RESUMO
Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal ß-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal ß-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/classificação , Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses.
Assuntos
Autofagia , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Phytophthora infestans/patogenicidade , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Solanum tuberosum/microbiologia , Doenças das Plantas/imunologia , Ligação Proteica , Solanum tuberosum/imunologiaRESUMO
Accelerated gene evolution is a hallmark of pathogen adaptation following a host jump. Here, we describe the biochemical basis of adaptation and specialization of a plant pathogen effector after its colonization of a new host. Orthologous protease inhibitor effectors from the Irish potato famine pathogen, Phytophthora infestans, and its sister species, Phytophthora mirabilis, which is responsible for infection of Mirabilis jalapa, are adapted to protease targets unique to their respective host plants. Amino acid polymorphisms in both the inhibitors and their target proteases underpin this biochemical specialization. Our results link effector specialization to diversification and speciation of this plant pathogen.
Assuntos
Mirabilis/enzimologia , Mirabilis/microbiologia , Phytophthora infestans/patogenicidade , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/microbiologia , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Evolução Molecular , Filogenia , Phytophthora infestans/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Polimorfismo Genético , Proteínas Secretadas Inibidoras de Proteinases/classificação , Proteínas Secretadas Inibidoras de Proteinases/genética , Especificidade da EspécieRESUMO
Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8(R469A;D470A) resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.