Mutants Of Ustilago Maydis Research Articles

A non-Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis.

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.

Genetic Control of Resistance to the Piperidine Fungicide Fenpropidin in Ustilago maydis

Mutants of Ustilago maydis (DC.) Corda, resistant to the piperidine fungicide fenpropidin, were isolated in a mutation frequency of 3.2 × 10–5, after UV‐irradiation and selection on media containing 75 μg/ml fenpropidin. Genetic analysis with 15 such mutant isolates resulted in the identification of two unlinked chromosomal loci, U/fpd‐1 and U/fpd‐2. The U/fpd mutations are responsible for moderate resistance levels to fenpropidin (Rf: 42–56 or 15 based on effective concentration causing a 50% reduction in the growth rate (EC50) or minimal inhibitory concentration (MIC) values, respectively). Haploid strains carrying both U/fpd mutations do not exhibit higher levels of resistance to fenpropidin, indicating no additivity of gene effect between non‐allelic genes. Cross‐resistance studies with other Sterol Biosynthesis inhibitors (SBIs) showed that the U/fpd‐mutant isolates exhibited a positive cross‐resistance to the piperidine piperalin and to the related morpholine fungicides fenpropimorph and tridemorph, but not to the inhibitors of C‐14 demethylase and squalene epoxidase. Crosses between mutants carrying the U/fpd‐genes with compatible isolates carrying the U/fpm or U/tdm mutations, which have been identified in previous genetic studies for resistance to morpholine fungicides fenpropimorph and tridemorph, yielded, with the exception of U/fpd‐2 × U/fpm‐2 crosses, a large number of recombinants with wild‐type sensitivity, indicating that the mutant genes involved were not allelic. Analysis of progeny from crosses between U/fpd‐2 and U/fpm‐2 mutants yielded no recombinants with wild‐type sensitivity, but a 1 : 1 progeny segregation was observed at the MIC for the U/fpd‐2 isolates, indicating that these genes are alleles of the same locus. A study of the fitness of fenpropidin‐resistant isolates showed that the U/fpd mutations do not affect the phytopathogenic fitness‐determining characteristics such as growth in liquid culture and pathogenicity on young corn plants.

Genetic control of resistance to tridemorph inustilago maydis

Mutants ofUstilago maydis with low resistance to tridemorph isolated in a mutation frequency of 7x 10-6 after UV-irradiation and selection on media containing 25 μg ml-1 tridemorph. Genetic analysis with nine such mutant isolates resulted in the identification of two unlinked chromosomal loci,U/tdm- 1 andU/tdm- 2. TheU/tdm mutations are responsible for low resistance levels to tridemorph (resistance factor, Rf, of 3 or 5 based on effective concentration causing a 50% reduction in the growth rate (EC50) or minimal inhibitory concentration (MIC) values, respectively) and low to moderate level of resistance to fenpropimorph (Rf 10 or 16 based on MIC or EC50, respectively) and fenpropidin (Rf 5 or 11 based on MIC or EC50, respectively). Haploid strains carrying bothU/tdm mutations exhibit higher levels of resistance to the above fungicides, indicating interallelic interaction between nonallelic genes. Crosses between mutants carrying theU/tdm- genes with compatible isolates carrying theU/fpm- 1 orU/fpm- 2 mutations, which were found in previous work to carry fenpropimorph resistance, yielded in all cases a large number of recombinants with wild-type sensitivity, indicating that the mutant genes involved were not allelic. Cross-resistance studies with the inhibitors of C-14 demethylase showed that. the U/tdm-mutations were responsible for increased sensitivity to the triazoles triadimefon, triadimenol, propiconazole and flusilazole, and to the pyridine pyrifenox. Study of gene effect on the fitness ofU. maydis showed thatU/tdm-mutations appeared to be pleiotropic, having more or less adverse effects on growth rate in liquid culture and pathogenicity on young corn plants.

The mechanism of resistance to sterol 14? demethylation inhibitors in a mutant (Erg 40) ofUstilago maydis

Resistance to DMI fungicides is a problem in both agriculture and medicine. Several mechanisms of resistance exist, but, as yet, few have been characterised in field resistant strains of plant pathogens. One approach to evaluating the role of mutations in the sterol 14α demethylase (14DM) target site requires cloning this gene and confirming its identity by complementation in an appropriate mutant. The azole-resistant mutant, Erg 40, of Ustilago maydis which is totally blocked at the 14α demethylation step in sterol biosynthesis seems to be suitable for such expression studies. Transformation of Erg 40 with a plasmid containing the yeast 14α demethylase (CYP51A1) gene removed the block in sterol biosynthesis and generated azole-sensitive transformants. Detailed analysis of these transformants failed to detect the presence of the yeast gene and suggested, instead, that changes in sterol biosynthesis resulted simply from the transformation protocol and not from the incorporation of extracellular DNA. Subsequent sequence analysis has revealed a mutation in the 14α demethylase gene of Erg 40. The results suggest that azole resistance in Erg 40 is not simply controlled by this mutation but involves some additional regulatory function, and consequently Erg 40 is not suitable for complementation studies with CYP51A1 genes. © 2000 Society of Chemical Industry

Carboxin-resistant mutant of ustilago maydis is impaired in its pathogenicity for zea mays

We analyzed the pathogenicity of chitin synthetase (chs) disruptants of Ustilago maydis obtained with the carboxin-resistant or the hygromycin-resistant cassettes. We found that only chitin synthetase (chs) mutants obtained by gene disruption with the carboxin resistance cassette lost their virulence to maize (Zea mays) seedlings. Carboxin is a systemic fungicide that inhibits respiration by preventing the oxidation of succinate. We demonstrated that carboxin-resistant transformants were affected in the levels of succinate dehydrogenase and respiratory activities when compared with hygromycin-resistant disruptants. We propose that loss of virulence in the carboxin-resistant transformants is owing to loss of respiratory fitness, which probably represents an important component of virulence in this fungus.http://link. springer-ny.com/link/service/journals/00284/bibs/39n5p291.html</HEA

Genetic control of resistance to fenpropimorph in Ustilago maydis

Fenpropimorph‐resistant mutants of Ustilago maydis were obtained at high frequency (30 × 10−6) after UV‐irradiation followed by selection on media containing fenpropimorph (50 μg mL−1). Genetic analysis of 30 such mutants resulted in the identification of two unlinked chromosomal loci, the U/fpm‐1 locus with two allelic genes (U/fpm‐1A and U/fpm‐1B) and the U/fpm‐2 locus. The mutant genes U/fpm‐1A and U/fpm‐2 are responsible for high resistance levels (Rf: 75–100 or 257–286 based on MICs or ED50s, respectively), while the U/fpm‐1B mutation gives only a small reduction (approximately 7–10‐fold) in fenpropimorph sensitivity. Cross‐resistance studies with other SBIs showed that the major gene (U/fpm‐1A and U/fpm‐2) mutants were cross‐resistant to the related compound fenpropidin (Rf: 15–20 or 53–66 based on MICs or ED50s values, respectively) and to tridemorph (Rf: 5 or 7.1–9.5 based on MICs or ED50s values, respectively), but not to the inhibitors of steps of ergosterol biosynthesis preceding the Δ14‐reductase. The minor gene (U/fpm‐1B) mutants also had low‐level resistance (approximately 5‐fold) to tridemorph and to fenpropidin, but in contrast with the major gene mutants they were 2–10 times more sensitive to the triazoles studied (triadimefon, triadimenol, propiconazole and flusilazole) and to the pyridine, pyrifenox.Studies of the fitness of U. maydis mutants showed that in major gene mutants, resistance was not associated with changes in growth rate in liquid culture or pathogenicity on young maize plants. The minor gene mutation reduced significantly the growth rate in liquid culture and the pathogenicity, either in homozygous or heterozygous condition in dikaryotic mycelium.

Resistance to Sterol Demethylation Inhibitors inUstilago maydis. III. Cross-Resistance Patterns and Sterol Analyses

Two spontaneous triadimefon-resistant mutants of Ustilago maydis, 151ar/1 and 151ar/3, were investigated with regard to their extent of cross-resistance and their sterol composition to elicit indications about the specificity of the present resistance mechanisms. Testing resistance to various sterol biosynthesis inhibitors and toxicants with different modes of action, it could be demonstrated that, in the mutant 151ar/1, cross-resistance was limited to the sterol demethylation inhibitors (DMIs), whereas, in strain 151ar/3, resistance included most sterol biosynthesis inhibitors studied (DMIs, morpholines, piperidines, allylamines) as well as the unrelated compounds vinclozolin and cycloheximide. Sterol analyses showed that both mutants contained ergosterol as the main sterol component. In comparison with the sensitive reference strain, the mutant 151ar/1 had a slightly elevated content of C-14 methyl sterols, whereas in strain 151ar/3 the amount of ergosterol was increased. Triadimefon caused an accumulation of C-14 methyl sterols and a decrease in ergosterol content in the sensitive strain and the mutant 151ar/1, whereas the other strain 151ar/3 remained unaffected. The results indicate that several resistance mechanisms are probably operating in the two mutants.

Molecular Analysis of Azole Fungicide Resistance in a Mutant of Ustilago maydis

Molecular Analysis of Azole Fungicide Resistance in a Mutant of Ustilago maydis

A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis.

A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized. A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection. Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance. Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases.

Mitochondrial electron transport in a carboxin-resistant, antimycin A-sensitive mutant of Ustilago maydis

Succinoxidase activity of mitochondria from an antimycin A-sensitive ( ants) mutant of Ustilago maydis is approximately five times less sensitive to the fungicide carboxin than that of mitochondria from wild-type cells. The antimycin A sensitivity is due to the absence of an alternative electron transport pathway in mitochondria obtained from mutant cells grown in control medium. The same mutant, however, develops high rates of alternative respiration if grown in the presence of chloramphenicol. Substrate and oxygen affinity as well as resistance to hydroxamates indicate that this respiration is mediated by only one mitochondrial electron transport pathway, similar to the inducible system described earlier in wild-type mitochondria. Induction appears to be regulated by the activity of the cytochrome pathway. The absence of the constitutive system from the ants mutant mitochondria which are resistant to carboxin, a selective inhibitor of the succinate dehydrogenase complex, supports the view that this system must be related to succinic dehydrogenase.

Components of the genetic repair mechanism are not confined to the nucleus.

IT has been shown that radiation-sensitive mutants in separate genes, which presumably affect different steps in the repair of damaged DNA, can complement in Escherichia coli1, or yeast2 and Ustilago diploids. Could such strains complement each other's deficiencies in heterokaryotic cell ? Such complementation would not be expected if the repair mechanism was completely confined to the nucleus. Recently, mutants of Neurospora two or three times more sensitive to the lethal effect of ultraviolet light than wild type have been reported to complement in heterokaryons3. We have tested this further with three radiation sensitive (uvs) mutants of Ustilago maydis which are 10–30 times more sensitive to ultraviolet light than wild type cells, and which are believed to be deficient in ability to repair damaged DNA4. These mutants are recessive, unlinked and complement in diploid cells. Heterokaryotic cells were obtained using the conditions devised by Puhalla5 which allow strains of opposite mating type to fuse to form hyphal cells containing nuclei of opposite mating type, although we used a different medium (Ustilago liquid complete medium supplemented with 1.7 per cent Difco cornmeal agar). Groups of heterokaryotic cells were removed with a needle from compatible matings between strains with like or different uvs mutations, and spread on a plate of complete medium. The plate was irradiated with 960 ergs/mm2 ultraviolet light from a standard germicidal lamp. This dose kills about 20 per cent of wild type cells and at least 98 per cent of uvs cells. After overnight incubation, 150 heterokaryotic cells of each type were scored for viability under a low power microscope. On this medium viable heterokaryons grow to form a microcolony consisting largely of haploid cells. The parent strains each carry complementing auxotrophic markers, and so it is easy to show that the surviving colonies are not diploids by replica plating them to unsupplemented medium on which only diploids will grow. The results of the tests are shown in Table 1.