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Keywords:

  • Plant disease;
  • Non-specific toxin;
  • Active oxygen;
  • Pyridoxine;
  • Zinc cluster transcription factor

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

Plant pathogenic fungi utilize multiple strategies for infection of host plants. Among them is the production of toxins. Toxins produced by plant pathogenic fungi differ in structure as well as in their role in disease and mode of action[1]. Toxins play diverse roles in disease, from impacting symptom expression and disease progress to being absolutely required for pathogenesis. Some toxins are directly toxic, killing cells and allowing for infection of dead cells. Others interfere with induction of defense responses or induce programmed cell death-mediated defense responses in order to generate necrosis required for pathogenesis[2].

One very interesting group of toxins are the photoactivated perylenequinones. These toxins act by absorbing light energy and generating reactive oxygen species which damage host cells. Studies with toxin-deficient mutants as well as observations on the importance of light in symptom expression have documented the importance of these toxins in pathogenesis, and have led to investigations of toxin resistance as a means of generating resistance to disease. The production of highly reactive oxygen species and the autoresistance by the producing fungus have also led to the use of these fungi as models for understanding cellular resistance to singlet oxygen and photosensitizing compounds. This review focuses on our state of knowledge of these compounds, with an emphasis on cercosporin, the toxin produced by fungi in the genus Cercospora.

2Production and mode of action of fungal perylenequinone toxins

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

To date, all of the identified perylenequinone toxins are produced by members of the Ascomycota, the largest phylum within the fungal kingdom. Most producers are classified within the Loculoascomycetes; however, recent reports have documented production by a Pyrenomycete (Hypomyces) as well as a Discomycete lichen (Graphis) (Table 1). All of the perylenequinones identified to date share a basic 3,10-dihydroxy-4,9-perylenequinone chromophore and differ mainly in side chains (Fig. 1). These fungal compounds show close structural similarity and mode of action to the extended quinones produced by protozoans and plants, for example, hypericin (Fig. 1), the active compound produced by the medicinal herb St. John's wort[3].

Table 1. Production of perylenequinones by fungi
Fungal speciesEcological nicheCompound(s)References
Alternaria speciesDecay saprophytes on fruits and vegetables; pathogens of water hyacinth and spotted knapweedAlteichin [4–6]
  A. alternata Alterlosins 
  A. eichorniae Altertoxins 
  Stemphyltoxin III 
Cercospora speciesPlant pathogens (multiple host species)Cercosporin [7]
  Isocercosporin 
Cladosporium cucumerinum Saprophyte; pathogens of cucumber, sugar beet, and timothyCalphostin C [8–11]
C. cladosporioides  Cladochrome 
C. herbarum  Ent-isophleichrome 
C. phlei  Phleichrome 
Elsinoe speciesPathogens of citrus, other plant speciesElsinochromes [11]
Graphis hematites LichenIsohypocrellin [12]
Hypocrella bambusae Pathogen of bambooHypocrellins [11,13]
Hypomyces speciesPathogen of mushrooms, shelf fungiHypomycin A [14]
Scolecotrichum graminis Pathogen of orchardgrasssCercosporin [15]
  Isocercosporin 
  Acetylisocercosporin 
Shiraia bambusicola Pathogen of bambooShiraiachromes [16]
Stemphylium botryosum Saprophyte; plant pathogen (multiple host species)Stemphyltoxins [4]
  Altertoxins 
image

Figure 1. Structures of representative fungal perylenequinone toxins and the plant extended quinone hypericin.

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The similarity of the fungal perylenequinone structure to hypericin led to investigations of the fungal compounds as photosensitizers [17,18]. Photosensitizers are structurally diverse compounds classified together based on their common ability to be activated by visible and UV-A wavelengths of light and to generate active oxygen species[19]. Many biological molecules are photosensitizers. These include compounds important in human biology such as riboflavin and porphyrins, plant-derived compounds such as chlorophyll, coumarins, and acetylenes, and common dyes such as acridine orange, rose bengal, and methylene blue. In nature, photosensitizers play diverse roles as defense compounds in plants, pathogenesis determinants in fungi, and as molecules responsible for photomovement of protozoans[20].

Photosensitizers absorb light and are converted to an electronically active triplet state. Triplet state photosensitizers react in one of two ways. They may react by electron transfer (radical) reactions via a reducing substrate (type I reaction), leading to the production of a reduced sensitizer molecule. This molecule may react directly with cellular molecules or with oxygen, leading to the production of lipid free radicals and active oxygen species such as superoxide (O2), hydrogen peroxide (H2O2), and the hydroxyl radical (OH)[21]. Alternatively, the triplet sensitizer may react directly with oxygen by an energy transfer mechanism (type II reaction)[19] leading to the production of the non-radical, but highly toxic singlet oxygen (1O2). Almost all macromolecules in cells are susceptible to oxidative damage caused by photosensitizers. Most commonly, photosensitizers damage lipids, proteins, and DNA, with the type of damage being determined by where the photosensitizer molecule localizes in cells such as in the membranes, cytoplasm or nucleus[22].

3Cercosporin

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

Our laboratories study the perylenequinone toxin cercosporin (Fig. 1), produced by many members of the genus Cercospora. Cercospora species are a highly successful group of pathogens that cause disease on a diversity of plants world-wide, including corn, sugar beet, tobacco, coffee, and soybean, as well as many ornamental and weed species. Cercospora species cause major economic problems due to their world-wide distribution and wide host range, and also due to the lack of adequate levels of resistance in many hosts. One of the reasons for the success of these pathogens appears to be their production of cercosporin. Cercosporin is produced by many members of the genus and has near universal toxicity to plants[7]. For many years it was observed that high light intensities were required for symptom development on infected plants, as leaves that were shaded did not develop symptoms [23,24]. This effect was so striking that it led to the recommendation in the 1940s that bananas be grown under the shade of coconut palms to protect against disease[25]. Although these observations suggested that some type of light-activated compound was involved in disease development, it was not until the 1970s that cercosporin was identified as a phototoxin produced by the pathogen [26,27].

Cercosporin was first isolated in the 1950s as an interesting red pigment from Cercospora kikuchii, a soybean pathogen[28]. Cercosporin has since been isolated from a large number of Cercospora species and Cercospora-infected plants[7]. Its structure was determined in the 1970s[27]. Cercosporin is red in color, is sparingly soluble in water, but readily soluble in base and in several organic solvents. Because of its limited water-solubility, red crystals of cercosporin accumulate in culture and are readily visible (Fig. 2).

image

Figure 2. Production of cercosporin by mycelium of Cercospora nicotianae in culture. (a) Underside of mycelial colonies showing red pigmentation due to cercosporin. Arrow indicates cercosporin-deficient mutant. Panel a reprinted from Fig. 1 of Chung et al.[41] with kind permission of Springer Science and Business Media. (b) Microscopic view of Cercospora hyphae showing crystals of cercosporin excreted by the fungus. Hyphae of cercosporin-producing cultures emit a green fluorescence indicative of reduced cercosporin.

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Cercosporin's photosensitizing activity was first reported by Yamazaki et al.[18] who demonstrated that cercosporin toxicity to mice and bacteria was light-dependent and required oxygen. Subsequently, cercosporin was shown to generate active oxygen species. Cercosporin is a potent producer of singlet oxygen (1O2) with measured 1O2 quantum yields (yield of 1O2 generated per photon of light absorbed by the photosensitizer) of 0.81–0.97 [29,30]. Cercosporin is also capable of producing superoxide (O2) when incubated in the presence of reducing agents[31]; however, it is not a strong type I sensitizer. The importance of 1O2 production in vivo was demonstrated by the efficient protection of cultured plant cells by quenchers of 1O2[17]. The role of radical and reduced forms of oxygen (O2, H2 O2) in cercosporin toxicity in vivo is less clear.

Due to its production of active oxygen species, cercosporin is almost universally toxic. In addition to plants, cercosporin toxicity has been demonstrated against mice, gram-positive and gram-negative bacteria, fungi, and Oomycetes [18,32]. Cercosporin has also been shown to have antiviral activity and to be an efficient inhibitor of protein kinase C [33,34]. In plants, treatment of plant cells and tissues with cercosporin in the light results in very rapid (1–2 min) damage to membranes assayed by measuring leakage of electrolytes from tissues and the bursting of protoplasts[35]. Cells damaged by cercosporin show an accumulation of lipid peroxidation products, a marked increase in the ratio of saturated to unsaturated fatty acids, and a decrease in plant protoplast membrane fluidity, changes characteristic of membranes damaged by lipid peroxidation[36].

4Biosynthesis of cercosporin

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

The first studies of cercosporin biosynthesis were done in the 1970s by Okubo et al.[37], who demonstrated that synthesis begins with the condensation of acetate and malonate and proposed that cercosporin was synthesized via the polyketide pathway. In recent years, considerable efforts have focused on studying the regulation of the biosynthetic pathway and on identification of pathway and regulatory genes. Cercosporin production is regulated in culture. Light is absolutely required, not only for activity, but also for cercosporin production[38]. Cercosporin biosynthesis is repressed at high temperatures (30 °C) and is also regulated by nutrients, though nutritional regulation varies with the species[39]. Cercosporin is produced only in vegetative cultures, and is suppressed under conditions that induce sporulation.

Thus far the genes encoding the first and last steps in the pathway as well as several regulatory genes have been identified. Upchurch and co-workers identified the gene required for export of cercosporin out of the fungal hyphae by taking advantage of the requirement of light for cercosporin biosynthesis. They used subtractive hybridization to identify genes that are upregulated in light-grown cultures[38]. They identified cercosporin facilitator protein (CFP), a gene encoding a 65.4 kDa protein with homology to the major facilitator superfamily (MFS) of membrane transport proteins[40]. Mutants disrupted for CFP produce less than 5% of the cercosporin produced by the wild type strain.

To identify additional biosynthetic genes, we adapted an insertional (REMI) mutagenesis strategy and screened for toxin-deficient mutants (Fig. 2a)[41]. Recovery of flanking sequences from a REMI mutant completely deficient in cercosporin production led to the identification of a cercosporin toxin biosynthesis gene (CTB1) encoding the polyketide synthase responsible for cercosporin biosynthesis[42]. CTB1 encodes a predicted protein of 2196 amino acids with keto synthase, acyltransferase, thioesterase/claisen cyclase, and two acyl carrier protein domains, and is highly homologous to many fungal type I polyketide synthases. CTB1 expression was shown to be highly regulated by light and medium composition, correlating with conditions required for cercosporin production.

Efforts are currently underway to identify possible genes required for the intermediate steps between CTB1 and CFP by sequence analysis of genes clustered with CTB1 (K.R. Chung, unpublished). To date, nine putative open reading frames with amino acid similarities to methyltransferases, FAD/FMN, oxygen-dependent oxidoreductases, NADPH-dependent oxidoreductases, MFS transporters, transcription activators, and signal peptidases have been identified. Functional characterization of these genes in relation to cercosporin biosynthesis are in progress.

Two regulatory genes important in cercosporin production have also been identified. CRG1, a gene encoding a putative zinc cluster transcription factor, was initially identified by complementation of a cercosporin-sensitive Cercospora mutant, and thus was identified as a gene involved in cercosporin resistance[43]. Mutants disrupted for CRG1 are also delayed in cercosporin production, indicating a link to biosynthesis. In addition, Shim and Dunkle[44] used suppression subtractive hybridization to identify transcripts specific to cercosporin-producing cultures of C. zeae-maydis and identified CZK3, encoding a map kinase kinase kinase. CZK3 disruption mutants are deficient in both cercosporin production and in conidiation.

5Role of cercosporin and other perylenequinones in plant disease

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

Several lines of evidence document an important role for cercosporin and other perylenequinones in disease. Cercosporin is produced by many members of the genus and can be isolated from lesions on infected plants, confirming production during disease development. Ultrastructural studies of cellular damage occurring in Cercospora-infected sugar beet leaves show membrane damage as the primary ultrastructural symptom, consistent with the membrane-damaging activity of cercosporin[45]. Studies on infection of plants by Cercospora, Mycosphaerella, and Alternaria species have also shown strong correlations between disease severity and both high light intensities and day length. For example, fewer lesions are produced by C. coffeicola on shaded coffee leaves[24]. Disease ratings are reduced and symptom expression is delayed on C. beticola-infected sugar beet leaves when plants are grown under low light[23]. Banana plants infected with Mycosphaerella musicola develop lesions only on leaves exposed to high light intensities[46]. In these latter two cases, light did not affect penetration of the fungus into leaves, but was required for lesion development following penetration. Exposure to bright sunlight also increased lesion number and percent infected leaf area and decreased symptomless infection in cotton infected by Alternaria alternata[47].

With cercosporin, studies with toxin-deficient mutants have consistently confirmed an important role in disease development. Mutants of C. kikuchii disrupted for the CFP cercosporin transporter gene were significantly reduced in both cercosporin production and in symptom development on soybean[40]. Disruption mutants of the CZK3 MAPKKK in C. zeae-maydis, which are deficient in both cercosporin biosynthesis and conidiation, caused only limited small chlorotic lesions on corn as compared to the large necrotic lesions generated by the wild type strain[44]. In our work, we have tested both UV-generated and ctb1 polyketide synthase disruption mutants (Fig. 3). UV mutagenesis was used to generate C. kikuchii mutants deficient in cercosporin production both in vitro and in vivo; these mutants produced only few pinpoint lesions when inoculated onto soybean as compared to the numerous, larger necrotic lesions with accompanying chlorosis produced by wild type[48]. Isolation of CTB1 allowed for the isolation of C. nicotianae cercosporin-minus mutants by targeted gene disruption[42]. These mutants produced significantly fewer and smaller lesions on tobacco as compared to wild type, and also lacked the coalescing of lesions that leads to blighting symptoms on the leaves. Taken together these results firmly support the conclusion that cercosporin is an important virulence factor for Cercospora pathogens.

image

Figure 3. (a) Symptoms caused by Cercospora kikuchii wild type (left) and UV-mutagenized cercosporin-minus mutant (right) on soybean. (b) Symptoms caused by Cercospora nicotianae wild type (left) and cercosporin polyketide synthase disruption mutant (right) on tobacco. Toxin-deficient mutants produce significantly fewer and smaller lesions than do the wild type strains. Panel b reprinted from Choquer et al.[42].

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Although it is clear cercosporin is required for normal disease development, the specific manner in which it does so is not completely known. Cercosporin's pronounced toxicity and documented mode of action led us to hypothesize that cercosporin kills the cells of the host and allows access to nutrients through peroxidation of membrane lipids, leading to leakage of nutrients into the intercellular spaces where the fungal hyphae grow[7]. Dickman et al.[49], however, showed that expression of animal anti-apoptotic genes in tobacco provides protection against necrotrophic pathogens including C. nicotianae. They hypothesized that necrotrophs, rather than directly killing the cells of the host, induce the host's own apoptotic pathways to cause the host cell death required for colonization by the pathogen. Thus whether cercosporin exerts its activity by direct toxicity or functions by somehow inducing plant apoptotic pathways remains to be determined.

6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

Although photosensitizers are commonly-occurring compounds, surprisingly little is known about the cellular basis of resistance and the genes that encode resistance traits. What is known in general about defense against photosensitization is focused in two major areas: mechanisms used by insects and protozoans that are exposed to photosensitizers, and chemical compounds that quench 1O2. Protozoans synthesize photosensitizers to serve as receptor pigments for photomovement; when exposed to high light intensities, these organisms protect themselves by photophobic responses, shading under debris, and irreversible detoxification of the compounds[50]. Insects that feed on photosensitizer-containing plants have evolved light-protective responses such as feeding within rolled leaves or secreting protective webs[51]. Others, e.g., insects that feed on furanocumarin-containing plants, degrade the compounds via a cytochrome P450 enzyme[52].

The other major area of study on photosensitizer defense focuses on the identification and characterization of compounds that quench 1O2. Many 1O2 quenchers have been identified through chemical studies[53]. Only a few of these compounds are found in biological systems, and for those that are, few have been documented to play a role in cell defense. The best characterized compounds responsible for photosensitizer protection are carotenoids [54,55]. Carotenoids quench both 1O2 and the triplet state of photosensitizers and are the most efficient quenchers identified of those that exist in biological systems. Carotenoid pigments are the major means used by plants to protect chloroplasts against 1O2 that is an unavoidable by-product of photosynthesis[56]. Other less effective biological quenchers of 1O2 include thiols, amino acids such as histidine, methionine, and tryptophan, and some peptides, amines, and phenols[53]. We have recently shown that pyridoxine (vitamin B6) and its vitamers quench 1O2[57]. For most compounds, quenching activity is tested in vitro, however, and there is little evidence for a role in cells. Other than the importance of carotenoids in protection of chloroplasts against chlorophyll-produced 1O2, the basis of resistance in plants to endogenous photosensitizers (e.g., hypericin, furanocoumarins, thiophenes, acetylenes) is not understood.

We have been studying the resistance of Cercospora fungi to cercosporin as a model for understanding cellular resistance to photosensitizers and to 1O2. Cercosporin is toxic to mice, plants, bacteria, oomycetes, and many fungi. The only organisms that have been repeatedly shown to have resistance to cercosporin are Cercospora species themselves and other perylenequinone-producing fungi such as Alternaria and Cladosporium[32]. Cercospora species can produce up to mM concentrations of cercosporin in light-grown cultures without any measurable decrease in growth. Cercospora species also grow normally on medium supplemented with high concentrations (10–100 μM) of other structurally diverse 1O2-generating photosensitizers including porphyrins and xanthene and thiazine dyes [58,59]. This resistance is surprising given the toxicity of 1O2, and provides a unique system for understanding the cellular and genetic basis of 1O2 resistance.

Early research investigated and discarded some possible resistance mechanisms (for review see[7]). The fungus secretes the compound and utilizes the active molecule for infection of plants; thus, irreversible detoxification or compartmentalization of cercosporin cannot be a mechanism of resistance. Membranes of Cercospora fungi contain fatty acids susceptible to peroxidation. Levels and activity of antioxidant enzymes (superoxide dismutase, catalase, general peroxidase activity) and reducing substances (ascorbate, cysteine, reduced glutathione, total soluble and protein thiols) do not differ between cercosporin-resistant and sensitive fungi. Further, carotenoid production is not required for resistance in Cercospora. Although resistance of Neurospora and Phycomyces carotenoid-deficient and overproduction mutants can be correlated with carotenoid content, carotenoid-deficient mutants of C. nicotianae disrupted for phytoene dehydrogenase, a key enzyme in the carotenoid biosynthetic pathway, are unaltered in their resistance to cercosporin or to other photosensitizers. Based on these studies, we concluded that Cercospora species have novel and undefined mechanisms of 1O2 and photosensitizer resistance. Efforts in our labs and others over many years suggest that resistance is complex and due to multiple factors. These include toxin export, transient reduction and detoxification, and the role of quenchers.

6.1Toxin export

A number of studies have identified a role for membrane transport proteins in cercosporin resistance. Most work has been done by Upchurch and co-workers on CFP, the cercosporin transporter described earlier. In addition to its requirement for cercosporin production, CFP appears to play a role in cercosporin resistance. Growth of cfp disruption mutants on cercosporin-containing medium was inhibited by approximately 40% as compared to wild type[40]. Further, expression of CFP in the cercosporin-sensitive fungus Cochliobolus heterostrophus decreased the sensitivity of this fungus to cercosporin[60]. Other transport proteins have also been implicated in cercosporin resistance. In yeast, over-expression of Snq2p, a gene encoding a multidrug ATP-binding cassette (ABC) efflux protein, conferred resistance to cercosporin[61]. Botrytis cinerea mutants disrupted for the MFS transporter gene Bcmfs1 showed increased sensitivity to cercosporin[62]. In preliminary work (Herrero and Daub, unpublished), we have also identified two transporter homologues (an MFS and an ABC) in a C. nicotianae subtractive library representing transcripts differentially expressed between wild type and a cercosporin-sensitive mutant (see Section 7 below).

6.2Reductive detoxification

Extensive studies also support a role for transient reduction and detoxification of cercosporin as a major mechanism of cercosporin resistance. These studies were based on the observation that hyphae of cultures of cercosporin-resistant fungi producing or treated with cercosporin emit a green fluorescence distinct from the red fluorescence characteristic of cercosporin (Fig. 2b), suggesting that cercosporin in contact with hyphae of these species was derivatized in some way. Confocal and fluorescence microscopy using specific band-width filters demonstrated that the green fluorescence emitted by hyphae of Cercospora spp. and Alternaria alternata was consistent with a reduced form of cercosporin [30,58]. By contrast, cercosporin-treated cultures of a sensitive fungus (Aspergillus flavus) and non-viable cultures of Cercospora (killed by heat, chloroform vapor or UV light) emitted red fluorescence typical of cercosporin. Reduced cercosporin is very labile and readily re-oxidizes upon aeration or extraction away from strong reducing agents. Characterization of stable methylated and acetylated reduced derivatives demonstrated that they are significantly decreased in light absorption and are also poor generators of 1O2. Singlet oxygen quantum yields from reduced derivatives are very low, ranging from 0.02 to 0.18 vs. 0.87 to 0.97 for cercosporin, depending on the solvent [30,63]. The lowest yields were in aqueous solvents (D2O). Thus reduced cercosporin is a very poor photosensitizer, particularly in an aqueous environment such as in the cell.

Estimates of cell surface reducing activity (based on differential ability to reduce tetrazolium dyes spanning a range of redox potentials) support the hypothesis of reductive detoxification as a mechanism of resistance. Cercosporin-resistant fungi (Cercospora spp., A. alternata) were able to reduce significantly more dyes and dyes that are harder to reduce than were the cercosporin-sensitive fungi A. flavus and Neurospora crassa[64]. Measurement of cercosporin's formal redox potential by cyclic voltammetry identified a potential of −0.14 V[58], a value indicating that cercosporin is difficult to reduce, but is nonetheless a potential well within the capability of the normal reducing ability of cells.

Based on these studies we proposed[58] that cercosporin resistance is mediated, at least in part, by the ability of Cercospora species to transiently reduce cercosporin in contact with hyphae. Since reduction is not stable, cercosporin excreted by the cell would spontaneously re-oxidize into a form that is photoactive and toxic to host cells. Interestingly, Ververidis et al.[61] have identified a FAD-dependent pyridine nucleotide reductase homolog in yeast that, when over-expressed, imparts resistance to cercosporin and to other 1O2-generating photosensitizers, supporting a role for reduction in resistance.

6.3Quenching by pyridoxine

Isolation of genes required for resistance to cercosporin in C. nicotianae identified a novel role for pyridoxine (vitamin B6) as a quencher involved in cercosporin resistance. Putative resistance genes were identified by complementing UV-generated cercosporin-sensitive mutants with a genomic library from the wild-type strain[59]. Two of the genes recovered showed strong homology to sequences from organisms across multiple kingdoms and were subsequently determined to encode steps in the pyridoxine biosynthesis pathway[65]. Pyridoxal-5′-phosphate, the active form of vitamin B6, is recognized as an essential enzyme cofactor for transamination and other reactions, but had not been shown to be a cellular antioxidant. However, we showed that 1O2 quenching rate constants (Kq) calculated for pyridoxine, pyridoxal, pyridoxal phosphate, and pyridoxamine were in the range of 107–108 M−1 s−1, a rate comparable to those of vitamins C and E, two of the most efficient biological antioxidants known [57,65]. Pyridoxine also quenches superoxide[66].

7Identification of other resistance genes

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

Current efforts are focused on the identification of additional genes and mechanisms involved in cercosporin resistance by characterizing genes regulated by a transcription factor required for resistance. Cercosporin resistance gene 1 (CRG1) was identified in C. nicotianae and encodes a putative zinc cluster transcription factor[43]. Mutants disrupted for crg1 show significantly reduced growth when plated on medium containing cercosporin, and we hypothesize that the CRG1 protein regulates genes required for cercosporin resistance. Suppression subtractive hybridization (SSH) was used to identify cDNA's differentially expressed between the wild type C. nicotianae and a crg1 disruption mutant (Herrero and Daub, unpublished). A total of 206 unique sequences were identified and assigned to functional categories. Among those of interest are several assigned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) category of “biodegradation of xenobiotics”, including homologues to a cyanide hydratase, several ABC and MFS transporters, and several oxidative stress-related genes. Also of interest is a putative activator of CRG1 that has homology to AFLJ, a gene encoding an activator of the aflatoxin biosynthesis regulator AFLR. The importance of these genes in cercosporin resistance is being determined by regulation studies and targeted gene disruption experiments.

In addition to the subtractive hybridization studies, we are also investigating if genes found with the CTB1 biosynthetic cluster (described in Section 4 above) may play a role in resistance (K.R. Chung, unpublished). Previous studies have shown that cercosporin biosynthesis and resistance are linked. For example, both the CRG1 regulator and the CFP transporter are required for resistance in addition to production. We hypothesize that important resistance genes may be found in the biosynthetic cluster.

8Summary and conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References

Several highly successful genera of plant pathogenic fungi use photosensitizing perylenequinone toxins for plant pathogenesis. As such, they have evolved a uniquely clever strategy for parasitism of plants, which require light for growth and survival. Studies on the cercosporin biosynthetic pathway have shown that it is produced via the polyketide pathway, an important pathway of secondary metabolism in fungi. Significant progress is being made in identifying biosynthetic and regulatory genes, which is contributing to our knowledge of polyketide synthesis and regulation in fungi. In addition, studies of the resistance of these fungi to cercosporin have led to a broader understanding of cellular resistance to photosensitizers and to singlet oxygen, discoveries which will impact medicine and biology in addition to agriculture. Our goal is to utilize an understanding of these toxins, their mode of action, and mechanisms of resistance, to devise novel strategies for control of these important pathogens.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Production and mode of action of fungal perylenequinone toxins
  5. 3Cercosporin
  6. 4Biosynthesis of cercosporin
  7. 5Role of cercosporin and other perylenequinones in plant disease
  8. 6Cercosporin as a model for understanding cellular resistance to photosensitizers and singlet oxygen
  9. 7Identification of other resistance genes
  10. 8Summary and conclusions
  11. Acknowledgements
  12. References
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