc

Plant defensins belong to the wide family of antimicrobial peptides implied in innate immunity. They are characterised by their richness in cysteines and disulphide bridges as well as their antifungal properties. However, other biological activities have also been described for some plant defensins. This work continues the study of one of these additional activities : the property to confer cellular tolerance to zinc excess when a defensin is expressed in yeast. Arabidopsis halleri defensin AhPDF1.1b is taken as a model to study the mode of action of defensins in this particular activity. Firstly, the possibility that recombinant AhPDF1.1b could chelate zinc was researched. This study has required the production of the recombinant protein. Its production in Pichia pastoris was set up. We showed by fluorimetry and ESI-MS in native conditions that it is indeed possible for the defensin AhPDF1.1b to fix several zinc atoms, particularly after reduction of the protein. However the stability of these complexes as well as zinc coordination remain to be elucidated. Secondly, a study of the cellular response of yeast to the expression of AhPDF1.1b under zinc excess was led. When it is expressed in yeast, AhPDF1.1b is not secreted but accumulated in the endoplasmic reticulum (ER) and in pre-vacuolar compartments, which led us to study the influence of AhPDF1.1b expression and zinc excess on ER functioning. It has been shown that the control of the ER quality, and especially the UPR “Unfolded Protein Response” and the ERAD “ER-associated degradation” boosted by the expression of AhPDF1.1b during a zinc overload what probably triggers the mechanisms of tolerance. Eventually, a transcriptomic analysis on yeast expressing AhPDF1.1b under zinc excess was developed. The expression of defensin alone does not disturb the gene expression profile. However, under zinc excess conditions, it allows a limitation of the deregulations of gene expression provoked by zinc overload. In the conditions of AhPDF1.1b expression and zinc excess, there is an induction of an hypoxic response and surprisingly, a great production of ergosterol.Les défensines de plantes appartiennent à la grande famille des peptides antimicrobiens impliqués dans l’immunité innée. Elles se caractérisent par leur richesse en cystéines et ponts disulfures et par leurs propriétés antifongiques. Cependant, d’autres activités biologiques ont également été décrites chez certaines défensines de plante. Ce travail poursuit l’étude d’une de ces activités annexes et notamment la propriété de conférer une tolérance cellulaire à un excès de zinc lorsqu’une défensine est exprimée dans la levure. La défensine d’Arabidospsis halleri, AhPDF1.1b est prise comme défensine modèle pour étudier le mode d’action des défensines dans cette activité très particulière. Dans un premier temps, la possibilité de chélation du zinc par AhPDF1.1b a été recherchée. Cette étude a nécessité la production de la protéine recombinante. La mise au point de cette production chez Pichia pastoris a été entreprise. Nous avons montré par des études de fluorimétrie et d’ESI-MS en conditions natives qu’il est en effet possible que la défensine AhPDF1.1b recombinante fixe plusieurs atomes de zinc, en particulier après réduction. Cependant la stabilité de ces complexes et la coordination du zinc restent à élucider. Dans un deuxième temps, une étude sur la réponse cellulaire de la levure à l’expression de AhPDF1.1b en condition d’excès de zinc a été menée. Lorsqu’elle est exprimée dans la levure, AhPDF1.1b n’est pas sécrétée mais accumulée dans le réticulum endoplasmique (RE) et des compartiments pré-vacuolaires, ce qui nous a conduit à étudier l’influence de l’expression de AhPDF1.1b et d’un excès de zinc sur le fonctionnement du RE. Il a été montré que le contrôle qualité du RE notamment l’ UPR « Unfolded Protein Response » et l’ ERAD « ER-associated degradation» sont augmentés lors de l’expression de AhPDF1.1b et d’une surcharge en zinc, ce qui concourt au mécanisme de tolérance au zinc. Enfin, dans un troisième temps, une analyse transcriptomique sur des levures exprimant AhPDF1.1b en conditions d’excès de zinc a été développée. L’expression de la défensine seule ne perturbe pas le profil d’expression des gènes. Par contre, en conditions de surcharge en zinc, elle permet de limiter certaines dérégulations de l’expression des gènes provoquées par l’excès de zinc. En conditions d’expression de AhPDF1.1b et d’excès de zinc, il y a une induction d’une réponse hypoxique et de façon surprenante, une surproduction très importante de l’ergostérol. Bien que beaucoup de choses restent à découvrir concernant le mode d’action de la défensine AhPDF1.1b, les résultats présentés dans cette thèse constituent un premier pas vers son élucidatio

Procédé de production d’un composé de la voie de biosynthèse des stérols chez un organisme eucaryote

Procédé de production d’un composé de la voie de biosynthèse des stérols chez un organisme eucaryot

Genome-wide transcriptomic response to zinc overload in the yeast Saccharomyces cerevisiae expressing or not a plant defensin

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The awr gene family encodes a novel class of Ralstonia solanacearum type III effectors displaying virulence and avirulence activities

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophanarginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (¿awr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts

The nuclear magnetic resonance solution structure of the synthetic AhPDF1.1b plant defensin evidences the structural feature within the γ-motif

International audiencePlant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix β-sheet motif and contains the γ-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the γ-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc

The awr gene family encodes a novel class of Ralstonia solanacearum type III effectors displaying virulence and avirulence activities

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophanarginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (¿awr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts