RESUMO
The mechanism of translocation of RxLR effectors from plant pathogenic oomycetes into the cytoplasm of their host is currently the object of intense research activity and debate. Here, we report the biochemical and thermodynamic characterization of the Phytophthora infestans effector AVR3a in vitro. We show that the amino acids surrounding the RxLR leader mediate homodimerization of the protein. Dimerization was considerably attenuated by a localized mutation within the RxLR motif that was previously described to prevent translocation of the protein into host. Importantly, we confirm that the reported phospholipid-binding properties of AVR3a are mediated by its C-terminal effector domain, not its RxLR leader. However, we show that the observed phospholipid interaction is attributable to a weak association with denatured protein molecules and is therefore most likely physiologically irrelevant.
Assuntos
Fosfolipídeos/metabolismo , Phytophthora infestans/metabolismo , Multimerização Proteica , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Mutação , Fosfolipídeos/química , Phytophthora infestans/genética , Doenças das Plantas/microbiologia , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Solanum tuberosum/microbiologia , Fatores de Virulência/genéticaRESUMO
The oomycete potato late blight pathogen, Phytophthora infestans, and the apicomplexan malaria parasite Plasmodium falciparum translocate effector proteins inside host cells, presumably to the benefit of the pathogen or parasite. Many oomycete candidate secreted effector proteins possess a peptide domain with the core conserved motif, RxLR, located near the N-terminal secretion signal peptide. In the Ph. infestans effector Avr3a, RxLR and an additional EER motif are essential for translocation into host cells during infection. Avr3a is recognized in the host cytoplasm by the R3a resistance protein. We have exploited this cytoplasmic recognition to report on replacement of the RxLR-EER of Avr3a with the equivalent sequences from the intracellular effectors ATR1NdWsB and ATR13 from the related oomycete pathogen, Hyaloperonospora parasitica, and the host targeting signal from the Pl. falciparum virulence protein PfHRPII. Introduction of these chimeric transgenes into Ph. infestans and subsequent virulence testing on potato plants expressing R3a demonstrated the alternative motifs to be functional in translocating Avr3a inside plant cells. These results suggest common mechanisms for protein translocation in both malaria and oomycete pathosystems.
Assuntos
Proteínas de Algas/metabolismo , Motivos de Aminoácidos , Phytophthora/metabolismo , Plasmodium falciparum/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Algas/química , Proteínas de Algas/genética , Sequência de Aminoácidos , Animais , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Oomicetos/genética , Oomicetos/metabolismo , Phytophthora/genética , Phytophthora/patogenicidade , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Solanum tuberosum/microbiologia , Transformação Genética , VirulênciaRESUMO
Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.