Robert M. Hanau

Phytoalexin accumulation in sorghum: identification of an apigeninidin acyl ester

Abstract The accumulation of a new 3-deoxyanthocyanidin phytoalexin in sorghum leaves and mesocotyls was demonstrated to occur following inoculation with an isolate of Colletotrichum graminicola pathogenic to sorghum, an isolate of C. graminicola pathogenic only to corn, and an isolate of Helminthosporum maydis (a nonpathogen of sorghum). The compound was found to be a caffeic acid ester of arabinosyl-5-O-apigeninidin and was never present in uninoculated tissues. Comparison of the fungitoxicity of the compound with that of two phytoalexins identified previously (apigeninidin and luteolinidin) showed that all three of the compounds exhibited toxicity to C. graminicola at concentrations of less than 10 μ m .

Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA-γ

Abstract The complete nucleotide sequences of RNAγ from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5′-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3′-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNAγ ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNAγ RF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNAγ ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3′-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984 , Virology 139, 375–383). In the genomic RNAγ, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.

Genetic and morphological comparisons ofGlomerella (Colletotrichum) isolates from maize and from sorghum

Abstract Various morphological and genetic characteristics were compared among six isolates of Colletotrichum from maize and six from sorghum. For the first time, a teleomorph was induced in sorghum isolates by pairing them on autoclaved sorghum leaves in a humidity chamber. The sorghum teleomorph was morphologically similar to Glomerella graminicola and Glomerella tucumanensis , the teleomorphs of Colletotrichum isolates from maize and from sugarcane, respectively. Mating tests demonstrated that Glomerella isolates from maize and sorghum were not interfertile. Several small but consistent differences in the morphologies of the isolates from maize and from sorghum were observed which agreed with earlier reports. DNA fingerprints detected as restriction fragment length polymorphisms of mitochondrial DNA and random polymorphic DNA (RAPD) produced from nuclear DNA by the polymerase chain reaction could be used to reliably and unambiguously distinguish members of the two groups of isolates. Results of a statistical analysis of similarity of the RAPD fingerprints suggested that maize and sorghum isolates of Colletotrichum are only about 45% similar (±10%) and represent two distinct and separate genetic lineages. We conclude that isolates of Colletotrichum from maize and sorghum are sibling species since they are morphologically very similar but reproductively completely isolated.

Characterization of two divergent β-tubulin genes from Colletotrichum graminicola.

We have cloned and sequenced two beta-tubulin genes, TUB1 and TUB2, from the phytopathogenic fungus, Colletotrichum graminicola. The nucleotide sequences of the coding regions of the two genes are only 72.8% homologous. This divergence is reflected in the deduced amino acid (aa) sequences which differ at 94 aa residues. Comparison with the aa sequences of other fungal beta-tubulins indicates that the C. graminicola TUB2 gene encodes a conserved isotype, whereas the C. graminicola TUB1 product is highly divergent. Both genes contain six identically placed introns and the position of each intron is conserved in other fungal beta-tubulin genes. Also typical of other fungal beta-tubulin genes, there is a pronounced bias in codon usage in the C. graminicola TUB2 gene; there is a lesser codon bias in TUB1 from C. graminicola. Both C. graminicola beta-tubulin genes are transcribed and yield similar sized messages.

The SOD2 gene, encoding a manganese-type superoxide dismutase, is up-regulated during conidiogenesis in the plant-pathogenic fungus Colletotrichum graminicola

The SOD2 gene, encoding a manganese-type superoxide dismutase (MnSOD), was identified from Colletotrichum graminicola among a collection of cDNAs representing genes that are up-regulated during conidiogenesis. The SOD2 gene consists of a 797-bp open reading frame that is interrupted by three introns and is predicted to encode a polypeptide of 208 amino acids. All conserved residues of the MnSOD protein family, including four consensus metal binding domains, are present in the predicted SOD2 protein. However, the predicted protein does not appear to contain a signal peptide that would target it to the mitochondria. Northern hybridizations revealed that expression of the approximately 900-bp SOD2 transcript is closely associated with differentiation of both oval and falcate conidia. Southern analysis indicated that there is only a single copy of the gene. SOD2 disruption strains were morphologically and pathogenically indistinguishable from wild-type strains. The dispensability of the MnSOD enzyme may be due to the activities of two other SOD enzymes, a highly expressed iron-type superoxide dismutase and a much less abundant copper/zinc type, that were also detected in C. graminicola.

Protection against phenol toxicity by the spore mucilage of Colletotrichum graminicola, an aid to secondary spread

Abstract Phenolic compounds inhibitory to the germination of spores of Colletotrichum graminicola were shown to leach from necrotic lesions on corn leaves caused by the fungus. Primary components of the phenolic mixture were identified as esters and glycosides of p -coumaric and ferulic acids as well as the free compounds themselves. Spores of C. graminicola produced in acervuli on infected leaves were shown to be surrounded by a mucilaginous matrix as is the case when the fungus is cultured in vitro . It is suggested that the mucilage protects spores from the inhibitory effects of the phenols by the presence of proline-rich proteins that have been shown to have a high binding affinity for a variety of phenols.

Genetic analysis of cross fertility between two self-sterile strains of Glomerella graminicola.

The ascomycete Glomerella cingulata has an unusual and complex mating system which is con- trolled by multiple, multiallelic loci. Cross fertility be- tween different isolates occurs via complementation of mutated fertility genes, a process known as unbal- anced heterothallism. We have examined the herita-

CONIDIAL DIMORPHISM IN COLLETOTRICHUM GRAMINICOLA

Colletotrichum graminicola produced two types of conidia in culture and during infection of corn leaves. One was lunate to falcate and was produced blastically from morphologically distinct conidi? ogenous cells. The second type was oval to elliptic, variable in size but smaller than falcate conidia, and was produced blastically from hyphae that lacked distinct conidiogenous cells. Falcate conidia each contained one nucleus, whereas the oval type contained one to four nuclei. A difference in cell wall structure was suggested by greater sensitivity of oval conidia to cell-wall degrading enzymes. Oval conidia were the only type produced in shake culture grown in the dark. Both types of conidia were produced by cultures on agar media and by standing cultures in small volumes of liquid medium but, unlike oval conidia, development of falcate conidia was light-dependent When used to inoculate susceptible corn seedlings, both types of conidia caused symptoms comparable over a range of inoculum concentrations. Regardless ofthe type of conidia used to inoculate seedlings, both were found in lesions of infected leaves.

Nitrate-nonutilizing mutants used to study heterokaryosis and vegetative compatibility in Glomerella graminicola (Colletotrichum graminicola)

Nitrate-nonutilizing (nit) mutants of Glomerella graminicola were recovered by selecting chlorate-resistant sectors. Heterokaryons were formed by complementation between two different classes of nit mutants. Complementation groups were distinguished in nitrogen feeding tests and segregated as two, unlinked genes among random progeny of sexual crosses. The two genes are comparable to those encoding the nitrate reductase enzyme and one of a series of molybdenum cofactors in Aspergillus nidulans and Neurospora crassa . Heterokaryon tests were reliable indicators of allelic and dominance relationships between mutations with similar phenotypes. Vegetative compatibility (VC) between two strains of G. graminicola appeared to be regulated by approximately five unlinked VC loci, analogous to those described for other fungi.

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis

SummaryA spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.