Olive Twig and Branch Dieback: Etiology, Incidence, and Distribution in California.

Eighteen different fungal species were isolated from symptomatic wood of olive trees (Olea europaea) affected by twig and branch dieback in California and identified by means of morphological characters and multigene sequence analyses of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequence of the ß-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). These species included Diaporthe viticola, Diatrype oregonensis, Diatrype stigma, Diplodia mutila, Dothiorella iberica, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Phomopsis sp. group 1, Phomopsis sp. group 2, and Schizophyllum commune, which are for the first time reported to occur in olive trees; Eutypa lata, Neofusicoccum luteum, Neofusicoccum vitifusiforme, and Phaeoacremonium aleophilum, which are for the first time reported to occur in olive trees in the United States; and Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum mediterraneum, and Trametes versicolor, which have been previously reported in olive trees in California. Pathogenicity studies conducted in olive cultivars Manzanillo and Sevillano showed N. mediterraneum and Diplodia mutila to be the most virulent species and Diatrype stigma and D. oregonensis the least virulent when inoculated in olive branches. Intermediate virulence was shown for the rest of the taxa. This study demystifies the cause of olive twig and branch dieback and elucidates most of the fungal pathogens responsible for this disease in California.

Expression of rab GTP-binding proteins during oligodendrocyte differentiation in culture.

Oligodendrocytes (OLs) synthesize and transport vast amounts of proteins and lipids from the cell body to the morphologically and biochemically distinct domains of the myelin membrane. From our prediction that regulators of vesicular transport should be up-regulated at the time of myelin production, we hypothesized that the up-regulated and unidentified small GTPases found by Huber et al. [1994a] may be Rab proteins. We have analyzed the mRNA expression of rabs in OLs, and have detected rabs 10, 11b, 18, 24, 26, and 28 in addition to rabs that were found previously. Our data show that among the Rabs so far detected during differentiation, only Rabs 5a and 8a exhibited up-regulation in addition to the previously published Rab3a (Madison et al. [1999], J. Neurochem. 72:988-998). We discuss the limited extent of up-regulation of rabs in the context of the presumed necessity for an increase in Rab activity during myelin assembly.

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes.

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.

Transient transfection of oligodendrocyte progenitors by electroporation.

The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10-15% of the surviving cells, and provides approximately 10(4) surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.

TPO1, a member of a novel protein family, is developmentally regulated in cultured oligodendrocytes.

Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5-kb mRNA that is strongly up-regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5'-termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51.8 kDa with a predicted pI of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subcellular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myelin biogenesis.

Cloning and characterization of MVP17: a developmentally regulated myelin protein in oligodendrocytes.

The remarkable quantities of myelin membrane produced by oligodendrocytes has led us to examine the mechanisms involved in the sorting and transport of proteins and lipids during myelinogenesis. Noting that it has been proposed that proteins destined for the apical surface of polarized epithelial cells co-cluster with glycolipid-rich microdomains during sorting and transport from the trans-Golgi network (Simons and van Meer: Biochemistry 27:6197-6202, 1988; Simons and Wandinger-Ness: Cell 62:207-210, 1990), we hypothesized that the glycolipid-rich oligodendrocytes may adopt this mechanism for myelinogenesis. Protein-lipid complexes from oligodendrocytes and myelin were isolated utilizing detergent insolubility and two-dimensional gel electrophoresis. A developmentally regulated protein, MVP17 (myelin vesicular protein of 17 kDa), was identified. Microsequencing of the N-terminal peptide revealed a high homology to human T-cell MAL protein (Alonso and Weissman: Proc Natl Acad Sci USA 84:1997-2001, 1987). The corresponding MVP17 cDNA was isolated from an oligodendrocyte cDNA library. The predicted protein sequence showed 88.9% identity with MAL, and the hydrophobicity profile suggested four transmembrane domains. In vitro translation demonstrated a signal at the deduced Mr of approximately 17 kDa. Northern analyses indicated that MVP17 mRNA expression is restricted to brain and kidney and that this expression is up-regulated in oligodendrocytes and brain during the period of active myelination. These data suggest that MVP17 is involved in myelin biogenesis and/or myelin function.