Phytopathogenic Cercosporoid Fungi-From Taxonomy to Modern Biochemistry and Molecular Biology.

Phytopathogenic cercosporoid fungi have been investigated comprehensively due to their important role in causing plant diseases. A significant amount of research has been focused on the biology, morphology, systematics, and taxonomy of this group, with less of a focus on molecular or biochemical issues. Early and extensive research on these fungi focused on taxonomy and their classification based on in vivo features. Lately, investigations have mainly addressed a combination of characteristics such as morphological traits, host specificity, and molecular analyses initiated at the end of the 20th century. Some species that are important from an economic point of view have been more intensively investigated by means of genetic and biochemical methods to better understand the pathogenesis processes. Cercosporin, a photoactivated toxin playing an important role in Cercospora diseases, has been extensively studied. Understanding cercosporin toxicity in relation to reactive oxygen species (ROS) production facilitated the discovery and regulation of the cercosporin biosynthesis pathway, including the gene cluster encoding pathway enzymes. Furthermore, these fungi may be a source of other biotechnologically important compounds, e.g., industrially relevant enzymes. This paper reviews methods and important results of investigations of this group of fungi addressed at different levels over the years.

Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes.

BACKGROUND: Mycosphaerella fijiensis, causative agent of the black Sigatoka disease of banana, is considered the most economically damaging banana disease. Despite its importance, the genetics of pathogenicity are poorly understood. Previous studies have characterized polyketide pathways with possible roles in pathogenicity. To identify additional candidate pathogenicity genes, we compared the transcriptome of this fungus during the necrotrophic phase of infection with that during saprophytic growth in medium. RESULTS: Transcriptome analysis was conducted, and the functions of differentially expressed genes were predicted by identifying conserved domains, Gene Ontology (GO) annotation and GO enrichment analysis, Carbohydrate-Active EnZymes (CAZy) annotation, and identification of genes encoding effector-like proteins. The analysis showed that genes commonly involved in secondary metabolism have higher expression in infected leaf tissue, including genes encoding cytochrome P450s, short-chain dehydrogenases, and oxidoreductases in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Other pathogenicity-related genes with higher expression in infected leaf tissue include genes encoding salicylate hydroxylase-like proteins, hydrophobic surface binding proteins, CFEM domain-containing proteins, and genes encoding secreted cysteine-rich proteins characteristic of effectors. More genes encoding amino acid transporters, oligopeptide transporters, peptidases, proteases, proteinases, sugar transporters, and proteins containing Domain of Unknown Function (DUF) 3328 had higher expression in infected leaf tissue, while more genes encoding inhibitors of peptidases and proteinases had higher expression in medium. Sixteen gene clusters with higher expression in leaf tissue were identified including clusters for the synthesis of a non-ribosomal peptide. A cluster encoding a novel fusicoccane was also identified. Two putative dispensable scaffolds were identified with a large proportion of genes with higher expression in infected leaf tissue, suggesting that they may play a role in pathogenicity. For two other scaffolds, no transcripts were detected in either condition, and PCR assays support the hypothesis that at least one of these scaffolds corresponds to a dispensable chromosome that is not required for survival or pathogenicity. CONCLUSIONS: Our study revealed major changes in the transcriptome of Mycosphaerella fijiensis, when associating with its host compared to during saprophytic growth in medium. This analysis identified putative pathogenicity genes and also provides support for the existence of dispensable chromosomes in this fungus.

Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin.

The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants.

Genetic Characteristics and Metabolic Interactions between Pseudocercospora fijiensis and Banana: Progress toward Controlling Black Sigatoka.

The international importance of banana and severity of black Sigatoka disease have led to extensive investigations into the genetic characteristics and metabolic interactions between the Dothideomycete Pseudocercospora fijiensis and its banana host. P. fijiensis was shown to have a greatly expanded genome compared to other Dothideomycetes, due to the proliferation of retrotransposons. Genome analysis suggests the presence of dispensable chromosomes that may aid in fungal adaptation as well as pathogenicity. Genomic research has led to the characterization of genes and metabolic pathways involved in pathogenicity, including: secondary metabolism genes such as PKS10-2, genes for mitogen-activated protein kinases such as Fus3 and Slt2, and genes for cell wall proteins such as glucosyl phosphatidylinositol (GPI) and glycophospholipid surface (Gas) proteins. Studies conducted on resistance mechanisms in banana have documented the role of jasmonic acid and ethylene pathways. With the development of banana transformation protocols, strategies for engineering resistance include transgenes expressing antimicrobial peptides or hydrolytic enzymes as well as host-induced gene silencing (HIGS) targeting pathogenicity genes. Pseudocercospora fijiensis has been identified as having high evolutionary potential, given its large genome size, ability to reproduce both sexually and asexually, and long-distance spore dispersal. Thus, multiple control measures are needed for the sustainable control of black Sigatoka disease.

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis.

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.

A polyketide synthase gene cluster required for pathogenicity of Pseudocercospora fijiensis on banana.

Pseudocercospora fijiensis is the causal agent of the highly destructive black Sigatoka disease of banana. Previous research has focused on polyketide synthase gene clusters in the fungus, given the importance of polyketide pathways in related plant pathogenic fungi. A time course study of expression of the previously identified PKS7-1, PKS8-2, and PKS10-2 gene clusters showed high expression of all three PKS genes and the associated clustered genes in infected banana plants from 2 weeks post-inoculation through 9 weeks. Engineered transformants silenced for PKS8-2 and PKS10-2 were developed and tested for pathogenicity. Inoculation of banana plants with silencing transformants for PKS10-2 showed significant reduction in disease symptoms and severity that correlated with the degree of silencing in the conidia used for inoculation, supporting a critical role for PKS10-2 in disease development. Unlike PKS10-2, a clear role for PKS8-2 could not be determined. Two of four PKS8-2 silencing transformants showed reduced disease development, but disease did not correlate with the degree of PKS8-2 silencing in the transformants. Overall, the degree of silencing obtained for the PKS8-2 transformants was less than that obtained for the PKS10-2 transformants, which may have limited the utility of the silencing strategy to identify a role for PKS8-2 in disease. Orthologous PKS10-2 clusters had previously been identified in the related banana pathogens Pseudocercospora musae and Pseudocercospora eumusae. Genome analysis identified orthologous gene clusters to that of PKS10-2 in the newly sequenced genomes of Pseudocercospora fuligena and Pseudocercospora cruenta, pathogens of tomato and cowpea, respectively. Our results support an important role for the PKS10-2 polyketide pathway in pathogenicity of Pseudocercospora fijiensis, and suggest a possible role for this pathway in disease development by other Pseudocercospora species.

Engineering Cercospora disease resistance via expression of Cercospora nicotianae cercosporin-resistance genes and silencing of cercosporin production in tobacco.

Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.

The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

A novel polyketide synthase gene cluster in the plant pathogenic fungus Pseudocercospora fijiensis.

Pseudocercospora fijiensis, causal agent of black Sigatoka of banana, produces polyketide synthase (PKS) pathways shown to be important in disease development by related Dothideomycete fungi. Genome analysis of the P. fijiensis PKS8-1 gene identified it as part of a gene cluster including genes encoding two transcription factors, a regulatory protein, a glyoxylase/beta-lactamase-like protein, an MFS transporter, a cytochrome P450, two aldo/keto reductases, a dehydrogenase, and a decarboxylase. Genome analysis of the related pathogens Pseudocercospora musae, Pseudocercospora eumusae, and Pseudocercospora pini-densiflorae, identified orthologous clusters containing a nearly identical combination of genes. Phylogenetic analysis of PKS8-1 identified homology to PKS proteins in the monodictyphenone and cladofulvin pathways in Aspergillus nidulans and Cladosporium fulvum, respectively. Analysis of clustered genes showed that the PKS8-1 cluster shares genes for enzymes involved in the production of the emodin intermediate in the monodictyphenone and cladofulvin pathways, but differs in many genes, suggesting production of a different metabolic product. Time course analysis of gene expression in infected banana showed up-regulation of PKS8-1 and four of eight clustered genes as early as 2 weeks post-inoculation and remaining high through 9 weeks. Overexpression of the pathway through constitutive expression of an aflR-like transcription factor gene in the cluster resulted in increased expression in culture of PKS8-1 as well as the four clustered genes that are up-regulated in infected plants. No differences were seen in timing or severity of disease symptoms with the overexpression strains relative to controls, however gene expression analysis showed no difference in expression in planta by an overexpression strain relative to controls. Thus constitutive expression of the aflR-like gene is not sufficient to upregulate the pathway above normal expression in planta. Pathway expression during all phases of disease development and conservation of the pathway in related Pseudocercospora species support a role for this pathway in disease.

A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.

Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata.

Mannitol has been hypothesized to play a role in antioxidant defense. In previous work, we confirmed the presence of the two mannitol biosynthetic enzymes, mannitol dehydrogenase (MtDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH), in the fungus Alternaria alternata and created disruption mutants for both enzymes. These mutants were used to investigate the role of mannitol in pathogenicity of A. alternata on its host, tobacco. Conidia of all mutants were viable and germinated normally. GC-MS analysis demonstrated elevated levels of trehalose in the mutants, suggesting that trehalose may substitute for mannitol as a storage compound for germination. Tobacco inoculation showed no reduction in lesion severity caused by the MtDH mutant as compared with wild type; however, the MPDH mutant and a mutant in both enzymes caused significantly less disease. Microscopy analysis indicated that the double mutant was unaffected in the ability to germinate and produce appressoria on tobacco leaves and elicited a defense response from the host, indicating that it was able to penetrate and infect the host. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for spore germination either in vitro or in planta or for initial infection.

Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains.

Plant pathogens from the genus Cercospora produce cercosporin, a photoactivated fungal toxin that generates toxic reactive oxygen species. Mechanisms governing toxin auto-resistance in Cercospora spp. are poorly understood. In this work, suppressive subtractive hybridization was used to identify genes differentially expressed between the cercosporin-resistant wild-type (WT) Cercospora nicotianae and a sensitive strain lacking a transcription factor (CRG1) that regulates resistance. Out of 338 sequences recovered, 185 unique expressed sequence tags (ESTs) were obtained and classified into functional categories. The majority of genes showed predicted expression differences, and 38.5% were differentially expressed at least twofold between the WT and mutant strain. ESTs were recovered with homology to genes involved in detoxification of noxious compounds, multidrug membrane transporters and antioxidant and polyketide biosynthetic enzymes as well as to ATPases and ATP synthases. The findings suggest that CRG1 regulates genes involved in pH responses in addition to those involved in toxin resistance and biosynthesis.

Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress.

Vitamin B(6) (pyridoxine and its vitamers) plays an essential role as a co-factor for enzymatic reactions and has also recently been implicated in defense against cellular oxidative stress. The biosynthetic pathway was thoroughly characterized in Escherichia coli, however most organisms, including plants, utilize an alternate pathway involving two genes, PDX1 and PDX2. Arabidopsis thaliana contains one copy of PDX2, but three full-length copies of PDX1, one each on chromosomes 2, 3, and 5 (referred to as PDX1.1, PDX1.2, and PDX1.3, respectively). Phylogenetic analysis of the PDX1 homologues in A. thaliana showed that PDX1.1 and PDX1.3 clustered with the homologues from the other dicots, whereas PDX1.2 was more divergent, and did not cluster with either the dicots or monocots. Expression analysis using quantitative PCR showed that PDX1.1 and PDX1.3 were highly expressed in A. thaliana rosettes, while PDX1.2 showed only low level expression. All three PDX1 genes and PDX2 were responsive to abiotic stressors including high light, chilling, drought, and ozone, however, the response of PDX1.2 was disparate from that of the other PDX genes, showing a lessened response to high light, chilling, and drought, but an increased response to ozone. Green fluorescent protein fusion studies demonstrated that PDX2 localizes in the nucleus and membranes of cells, consistent with recent published data for PDX1. Insight into regulation of the biosynthetic genes during abiotic stress could have important applications in the development of stress-tolerant crops.

Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode polyketides important in pathogenicity.

The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae.

Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.

Photoactivated perylenequinone toxins in fungal pathogenesis of plants.

Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.

Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin.

Functional complementation between the PDX1 vitamin B6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli.

The pathway for de novo vitamin B(6) biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2, have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae. Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B(6) pathway encodes a protein responsible for synthesis of the pyridoxine ring.

Direct detection of singlet oxygen via its phosphorescence from cellular and fungal cultures.

In summary, we have developed methods to detect 1O2 spectrally from viable keratinocytes and fungal mycelial cultures. Both time-resolved and steady-state spectrophotometers were used, providing complementary information on optimal conditions for the unambiguous detection of 1O2 phosphorescence. By using our techniques, we were able to confirm that photosensitizers in contact with living cells indeed generate 1O2. The model systems and the procedures described can be adopted for other studies involving 1O2 and oxidative stress in living cells.