Deletion of a MFS transporter-like gene in Cercospora nicotianae reduces cercosporin toxin accumulation and fungal virulence

Many phytopathogenic Cercospora species produce a host-nonselective polyketide toxin, called cercosporin, whose toxicity exclusively relies on the generation of reactive oxygen species. Here, we describe a Cercospora nicotianae CTB4 gene that encodes a putative membrane transporter and provide genetic evidence to support its role in cercosporin accumulation. The predicted CTB4 polypeptide has 12 transmembrane segments with four conserved motifs and has considerable similarity to a wide range of transporters belonging to the major facilitator superfamily (MFS). Disruption of the CTB4 gene resulted in a mutant that displayed a drastic reduction of cercosporin production and accumulation of an unknown brown pigment. Cercosporin was detected largely from fungal hyphae of ctb4 disruptants, but not from the surrounding medium, suggesting that the mutants were defective in both cercosporin biosynthesis and secretion. Cercosporin purified from the ctb4 disruptants exhibited toxicity to tobacco suspension cells, insignificantly different from wild-type, whereas the disruptants formed fewer lesions on tobacco leaves. The ctb4 null mutants retained normal resistance to cercosporin and other singlet oxygen-generating photosensitizers, indistinguishable from the parental strain. Transformation of a functional CTB4 clone into a ctb4 null mutant fully revived cercosporin production. Thus, we propose that the CTB4 gene encodes a putative MFS transporter responsible for secretion and accumulation of cercosporin.     

Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea,provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.     

The AVR4 effector is involved in cercosporin biosynthesis and likely affects the virulence of Cercospora cf. flagellaris on soybean

One of the most devastating fungal diseases of soybean in the southern USA is Cercospora leaf blight (CLB), which is caused mainly by Cercospora cf. flagellaris. Recent studies found that the fungal effector AVR4, originally identified in Cladosporium fulvum as a chitin-binding protein, is highly conserved among other Cercospora species. We wanted to determine whether it is present in C. cf. flagellaris and, if so, whether it plays a role in the pathogen infection of soybean. We cloned the Avr4 gene and created C. cf. flagellaris ∆avr4 mutants, which produced little cercosporin and significantly reduced expression of cercosporin biosynthesis genes. The ∆avr4 mutants were also more sensitive to chitinase and showed reduced virulence on soybean compared to the wild-type. The observed reduced virulence of C. cf. flagellaris ∆avr4 mutants on detached soybean leaves is likely due to reduced cercosporin biosynthesis. The phenotypes of reduced cercosporin production and cercosporin pathway gene expression, similar to those of the ∆avr4 mutants, were reproduced when wild-type C. cf. flagellaris was treated with double-stranded RNA targeting Avr4 in vitro. These two independent approaches demonstrated for the first time the direct involvement of AVR4 in the biosynthesis of cercosporin.     

CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis,regulates cercosporin biosynthesis,fungal development,and pathogenesis

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.     

Temperature,incubation time and virulence of Cercospora coffeicola in the production of cercosporin

This study considers the influence of temperature, incubation time and virulence in the production of the toxin cercosporin by Cercospora coffeicola, the causal agent of brown eye spot in coffee. The area under the progress curve of cercosporin production (AUPCCP) was also evaluated. A pathogenicity test was performed in a group with 58 isolates of C. coffeicola, which allowed the selection of four isolates, two representing the highest (LFP 12 and LFP 59) and two the lowest (LFP 24 and LFP 43) severity groups. The four isolates were cultivated in potato dextrose agar (PDA) and incubated in growth chambers at different temperatures (15, 20, 25 and 30°C) for 20 days. The experiments were conducted in a randomized complete block design with four replications. The cercosporin production decreased in all tested isolates at 30°C, resulting in a lower AUPCCP. The higher values of cercosporin were obtained at 20 and 25°C between 8 and 12 days of incubation. The isolates that produced the highest and lowest cercosporin concentrations were LFP 12 and LFP 43, respectively. After 15 days of incubation, toxin production is practically null in all isolates independently of the incubation temperature. Thus, the hypothesis for quantification of cercosporin production as a variability parameter within the species is suggested.     

Involvement of calcium/calmodulin signaling in cercosporin toxin biosynthesis by Cercospora nicotianae

Cercosporin is a non-host-selective, perylenequinone toxin produced by many phytopathogenic Cercospora species. The involvement of Ca(2+)/calmodulin (CaM) signaling in cercosporin biosynthesis was investigated by using pharmacological inhibitors. The results suggest that maintaining endogenous Ca(2+) homeostasis is required for cercosporin biosynthesis in Cercospora nicotianae. The addition of excess Ca(2+) to the medium slightly increased fungal growth but resulted in a reduction in cercosporin production. The addition of Ca(2+) chelators [EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] also reduced cercosporin production. Ca(2+) channel blockers exhibited a strong inhibition of cercosporin production only at higher concentrations (>2 mM). Cercosporin production was reduced greatly by Ca(2+) ionophores (A23187 and ionomycin) and internal Ca(2+) blocker [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester]. Phospholipase C inhibitors (lithium, U73122, and neomycin) led to a concentration-dependent inhibition of cercosporin biosynthesis. Furthermore, the addition of CaM inhibitors (compound 48/80, trifluoperazine, W-7, and chlorpromazine) also markedly reduced cercosporin production. In contrast to W-7, W-5, with less specificity for CaM, led to only minor inhibition of cercosporin production. The inhibitory effects of Ca(2+)/CaM inhibitors were partially or completely reversed by the addition of external Ca(2+). As assessed with Fluo-3/AM (a fluorescent Ca(2+) indicator), the Ca(2+) content in the cytoplasm decreased significantly when fungal cultures were grown in a medium containing Ca(2+)/CaM antagonists, confirming the specificity of those Ca(2+)/CaM antagonists in C. nicotianae. Taken together, the results suggest that Ca(2+)/CaM signal transduction may play a pivotal role in cercosporin biosynthesis in C. nicotianae.     

Expression of the yeast cpd1 gene in tobacco confers resistance to the fungal toxin cercosporin

Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.     

Bcmfs1, a Novel Major Facilitator Superfamily Transporter from Botrytis cinerea, Provides Tolerance towards the Natural Toxic Compounds Camptothecin and Cercosporin and towards Fungicides

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.     

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.     

MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides

MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.     

Lipid peroxidation induced by cercosporin as a possible determinant of its toxicity

The photodynamic action of cercosporin was assayed in various kinds of natural and artificial membranes. Cerosporin induces lipoperoxidation of liposomes, rat liver and pea internode mitochondria and microsomes, estimated both as malondialdehyde (MDA) formation and O2 consumption. Cercosporin-induced lipoperoxidation is inhibited by either singlet oxygen quenchers, free radical trapping agents or EDTA. Superoxide anion (O2-), hydrogen peroxide and hydroxyl radicals (.OH) are not involved in the activity of cercosporin. In addition cercosporin, by chelating iron, lowers the lipoperoxidation induced by such a metal. Therefore cercosporin stimulates, through singlet oxygen production, the hydroperoxide formation but, at the same time, it inhibits the continuation of the iron-mediated free radical chain. The present results suggest that cellular lipid peroxidation has a certain relevance to toxic activity of cercosporin.