RESUMO
Plant 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 ß-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.
Assuntos
Proteínas de Arabidopsis/química , Defensinas/química , Fungicidas Industriais/química , Modelos Moleculares , Adaptação Fisiológica , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/farmacologia , Quelantes/química , Quelantes/metabolismo , Quelantes/farmacologia , Sequência Conservada , Defensinas/metabolismo , Defensinas/farmacologia , Fungicidas Industriais/metabolismo , Fungicidas Industriais/farmacologia , Fusarium/efeitos dos fármacos , Fusarium/fisiologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Prolina/química , Conformação Proteica , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Técnicas de Síntese em Fase Sólida , Zinco/metabolismo , Zinco/toxicidadeRESUMO
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-tryptophan-arginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (Δawr1-5) 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.
Assuntos
Genes Bacterianos/genética , Família Multigênica/genética , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/patogenicidade , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Morte Celular , Regulação da Expressão Gênica de Plantas/genética , Teste de Complementação Genética , Genômica , Interações Hospedeiro-Patógeno , Filogenia , Folhas de Planta/microbiologia , Pseudomonas syringae/genética , RNA de Plantas/genética , Ralstonia solanacearum/fisiologia , Deleção de Sequência , Nicotiana/microbiologia , Virulência/genéticaRESUMO
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.