RESUMO
A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.
Assuntos
Quitinases/genética , Proteínas de Ligação a DNA/genética , Pectobacterium carotovorum/fisiologia , Phytophthora/fisiologia , Proteínas de Plantas , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Fatores de Transcrição/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Parede Celular/metabolismo , Quitinases/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/fisiologia , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Dados de Sequência Molecular , Sinais de Localização Nuclear , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Solanum tuberosum/enzimologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Regulação para Cima , Dedos de ZincoRESUMO
A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.
Assuntos
Pectobacterium carotovorum/crescimento & desenvolvimento , Pectobacterium carotovorum/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Solanum tuberosum/microbiologia , Contagem de Colônia Microbiana , Escherichia coli/genética , Vetores Genéticos , Pectobacterium carotovorum/genética , Doenças das Plantas/microbiologia , Moldes GenéticosRESUMO
A potato cysteine protease (cyp) cDNA expressed at an early stage of an incompatible interaction with Phytophthora infestans was isolated. Both the nucleotide and deduced amino acid sequences are highly homologous to those of a tomato cysteine protease, CYP1. Striking protein similarity to all known cathepsins in animals, particularly cathepsin K, was also observed. However, unlike cathepsins, a granulin binding domain is located near the carboxyl terminus of the putative CYP protein. In animals, granulins bind to receptors in the plasma membrane and signal cell growth and division. A ribonuclease protection assay demonstrated that the cyp gene is tightly regulated and is induced 15 h post inoculation with P. infestans in potato leaves either with high field resistance or in which a resistance (R) gene is activated. We conclude that a common signaling pathway is activated in each form of resistance.