RESUMO
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
The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants.
Assuntos
Expressão Gênica , Proteínas de Plantas , Proteínas Secretadas Inibidoras de Proteinases , Solanum tuberosum/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Solanum tuberosum/genéticaRESUMO
A balanced proteolytic activity in the epidermis is vital to maintain epidermal homoeostasis and barrier function. Distinct protease-inhibitor systems are operating in different epidermal layers. In the uppermost layer, the stratum corneum, kallikrein-like proteases and their inhibitors are responsible for desquamation of the cornified keratinocytes, thus regulating the integrity of the epidermal barrier. Following discovery and characterisation of the human multidomain inhibitor LEKTI (lympho-epithelial Kazal-type-related inhibitor, encoded by hspink5), several new members of the Kazal-type inhibitor family have been identified. Here we describe expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2. Its expression was analysed by RT-PCR and immunohistochemistry revealing organ-specific pattern with high level of expression in the epidermis and several epithelia including the stomach, kidney and uterus. In addition, mSPINK12 expression in the epidermis of skin at footpads, where stratification is markedly pronounced, was several folds higher than in the abdominal epidermis. mSPINK12 mRNA levels were not affected by any cytokines tested while treatment of primary murine keratinocytes with the combination of calcium and sorbitol resulted in a strong increase in its mRNA. It appears that mspink12 is especially expressed in the epidermal areas with thick skin and that its regulation generally responds to differentiation signals. mrSPINK12 shows an inhibitory activity against murine keratinocyte-derived trypsin-like proteolytic activity, thus, the protein does appear orthologous to human LEKTI2 and may play an role in the regulation of epithelial cell functions.