Characterisation of the nitrite reductase gene (NII1) and the nitrate-assimilation gene cluster of Stagonospora (Septoria) nodorum.

By sequencing downstream of the cloned nitrate reductase gene (NIA1) in the phytopathogenic fungus Stagonospora (Septoria) nodorum, a second open reading frame was found. Further analysis revealed this to be the nitrite reductase gene (NII1). Both genes are transcribed in the same direction, and are separated by an intergenic region of 829-bp. The coding sequence of NII1 is interrupted by three small introns and corresponds to a predicted protein of 1141 amino acids in length. Consensus binding sites for regulatory proteins are present in the promoter region of NII1. There is no indication, however, from hybridisation or sequence analysis that the nitrate transporter gene is closely associated with the NIA1-NII1 cluster, as has been found for a number of fungi.

Disruption, replacement, and cosuppression of nitrate assimilation genes in Stagonospora nodorum.

We used Stagonospora (Septoria) nodorum to explore gene disruption as a general method of fungicide target validation. Nitrate reductase was chosen as a model target because the gene (NIA1) has been cloned from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting the NIA1 gene by both integration of an unselected vector during cotransformation and one-step gene replacement. Around 2% of transformants from the cotransformation approach became nitrate nonutilizing and Southern analysis confirmed disruption of the resident NIA1 gene. Half of the transformants with the gene replacement vector showed the nitrate nonutilizing phenotype expected from disruption. However, Southern analyses of 14 of these transformants showed that only 6 contained the expected NIA1 gene replacement. Of the remaining transformants, 6 had integrated multiple copies of the vector elsewhere in their genome and still had a functional nitrate reductase gene. Their inability to utilize nitrate was due to a lack of nitrite reductase activity. How this phenotype arose is not clear, but it might involve cosuppression of the nitrite reductase gene as the vector carried 1. 1 kb of the coding region and the complete 5' region of this gene which is adjacent to NIA1. Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.

Cloning of the nitrate reductase gene of Stagonospora (Septoria) nodorum and its use as a selectable marker for targeted transformation.

The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridisation with a PCR-generated probe. A 6. 7-kb fragment carrying the NIA1 gene was subcloned and partially characterised by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino-acid levels, with nitrate reductase genes of other filamentous fungi. Furthermore, consensus regulatory signals thought to be involved in the control of nitrogen metabolism are present in the 5' flanking region. The cloned NIA1 gene has been used to develop a gene-transfer system based on nitrate assimilation. Stable nia1 mutants of S. nodorum defective in nitrate reductase were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilisation with the wild-type S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of the integration of transforming DNA into the fungal genome; in all cases examined, integration was targeted to the homologous sequence.

Molecular analysis of the Septoria nodorum beta-tubulin gene and characterization of a benomyl-resistance mutation.

The complete nucleotide sequence of a benomyl-resistant allele of the Septoria nodorum beta-tubulin gene (tubAR) has been determined including 745 and 1024 nucleotides 5' and 3' to the tubAR coding region, respectively. tubAR encodes a 447 amino acid polypeptide which shows a high degree of homology with other fungal beta-tubulins. The gene contains three introns at codons 4, 12 and 53, uses 48 of the possible 61 sense codons and has a GC content of 59.1% in its coding region. S1 nuclease mapping has identified two transcriptional start sites 80 bp and 83 bp upstream of the translation start, and a transcriptional termination site 192 bp downstream of the stop codon. The two transcriptional start sites lie just 8 bp and 5 bp downstream of a CT motif consisting of 18 pyrimidine nucleotides interrupted by a single adenine. The wild-type allele tubA+ has been cloned using the polymerase chain reaction and the mutation producing the benomyl-resistant phenotype of tubAR mapped to a C to T transition at the first position of codon 6, resulting in a histidine to tyrosine amino acid substitution.

Variation in electrophoretic karyotype between strains of Septoria nodorum.

The electrophoretic karyotype of 11 strains of the phytopathogenic fungus Septoria nodorum has been established by pulsed field gel electrophoresis with the CHEF DRII system. Each strain had a similar overall karyotype with 14-19 chromosomes being resolved in the size interval between approximately 0.5 and 3.5 megabase pairs (Mb). However, there were clear differences in karyotype both between and within groups of strains adapted to wheat or to barley. Considerable karyotype variation was apparent even among 6 wheat-adapted strains isolated from the same population. Only 2 strains possessed identical karyotypes; these were isolated from the same leaf and were heterokaryon-compatible and are probably independent isolates of the same clone.

Selection for increased penicillin titre following hybridization of divergent lines of Aspergillus nidulans.

Sexual hybridization of two divergent lines of Aspergillus nidulans, which had been selected for increased penicillin titre through successive cycles of mutagenesis, released considerable variation for this character. The recovery of segregants with titres equivalent to that of the unselected ancestor suggested that mutations in different genes had been selected in the two lines. However, complementary segregants with substantially improved titres were not found, indicating interactions, probably of a duplicate nature, among the induced mutations. All the genetic variation released by hybridization was fixed following two generations of selection for high titre, but only a small gain over the initial selection lines was achieved. Hybridization of divergent strains has been widely advocated as a means of strain development. The failure to achieve the anticipated gains in this programme is attributed primarily to the unfavourable interactions amongst the induced mutations. Whether similar interactions occur generally in crosses between strains selected by mutagenesis remains to be established and will be an important factor in determining the contribution of recombinational approaches to yield improvement.

Genetics of penicillin titre in lines of Aspergillus nidulans selected through recurrent mutagenesis.

Genetic analyses of mutations for increased penicillin titre were carried out on two strains of Aspergillus nidulans isolated after independent programmes of recurrent mutation and selection, similar to those used industrially for strain improvement. Both selected strains were stable in terms of colony morphology and penicillins titre. Backcrosses to the unselected ancestor indicated that the increased yield of each strain was due to an induced polygenic system with both additive and non-additive gene action. The titre-increasing mutations in one strain (A6-9) were recessive to their wild-type alleles, while those in the other (B6-27) were either semi-dominant or showed ambidirectional dominance. The diploid between A6-9 and B6-27 had a titre less than either component haploid, suggesting that the mutations in the two strains involved different genes. Strain B6-27 carried a translocation between chromosomes III and VII which had been induced during the selection programme. This investigation complements previous work on the genetics of penicillin production in A. nidulans and Penicillium chrysogenum and the results are directly relevant to industrial strain improvement by hybridization of divergent strains.

Recurrent mutation and selection for increased penicillin titre in Aspergillus nidulans.

Recurrent mutation and selection has been used to increase penicillin titre in two closely related strains of Aspergillus nidulans. A selection programme was initiated from each of the two strains (programmes A and B) and continued through six cycles of mutation and selection. Near-ultraviolet light in conjuction with 8-methoxypsoralen was employed as the sole mutagen throughout programme A and ethyl methanesulphonate as the sole mutagen throughout programme B. Excluding the first cycle of A, where only 50 strains were assayed, the selection programmes were identical. In each programme, 100 survivors were assayed for penicillin titre after each mutagenic treatment and, on the basis of a single yield test, the best five strains were picked and carried forward to the next cycle. In both selection programmes, a near 300% increase in penicillin titre was achieved. This yield advance illustrates the effectiveness for strain development of experimental designs involving successive cycles of mutagenesis with a single-stage screen and the selection of the top few percent survivors in each cycle.

Induced quantitative variation for penicillin titre in clonal populations of Aspergillus nidulans.

The variation in penicillin titre within populations of cultures of Aspergillus nidulans derived from untreated conidia and from conidia treated with ethyl methanesulphonate (EMS), near-ultraviolet light in the presence of 8-methoxypsoralen (8MOP) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), each at several dose levels, was determined. Both mutagentreated and untreated populations showed a continuous distribution of pencillin titres. The population mean titre of the mutagenized populations was decreased and the range of titres was increased relative to those of the control populations. No differences between sister cultures could be detected in three untreated populations, but nine out of ten populations derived from mutagenized conidia showed significant variation for penicillin titre. In general the magnitude of this induced variation increased with increasing dosage of the mutagen. Comparisons at fixed survival levels indicate that 8MOP mutagenesis is less effective for the induction of variation in penicillin titre than EMS or NTG mutagenesis. A statistical procedure was adopted to classify the survivors as unchanged cultures ('0'), titre-increasing mutants ('+') or titre-decreasing mutants ("-"). The frequency of both '+' and '-' mutants increased following mutagenesis, with NTG being the most active of the three mutagens. Over all treatments, these two mutant classes were recovered with equal frequency. The frequency of "+" mutants was largely independent of mutagen dose, within the ranges used, and moderate treatments (around 10% survival) gave as high or higher frequencies than more extreme doses. All three mutagens, and in particular NTG, produced morphological mutants. These contained an increased frequency of titre-decreasing mutants, but increases in titre appeared to be independent of changes in colony morphology. Estimates based on the observed frequencies of penicillin titre mutants suggest that several hundred genes are potentially capable of affecting this continuous variable.

The inheritance of penicillin titre in wild-type isolates of Aspergillus nidulans.

In a sample of 52 wild-type isolates of Aspergillus nidulans, penicillin titre ranged from 0-0 to 14-4 units/ml. These differences in titre were under genetic control. Most of the variation between isolates was attributable to differences between heterokaryon-compatibility groups although significant differences were also found within groups. Genetic variation for penicillin titre was observed among the progeny in four of seven crosses between wild-type isolates. In these four crosses the variation was continuous, indicating that this is a polygenically determined character, and progeny with titres superior and inferior to those of the parents were produced. The genes determining penicillin production are predominantly additive in their action. Crosses between heterokaryon-incompatible isolates generated more genetic variation than those involving compatible parents, supporting the hypothesis that heterokaryon-compatible isolates are frequently closely related.