Mitotic instability in benomyl-resistant transformants of a fluffy strain of Neurospora crassa

The isolation of the beta-tubulin gene from a benomyl-resistant Neurospora crassa strain (Orbach et al. 1986 Mol. Cell. Biol. 6:2452-2461) has provided a dominant selectable marker usable in transformation experiments with N. crassa

New mutants of Neurospora crassa highly resistant to the microtubule inhibitor benomyl

Mutants of N. crassa highly resistant to benomyl with respect to hyphal growth were obtained by mutagenizing wild type strain St. Lawrence 74A or mutant Bml 511(r) which is moderately resistant to the fungicide. One of these, strain E1-91 that has a mutation mapping at the Bml (beta-tubulin) locus, showed temporary sensitivity to the benzimidazole

A genetically validated approach for detecting inorganic polyphosphates in plants

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Polyphosphates represent important stores of phosphate and energy, and are abundant in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP-binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabidopsis cells by heterologous expression of Escherichia coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme leads to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1-expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron-dense structures in transmission electron microscopy sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalcisomes in the green alga Chlamydomonas reinhardtii11Nsci

The Arabidopsis mature endosperm promotes seedling cuticle formation via release of sulfated peptides.

In Arabidopsis mature seeds, the onset of the embryo-to-seedling transition is nonautonomously controlled, being blocked by endospermic abscisic acid (ABA) release under unfavorable conditions. Whether the mature endosperm governs additional nonautonomous developmental processes during this transition is unknown. Mature embryos have a more permeable cuticle than seedlings, consistent with their endospermic ABA uptake capability. Seedlings acquire their well-sealing cuticles adapted to aerial lifestyle during germination. Endosperm removal prevents seedling cuticle formation, and seed reconstitution by endosperm grafting onto embryos shows that the endosperm promotes seedling cuticle development. Grafting different endosperm and embryo mutant combinations, together with biochemical, microscopy, and mass spectrometry approaches, reveal that the release of tyrosylprotein sulfotransferase (TPST)-sulfated CIF2 and PSY1 peptides from the endosperm promotes seedling cuticle development. Endosperm-deprived embryos produced nonviable seedlings bearing numerous developmental defects, not related to embryo malnutrition, all restored by exogenously provided endosperm. Hence, seedling establishment is nonautonomous, requiring the mature endosperm