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1.
Plant Physiol ; 162(3): 1510-28, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23660837

RESUMO

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.


Assuntos
Proteínas de Plantas/química , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/química , Solanum tuberosum/imunologia , Sequência de Aminoácidos , Animais , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Repetições Ricas em Leucina , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Potexvirus/metabolismo , Potexvirus/patogenicidade , Proteínas/química , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Sequências Repetitivas de Aminoácidos , Solanum tuberosum/microbiologia , Solanum tuberosum/virologia , Nicotiana/genética , Tylenchoidea/metabolismo , Tylenchoidea/patogenicidade
2.
Phytopathology ; 99(2): 194-202, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19245333

RESUMO

Plant-parasitic cyst nematodes secrete cell wall modifying proteins during their invasion of host plants. In this study, we used a monoclonal antibody to immunopurify and to sequence the N terminus of the most abundant cellulases in stylet secretions of preparasitic juveniles of Globodera rostochiensis. The N-terminal amino acid sequence perfectly matched the sequence of an expressed sequence tag of two nearly identical genes, named Gr-eng3 and Gr-eng4, which show relatively low similarity with the previously identified Gr-eng1 and Gr-eng2 (i.e., 62% similarity and 42% identity). The recombinantly produced proteins from Gr-eng3 and Gr-eng4 demonstrated specific activity on carboxymethylcellulose, indicating that these genes encode active cellulases. To date, the cellulases in cyst nematodes are comprised of three possible domain structure variants with different types of ancillary domains at the C terminus of the glycosyl hydrolase family 5 (GHF5) domain. We used Bayesian inference to show that the phylogeny of the GHF5 domain based on currently available data suggest that the extant nematode cellulases arose through reshuffling of the GHF5 domain with different types of ancillary domains as relatively independent units. Knocking-down Gr-eng3 and Gr-eng4 using RNA interference resulted in a reduction of nematode infectivity by 57%. Our observations show that the reduced infectivity of the nematodes can be attributed to poor penetration of the host's root system at the onset of parasitism.


Assuntos
Celulases/genética , Celulases/metabolismo , Nematoides/enzimologia , Sequência de Aminoácidos , Animais , Celulases/química , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/parasitologia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase , Solanum tuberosum/parasitologia
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