Reliability of statistical analyses for estimating relative specificity in quantitative resistance in a model host-pathogen system.

The reliability of analyses of variance for evaluating host cultivar x pathogen isolate specificity in resistance controlled by polygenes with additive effects was tested with combinations of hypothetical host and pathogen genotypes in a model system. In each test, varying numbers of host and pathogen genotypes were combined in all combinations, the resulting disease severities were calculated according to the model, and those data were subjected to analysis of variance. The percentage of total variance accounted for by host x pathogen interaction decreased with increasing numbers of host and pathogen genotypes per test. Simulated selection for virulence among randomly generated pathogen genotypes increased the percentage of variance attributable to host x pathogen genotype interaction, but simulated selection for resistance among host genotypes decreased it. The percentage of variance accounted for by interaction was greatest when selection of resistant host genotypes was followed by selection of the most virulent pathogen genotype on each selected host genotype. When gene frequencies were varied in the model, the interaction variance was greatest at low frequencies of resistance genes and high frequencies of virulence genes, but the number of matches between genes for specific virulence and specific resistance was greatest for high frequencies of both resistance and virulence genes. A simplified method of analysis was developed to estimate the amount of specific resistance in a set of host genotypes inoculated in all combinations with a set of pathogen genotypes. This method, based on the variance of disease severity adjusted to remove general virulence, proved consistently accurate with varying numbers of genotypes in the set, varying numbers of loci for resistance and virulence, and varying frequencies of genes for resistance and virulence. The variance method is of comparable accuracy and is much simpler than the previously proposed methods based on regression analysis. Simulated selection for resistance in the host and for virulence in the pathogen population increased the accuracy of both the variance method and the regression method.

Cell surface redox potential as a mechanism of defense against photosensitizers in fungi.

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.

Stability analyses for estimating relative durability of quantitative resistance.

Experiments in which a series of host cultivars are inoculated in all combinations with a series of pathogen isolates have been used to detect specificity in the host resistance. A theoretical model of polygenic resistance involving both general and specific interactions with pathogen virulence was developed to test the abilities of statistical analyses to discriminate between host genotypes with different levels of general and specific resistance. Estimates of levels of specific resistance could be obtained in regressions of disease severity scores for each host cultivar X pathogen isolate combination vs. the virulence index of each isolate. If the virulence index was based on the mean disease severity induced by the isolate over all host cultivars, the slopes of the regression lines were correlated with the levels of specific resistance in host cultivars. If the virulence index was based on the disease severity induced by the isolate on a host cultivar with a minimum of specific resistance, the mean squares for deviations from the regression were correlated with the levels of specific resistance in host cultivars. A method was developed to consistently choose host cultivars with minimum specific resistance. The two regression analyses gave estimates of specificity in randomly generated, model genotypes of approximately equal accuracy, although the second method appeared to be more accurate when the numbers of loci controlling resistance and virulence were small. The best estimates of numbers of genes for specific resistance were obtained by calculating a rating based on mean disease severity, the mean square for deviation from the regression on the virulence index based on disease severity on the cultivar with minimum specific resistance and the slope of the regression on the virulence index based on the mean disease severity. The best estimates of proportions of resistance genes that were specific were obtained by calculating a rating based on the above deviation mean square and slope alone.

SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms.

Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes.

Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae.

The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.

A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae.

Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.