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.     

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.     

Biodegradation of the polyketide toxin cercosporin

Cercosporin is a non-host-specific polyketide toxin produced by many species of plant pathogens belonging to the genus Cercospora. This red-pigmented, light-activated toxin is an important pathogenicity determinant for Cercospora species. In this study, we screened 244 bacterial isolates representing 12 different genera for the ability to degrade cercosporin. Cercosporin degradation was determined by screening for the presence of cleared zones surrounding colonies on cercosporin-containing culture medium and was confirmed by assaying the kinetics of degradation in liquid medium. Bacteria belonging to four different genera exhibited the cercosporin-degrading phenotype. The isolates with the greatest cercosporin-degrading activity belonged to Xanthomonas campestris pv. zinniae and X. campestris pv. pruni. Isolates of these pathovars removed over 90% of the cercosporin from culture medium within 48 h. Bacterial degradation of red cercosporin was accompanied by a shift in the color of the growth medium to brown and then green. The disappearance of cercosporin was accompanied by the appearance of a transient green product, designated xanosporic acid. Xanosporic acid and its more stable lactone derivative, xanosporolactone, are nontoxic to cercosporin-sensitive fungi and to plant tissue and are labile in the presence of light. Detailed spectroscopic analysis (to be reported in a separate publication) of xanosporolactone revealed that cercosporin loses one methoxyl group and gains one oxygen atom in the bacterial conversion. The resulting chromophore (4,9-dihydroxy-3-oxaperlylen-10H-10-one) has never been reported before but is biosynthetically plausible via oxygen insertion by a cytochrome P-450 enzyme.     

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus<Emphasis Type="Italic"> Cercospora nicotianae</Emphasis>

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.Communicated by E. Cerdá-Olmedo     

Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity

We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.     

The photoactivated toxin cercosporin as a tool in fungal photobiology

Cercospora species are a highly successful group of fungi which pathogenize diverse species of economically important plants. Many Cercospora species produce a unique photoactivated and photoinduced polyketide toxin, cercosporin, which has been implicated as a pathogenicity factor. Illuminated cercosporin interacts with molecular oxygen to produce highly toxic singlet oxygen. Although nearly all organisms tested, including plants, mice and most fungi, are susceptible to cercosporin-mediated cell damage, Cercospora species are resistant. In general, little is known about how organisms protect themselves against singlet oxygen. Studies on how Cercospora species avoid autotoxicity are proving to be a valuable model in understanding this process in other systems. Furthermore, advances are being made in the understanding of how light regulates gene expression and cercosporin synthesis in Cercospora species. These studies are helping to elucidate mechanisms of gene regulation and light signal transduction for an environmental signal important in numerous fungal developmental processes, including secondary metabolite production.     

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.