The Crystal Structure of Necrosis- and Ethylene-Inducing Protein 2 from the Causal Agent of Cacao's Witches' Broom Disease Reveals Key Elements for Its Activity

Autor(es):Mostrar menos -

Zaparoli, Gustavo ^{[1, 2]} ; de Oliveira Barsottini, Mario Ramos ^{[1, 2]} ; de Oliveira, Juliana Ferreira ^[1] ; Dyszy, Fabio ^[3] ; Pereira Lima Teixeira, Paulo Jose ^[2] ; Barau, Joan Grande ^[2] ; Garcia, Odalys ^[2] ; Costa-Filho, Antonio Jose ^[3] ; Berteli Ambrosio, Andre Luis ^[1] ; Guimaraes Pereira, Goncalo Amarante ^{[1, 2]} ; Gomes Dias, Sandra Martha ^[1]

Afiliação do(s) autor(es)

^[1] Assoc Brasileira Tecnol Luz Sincrotron, Lab Nacl Biociencias LNBio, BR-13083970 Campinas, SP - Brazil

^[2] IB UNICAMP, Dept Genet & Evolucao, BR-13083970 Campinas, SP - Brazil

^[3] Univ Sao Paulo, Grp Biofis Mol Sergio Mascarenhas, Inst Fis Sao Carlos, BR-13560970 Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOCHEMISTRY; v. 50, n. 45, p. 9901-9910, 2011.
• Citações Web of Science: 17

Resumo

The necrosis- and ethylene-inducing peptide 1 (NEP1)-like proteins (NLPs) are proteins secreted from bacteria, fungi and oomycetes, triggering immune responses and cell death in dicotyledonous plants. Genomic-scale studies of Moniliophthora perniciosa, the fungus that causes the Witches' Broom disease in cacao, which is a serious economic concern for South and Central American crops, have identified five members of this family (termed MpNEP1-5). Here, we show by RNA-seq that MpNEP2 is virtually the only NLP expressed during the fungus infection. The quantitative real-time polymerase chain reaction results revealed that MpNEP2 has an expression pattern that positively correlates with the necrotic symptoms, with MpNEP2 reaching its highest level of expression at the advanced necrotic stage. To improve our understanding of MpNEP2's molecular mechanism of action, we determined the crystallographic structure of MpNEP2 at 1.8 angstrom resolution, unveiling some key structural features. The implications of a cation coordination found in the crystal structure were explored, and we show that MpNEP2, in contrast to another previously described member of the NLP family, NLPpya from Pythium aphanidermatum, does not depend on an ion to accomplish its necrosis- and electrolyte leakage-promoting activities. Results of site-directed mutagenesis experiments confirmed the importance of a negatively charged cavity and an unforeseen hydrophobic beta-hairpin loop for MpNEP2 activity, thus offering a platform for compound design with implications for disease control. Electron paramagnetic resonance and fluorescence assays with MpNEP2 performed in the presence of lipid vesicles of different compositions showed no sign of interaction between the protein and the lipids, implying that MpNEP2 likely requires other anchoring elements from the membrane to promote cytolysis or send death signals. (AU)

• Processo FAPESP: 10/51891-4 - Smolbnet 2.0: determinação por cristalografia e ressonância magnética nuclear, da estrutura das proteínas das famílias NEP (Necrosis e Ethylene inducing Peptides) e taumatina, além da oxidase alternativa dos fungos Moniliopthora perniciosa e Moniliopthora roreri
• Beneficiário: Juliana Ferreira de Oliveira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 10/51884-8 - SMOLBnet 2.0: estudos estruturais de proteínas chave para as doenças fúngicas do cacau vassoura-de-bruxa e monilíase: desenvolvimento de estratégias de controle e entendimento de modelos de patogenicidade
• Beneficiário: Andre Luis Berteli Ambrosio
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 08/50995-0 - Caracterização dos mecanismos de ação de duas classes de proteínas indutoras de necrose (CPs e NLPs), presentes em fungos do gênero Moniliophthora, patogênicos de cacau
• Beneficiário: Gustavo Henrique Alcalá Zaparoli
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 09/50119-9 - Estudo integrado e comparativo de três doenças fúngicas do cacau: vassoura-de-bruxa, monilíase e mal do facão, visando à compreensão de mecanismos de patogenicidade para o desenvolvimento de estratégias de controle
• Beneficiário: Gonçalo Amarante Guimarães Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Temático