Genetic Control of Phosphate-Metabolizing Enzymes in NEUROSPORA CRASSA: Relationships among Regulatory Mutations

In Neurospora crassa, the phosphate-metabolizing enzymes are made during phosphate starvation, but not under phosphate sufficiency. The synthesis of these enzymes is controlled by three regulatory genes: pcon-nuc-2, preg and nuc-1, pcon-nuc-2 and preg are closely linked. A model of the hierarchical relationships among these regulatory genes is presented. Studies of double mutants and revertants confirm several predictions of the model. It has been found that nuc-2 (null) and pcon-c (constitutive) mutations reside in the same cistron. preg-c (constitutive) mutations are epistatic to nuc-2 mutations. nuc-1 (null) mutations are epistatic to all others.     

一个高亲和力水稻根系磷转运蛋白候选基因片段的克隆

磷是影响作物产量的主要限制因子之一,植物在缺磷条件下主要高亲和力的磷转蛋白对磷进行有效吸收,利用ＲＴ－ＰＣＲ技术,经过缺磷处理水稻京系１７（Oryza sativa L.ssp. japonica cv.Jingxi１７）的根系中的克隆到一个１１７８bp的磷转 蛋白基因片段ＯjPT1,测序后与ＧenBank中的已知序列进行氨基酸水平上的同源性比较,结果表明,该序列与拟南芥、马铃薯、番茄、苜蓿、长春花等植物的同源性分别在７０％左右,并且与酵母、ＶＡ菌和子囊属脉胞菌等的磷转运蛋白也表出出较高的同源性。通过ＲＴ－ＰＣＲ结果证明,该基因片段为诱导表达,该基因已被ＧenBan接收（收录号为ＡＦ２３９６１９）。     

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms.

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.     

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.     

A two-component histidine kinase of the rice blast fungus is involved in osmotic stress response and fungicide action

We isolated and characterized a histidine kinase gene (HIK1) from the rice blast fungus, Pyricularia oryzae (Magnaporthe grisea). The deduced amino acid sequence of HIK1 showed highest similarity (85.7%) to a hybrid-type histidine kinase, Os-1/Nik-1 of Neurospora crassa. Disruption of HIK1 caused no defect in cell growth on normal media and in pathogenicity to rice plants. The Deltahik1 strain acquired resistance to three groups of fungicides (phenylpyrroles, dicarboximides, and aromatic hydrocarbons) similar to os-1 mutants of N. crassa. The Deltahik1 strain showed increased sensitivity to high concentrations of sugars although its salt sensitivity was not elevated, suggesting that the rice blast fungus can distinguish osmostresses caused by high sugar concentrations and high salt concentrations. In contrast, os-1 mutants of N. crassa are sensitive to high concentrations of both salts and sugars. These findings suggest that P. oryzae and N. crassa partially differ in their os (osmosensitive) signal transduction pathway.     

Molecular comparison of the negative-acting nitrogen control gene,nmr, inNeurospora crassa and otherNeurospora and fungal species

In Neurospora crassa, the expression of unlinked structural genes which encode nitrogen catabolic enzymes is subject to genetic and metabolic regulation. The negative-acting nmr regulatory gene appears to play a role in nitrogen catabolite repression. Using the N. crassa nmr gene as a probe, homologous sequences were identified in a variety of other filamentous fungi. The polymerase chain reaction was used to isolate the nmr-like gene from the exotic Mauriceville strain of N. crassa and from the two related species, N. intermedia and N. sitophila. Sequence comparisons were carried out with a 1.7-kb DNA segment which includes the entire coding region of nmr plus 5' and 3' noncoding sequences. The size of the nmr coding region was identical in all three Neurospora species. Approximately 30 nucleotide base substitutions were found in the coding region of the nmr gene of each of the sister species when compared to the standard N. crassa sequence. However, most of the base changes occurred in third codon positions and were silent. The NMR proteins of N. sitophila and of N. intermedia display only three and four amino acid substitutions, respectively, from the N. crassa protein. Two regions of high variability, which include deletions and insertions of bases, were found in the 5' and 3' noncoding regions of the gene.     

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.     

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.     

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.     

Molecular Cloning of a Gene (Cfp) Encoding the Cytoplasmic Filament Protein P59nc and Its Genetic Relationship to the Snowflake Locus of Neurospora Crassa

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Isolation of Mutants Deficient in the Repressible Alkaline Phosphatase

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pcon(c), and preg(c). Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.     

Induction of multiple germ tubes in Neurospora crassa by antitubulin agents

The antitubulin fungicide benomyl suppressed the linear growth of Neurospora crassa wild type strain St. Lawrence 74 at micromolar concentrations. The rate of germination of macroconidia was not affected. Macroconidia exposed to 1.7 microM benomyl for 5 h formed multiple germ tubes. When germlings incubated for 4 h were exposed to 1.7 microM benomyl for 3 h, their germ tube stopped growing, swelled and emitted several branches. Normal linear growth was restored after removal of the fungicide. Linear growth of N. crassa was resistant up to 16 microM nocodazole. This drug induced multipolar germination at 8 microM, and griseofulvin only at 140 microM. The microtubule (MT) cytoskeleton of N. crassa could be revealed by indirect immunofluorescence with the monoclonal antibody YOL 1/34 directed against yeast alpha-tubulin. We detected no striking effects of the benomyl treatments on MT organization. The MT-stabilizing agents deuterium oxide (D2O) and cAMP have no antagonistic effects on the benomyl-induced multipolar germination. The positioning of nuclei and mitochondria was determined from the DAPI and Rhodamine 123 fluorescence patterns, respectively. Benomyl inhibited nuclear migration into multiple germ tubes. Quantitative scanning cytophotometry revealed a peak in the intensity of the mitochondria-associated Rhodamine 123 fluorescence near the apex of untreated germlings. This peak disappeared in multiple germ tubes. Benomyl-resistant mutant bml 511 (r), mutated in its beta-tubulin gene, germinated normally in the presence of the fungicide. This strongly suggests that multiple germ tube formation was due to the effect of benomyl on beta-tubulin. Benomyl-resistant strain 74-3, constructed by reintroducing the cloned mutant N. crassa beta-tubulin gene into the cells by transformation, displayed a partial resistance to benomyl with respect to multipolar germination. Its rate of germination was slow (50% germination reached after 4 h at 37 degrees C as compared to 2.5 h for the wild type). In contrast to N. crassa, the other ascomycete Aspergillus nidulans is nocodazole-sensitive (linear growth suppressed at 1.6 microM). It did not respond to the MT inhibitors benomyl and nocodazole with respect to the pattern of germ tube emergence. Our results suggest that microtubule or membrane beta-tubulin is involved in the maintenance of developmental polarity during germ tube emergence and growth of N. crassa.