Gene Action and Linkage of Avirulence Genes to DNA Markers in the Rust Fungus Puccinia graminis.

ABSTRACT Two strains of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, were crossed on barberry, and a single F(1) progeny strain was selfed. The parents, F(1), and 81 F(2) progeny were examined for virulence phenotypes on wheat differential cultivars carrying stem rust resistance (Sr) genes. For eight Sr differentials, phenotypic ratios are suggestive of single dominant avirulence genes AvrT6, AvrT8a, AvrT9a, AvrT10, AvrT21, AvrT28, AvrT30, and AvrTU. Avirulence on the Sr; (Sr 'fleck') differential showed phenotypic ratios of approximately 15:1, indicating epistatic interaction of two genes dominant for avirulence. Avirulence on Sr9d favored a 3:13 over a 1:3 ratio, possibly indicating two segregating genes-one dominant for avirulence and one dominant for avirulence inhibition. Linkage analysis of eight single dominant avirulence genes and 970 DNA markers identified DNA markers linked to each of these avirulence genes. The closest linkages between AvrT genes and DNA markers were between AvrT6 and the random amplified polymorphic DNA marker crl34-155 (6 centimorgans [cM]) AvrT8a and the amplified fragment length polymorphism marker eAC/mCT-197 (6 cM) and between AvrT9a and the amplified fragment length polymorphism marker eAC/mCT-184 (6 cM). AvrT10 and AvrTU are linked at distance of 9 cM.

Cloning and characterization of the Neurospora crassa cyt-5 gene. A nuclear-coded mitochondrial RNA polymerase with a polyglutamine repeat.

The Neurospora crassa mutants, cyt-5-1 and cyt-5-4 have a cytochrome b- and aa3-deficient phenotype, suggesting that they result from a deficiency in a nuclear-coded component of the mitochondrial gene expression apparatus (Bertrand, H., Nargang, F. E., Colllins, R. A., and Zagozeski, C. A. (1977) Mol. Gen. Genet. 153,247-257). The complementing wild-type gene has been cloned and and shown to encode a protein with significant sequence similarity to Saccharomyces cerevisiae mitochondrial RNA polymerase and bacteriophage RNA polymerases. There are remarkable differences between the N. crassa protein and its yeast homologue, including a region of very little homology near the N termini of the two gene products. The cyt-5 gene encodes a stretch of polyglutamine in this region of uniquesequence. In addition, an acidic insertion (86 amino acids, of which 24 are Asp or Glu and 10 are Arg or Lys) is present near the C terminus of the cyt-5 gene product. Transcript levels of the cytochrome b and cytochrome oxidase subunit III genes are severely reduced in cyt-5 mutants, suggesting a likely mechanism for the cytochrome-deficient phenotype. In contrast, mitochondrial rRNAs accumulate to nearly normal levels in cyt-5 mutants. However, mitochondrial rRNA levels are not indicative of the rate of transcription of the corresponding genes, since crude lysates of mitochondria from cyt-5 mutants exhibit greatly reduced transcriptional activity with a 19 S rRNA promoter. The cyt-5 gene is flanked by at least one gene whose product also may be involved in mitochondrial function.

High-GC primers are useful in RAPD analysis of fungi.

For genetic analysis of fungal DNAs, we have modified the RAPD method to use primers with G + C contents of 80-100%. In RAPD analysis of Puccinia graminis f. sp. tritici DNAs, these primers generated twice the number of both amplification products per primer and polymorphisms among isolates as compared to the standard 60-70% G + C primers. With respect to segregation and genetic similarity, RAPD markers generated by the high-GC primers behaved as do RAPD markers produced by the standard primers. These high-GC primers also yielded increased numbers of amplification products in RAPDs on the DNAs of a broad range of other fungi.

The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis.

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.

Identification of Neurospora mitochondrial promoters and analysis of synthesis of the mitochondrial small rRNA in wild-type and the promoter mutant [poky].

Using an in vitro transcription assay, we previously identified promoters in Neurospora mtDNA at the 5' ends of the genes encoding the mitochondrial (mt) small and large rRNAs and cob pre-mRNA. Here, we identified two additional promoters in mtDNA restriction fragment EcoRI-6, 3.8 and 5.5 kilobases upstream of the 5' end of the mt small rRNA. By comparing the two new promoters with the three identified previously, we derived a modified promoter consensus sequence (AT-rich)15-27TTAG(A/T)RR(G/T)(G/C)N(A/T). The mt small rRNA in Neurospora is transcribed from at least two promoters, a major promoter at the 5' end of the small rRNA and one or both of the newly identified promoters in EcoRI-6. The latter gives a series of putative pre-rRNAs that contain 5' end extensions of various sizes. The 5' ends of a number of these RNAs map at or near hairpin structures. The [poky] mutant, which is grossly deficient in the mt small rRNA, has a 4-base pair deletion in the major promoter at the 5' end of the mt small rRNA. The residual small rRNAs in [poky] appear to be synthesized via the upstream promoter(s), but are missing 37-44 nucleotides from their 5' ends, indicating either that pre-rRNAs are processed abnormally or that abnormal 5' RNA ends are unstable. The effect of the promoter mutation in [poky] on other transcripts suggests that the mt small rRNA is cotranscribed with downstream genes encoding tRNAs, coIII and ND6. Seven nonallelic nuclear suppressors of [poky] result in increased concentrations of the mt small rRNA and pre-rRNAs, but do not restore the ability to synthesize small rRNAs having the correct 5' ends. The suppressor mutations could act by increasing transcription, processing, or stability of the mt small rRNA or its precursors. The suppressors provide a genetic approach for identifying components that affect transcription and processing of the mt small rRNA.