Highly Sensitive End-Point PCR and SYBR Green qPCR Detection of Phymatotrichopsis omnivora, Causal Fungus of Cotton Root Rot.

Phymatotrichopsis omnivora, the causal pathogen of cotton root rot, is a devastating ascomycete that affects numerous important dicotyledonous plants grown in the southwestern United States and northern Mexico. P. omnivora is notoriously difficult to isolate from infected plants; therefore methods for accurate and sensitive detection directly from symptomatic and asymptomatic plant samples are needed for disease diagnostics and pathogen identification. Primers were designed for P. omnivora based on consensus sequences of the nuclear ribosomal internal transcribed spacer (ITS) region of geographically representative isolates. Primers were compared against published P. omnivora sequences and validated against DNA from P. omnivora isolates and infected plant samples. The primer combinations amplified products from a range of P. omnivora isolates representative of known ITS haplotypes using standard end-point polymerase chain reaction (PCR) methodology. The assays detected P. omnivora from infected root samples of cotton (Gossypium hirsutum) and alfalfa (Medicago sativa). Healthy plants and other relevant root pathogens did not produce PCR products with the P. omnivora-specific primers. Primer pair PO2F/PO2R was the most sensitive in end-point PCR assays and is recommended for use for pathogen identification from mycelial tissue and infected plant materials when quantitative PCR (qPCR) is not available. Primer pair PO3F/PO2R was highly sensitive (1 fg) when used in SYBR Green qPCR assays and is recommended for screening of plant materials potentially infected by P. omnivora or samples with suboptimal DNA quality. The described PCR-based detection methods will be useful for rapid and sensitive screening of infected plants in diagnostic laboratories, plant health inspections, and plant breeding programs.

Comparative fine structural distribution of endopeptidase 24.15 (EC3.4.24.15) and 24.16 (EC3.4.24.16) in rat brain.

Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.

Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.