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The spraygene was cloned, and wildtype and mutant alleles were sequenced. Spray+has a 3452-bp open reading frame plus seven introns. The spraymutant had a T → G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant sprayphenotype. Electrophysiological analysis showed that ion fluxes in the sprayplasma membrane are normal; furthermore, whereas the spraymutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spraymutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca2+via calcineurin.

Neurospora mitochondrial plasmids are ubiquitous in natural populations, yet many of them are lethal to their host strains or seem to impose a molecular genetic load. Five pairs of strains of Neurospora tetraspermaand N. crassawith and without kalilo-like plasmids were tested under a variety of situations. The purpose was to find possible beneficial effects of plasmids that might offset their disadvantages. We found that, in all cases tested, plasmids conferred an advantage to growth at temperatures close to the top of the range for this fungus. Also, the plasmids improved fertility, as measured by perithecial production. Negative results were obtained for heavy metal resistance and ascospore germination. The results generate the hypothesis that plasmids may have adaptive significance to their hosts.

The Neurospora crassamutant frosthas a hyperbranching phenotype that can be corrected by adding Ca2+, suggesting that characterization of this gene might clarify the mechanism of Ca2+-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia. RFLP analysis revealed that the cloned DNA fragment mapped to the frlocus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the SaccharomycescerevisiaeCDC1 protein, implicated in manganese homeostasis. The frmutant was sensitive to Mn2+, and a revertant allele whose product differs by one amino acid was tolerant to Mn2+. Mn2+depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The frmutant was also sensitive to calcineurin inhibitors. These results suggest that fris involved in Mn2+homeostasis and point to a role for Mn2+in Neurosporabranching.

The 8.6-kb kalilo linear mitochondrial plasmid of Neurospora intermediawas found to give rise to multiple transcripts of 8.6, 4.4, 4.0, 1.3. 1.2, and 0.9 kb. Plasmid transcripts were found in all the developmental stages tested and were present in cells with and without plasmid integration into mtDNA. The 4.4- and 4.0-kb transcripts corresponded to the two known ORFs in size, position, and polarity. An analysis of RNA fractions has confirmed the mitochondrial location of kalilo. The kalilo-specific RNA appears to be selectively unstable in affected strains of N. intermedia;this may be a general property of linear plasmid RNA. Transcripts are most likely initiated from near the termini of the linear plasmid. Primer extension and sequencing showed a clear 5′ RNA end at nucleotide 101. However, the sequence in this region does not show homology with any known mitochondrial, plasmid, or nuclear promoter. The sequence does show homology with the tip of the plasmid and marks the border of a long series of direct repeats. Possibly the plasmid RNA polymerase may be bifunctional, recognizing sequences at the plasmid tip as well as the promoter.

The kalilovariants of Neurospora contain a cytoplasmic genetic factor that causes senescence. This factor is a 9.0 kb transposable element (kaIDNA) that lacks nucleotide sequence homology with mtDNA and is inserted into the mitochondrial chromosome, often at sites located within the open reading frame in the intron-DNA of the mitochondrial 25S-rRNA gene. Genomes containing the “foreign” DNA insert accumulate during growth, and death occurs as the cells become deficient in functional large and small subunits of mitochondrial ribosomes. The kaIDNA transposon may be an “activator” element that causes breaks in mtDNA. Nonsenescing [+] strains of Neurospora do not contain kaIDNA.

We have investigated the horizontal transfer of two mitochondrial plasmids and the Kalilo senescence phenotype in the fungus Neurospora without the use of heterokaryon-forcing markers. The Kalilo senescent state was only transferred between fully-compatible N. crassa strains, but not between strains differing at any of the loci het-c, het-d, het-e or mating-type. However, the linear plasmid kalDNA and the circular plasmid Han-2 were transferred following incompatible vegetative interactions. Our data suggest that vegetative incompatibility due to allelic differences at het-c is more effective in preventing transfer than that due to het-d, het-e or mating-type. Based on these observations we have developed a novel test for assessing vegetative incompatibility between Kalilo and non-Kalilo field isolates of N. intermedia. In this procedure combinations of Kalilo and non-Kalilo field isolates of N. intermedia were grown together and tested for senescence. Compatibility is inferred if the young non-Kalilo strain dies along with the senescent Kalilo strain, whereas incompatibility is inferred when the Kalilo strain dies without imposing its senescent state onto the non-Kalilo strain. Our results suggest that each of the nine Kalilo strains tested is incompatible with each of 20 non-Kalilo isolates from the same N. intermedia population of the Hawaiian island of Kauai. However, the observed incompatibility did not completely prevent cytoplasmic exchange, and in several cases plasmid transfer could be detected.