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Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora.

Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of ⌬rrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, ⌬rrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, ⌬rrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in ⌬rrg-1 strains. OS-2-phosphate levels are also low in ⌬rrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1 D921N allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway. INTRODUCTIONTwo-component regulatory systems are signal transduction pathways found in bacteria, Archaea, slime molds, fungi, and plants (for reviews, see Wolanin et al., 2002). Twocomponent systems have been implicated in responses to light, osmolarity, cellular redox status, nutrient and oxygen levels, virulence, and other factors (for review, see Wolanin et al., 2002;Borkovich et al., 2004). A basic two-component system consists of a histidine kinase and a response regulator (for review, see West and Stock, 2001;Wolanin et al., 2002). Histidine kinases autophosphorylate on a conserved histidine residue in the histidine kinase domain. The phosphoryl group is then transferred to an aspartate residue in the receiver domain on the response regulator. Complex two-component signaling pathways (also termed phosphorelays) contain hybrid histidine kinases that have both a histidine kinase domain and a response regulator receiver domain in the same protein, thus facilitating an intramolecular phosphate transfer reaction. The phosphate group on the aspartate residue is transferred to a histidine phosphotransfer (HPT) protein. Subsequently, the histidine phosphotransfer protein phosphorylates the receiver domain on a response regulator. Phosphorelays are the major two-component pathways in eukaryotic systems; however, two-component systems are not present in animals, making them ideal candidate targets for antimicrobial drugs (for review, see Alex et al., 1998;Wolanin et al., 2002;Borkovich et al., 2004).Many response regulators contain a ...

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Circadian clocks are composed of central oscillators, input pathways that transduce external information to the oscillators, and output pathways that allow the oscillators to temporally regulate cellular processes. Little is known about the output pathways. In this study, we show that the Neurospora crassa osmosensing MAPK pathway, essential for osmotic stress responses, is a circadian output pathway that regulates daily rhythms in the expression of downstream genes. Rhythmic activation of the highly conserved stress-activated p38-type MAPK [Osmotically Sensitive-2 (OS-2)] by the N. crassa circadian clock allows anticipation and preparation for hyperosmotic stress and desiccation that begin at sunrise. These results suggest a conserved role for MAPK pathways in circadian rhythmicity.circadian output pathway ͉ circadian rhythm ͉ Neurospora crassa ͉ osmotic stress phosphorelay

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Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin P and 6 chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immuno-