RESUMO
Crown rust, caused by Puccinia coronata f. sp. avenae, is the most damaging disease of oat. Quantification of the disease can be done by visual or digital assessments of diseased leaf area, lesion number, lesion size, and latent period. Laborious measurements of sporulation can also be made. As an alternative to these methods, a new quantitative assay was developed. The method employs simple inoculum application, quantitative sampling from inoculated areas, a closed tube DNA extraction method restricting loss of tissue, and real-time polymerase chain reaction (PCR) using a pathogen-specific TaqMan primers/probe set. Image analyses of genotypes with varying levels of crown rust resistance were compared to fungal DNA (FDNA) estimations by the new assay. The moderately resistant genotype IA98822-2 was distinguished from susceptible genotypes at the seedling stage, and the moderately resistant genotype O × T 107 was distinguished from the resistant cultivar TAM-O-301 at seedling and adult plant stages using FDNA. These separations were not evident with digital image analysis. The new assay also detected fungal development earlier and more rapidly in genotypes with lower levels of resistance. The assay can consistently estimate disease and should be useful for studying many aspects of the crown rust host-pathogen interaction where precise assessment of pathogen development is needed.
RESUMO
Delayed rectifier K+ channels are involved in the electrical activity of all excitable cells. The relationship between native K+ currents recorded from these cells and cloned K+ channel cDNAs has been difficult to ascertain partly because of contradictions in pharmacological characteristics between native and expressed currents. Through the study of the charybdotoxin (CTX) pharmacology of two cloned smooth muscle delayed rectifier K+ channels (cKv 1.2 and cKv1.5) expressed in oocytes, evidence for heterotetramer formation was obtained. We have shown that the presence of even a single CTX-insensitive subunit renders the heterotetrameric channel insensitive to CTX. The two K+ channel clones differ in an amino acid at the mouth of the pore region, which may be in a position to block the access of CTX to its binding site and hence determine CTX sensitivity of the heterotetrameric channel. These results may explain discrepancies reported between native and cloned smooth muscle K+ channels.
Assuntos
Músculo Liso/metabolismo , Canais de Potássio/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Charibdotoxina , Clonagem Molecular , Feminino , Dados de Sequência Molecular , Canais de Potássio/genética , Canais de Potássio/fisiologia , Venenos de Escorpião/metabolismo , Xenopus laevisRESUMO
We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.
Assuntos
Clonagem Molecular , Sistema Digestório/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , DNA/genética , DNA Complementar/genética , Cães , Eletrofisiologia , Feminino , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/fisiologia , Xenopus laevisRESUMO
A cDNA (CSMK1) encoding a delayed rectifier K+ channel of the Kv1.2 class was cloned from canine colonic circular smooth muscle and expressed in Xenopus oocytes. These channels appear to be uniquely expressed in gastrointestinal muscles and may participate in the electrical slow wave activity. Functional expression of CSMK1 in Xenopus oocytes demonstrated a K+ current that activated in a voltage-dependent manner upon depolarization. This current was highly sensitive to 4-aminopyridine (IC50, 74 microM). A low-conductance K+ channel was identified in inside-out patches from oocytes injected with CSMK1. This channel displayed a linear current-voltage relation with a slope conductance of 14 pS. The channels were blocked in a concentration-dependent manner by 4-aminopyridine. Northern blot analysis demonstrated that CSMK1 is expressed in a wide variety of gastrointestinal smooth muscles. Portal vein, renal artery, and uterus do not express CSMK1, suggesting that, among smooth muscles, expression of this K+ channel may be restricted to gastrointestinal smooth muscles. CSMK1 is 91% homologous to RAK, a delayed rectifier K+ channel cloned from rat heart, but displays unique pharmacological properties and tissue distribution.