The CTB1 Gene Encoding a Fungal Polyketide Synthase Is Required for Cercosporin Biosynthesis and Fungal Virulence of Cercospora nicotianae

Abstract

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.

Determination of a transcriptional regulator-like gene involved in biosynthesis of elsinochrome phytotoxin by the citrus scab fungus, Elsinoë fawcettii

“…The plates were placed approximately 45 cm away from the light source. When preparation of protoplasts was desired, fungal isolates were grown in 50 ml potato dextrose broth (PDB, Difco) for 7 days, ground, mixed with fresh PDB (200 ml), and incubated for an additional 15 h. For DNA or RNA isolation, fungal isolates were grown on media with a layer of sterile cellophane (Choquer et al, 2005). Screening of elsinochrome-deficient mutants was performed on thin PDA as previously described (Choquer et al, 2005;Liao & Chung, 2008a).…”

“…The NruI site is approximately 1200 nt downstream from the predicted TSF1 start codon. A split-marker strategy was applied to promote double crossing-over recombination as previously described (Choquer et al, 2005). Briefly, a 4.4 kb DNA fragment containing truncated 59 TSF1 fused with 39 HYG and a 4.2 kb fragment encompassing 39 TSF1 linked to 59 HYG were amplified with, respectively, primers efup11/ hyg3 (59-ggatgcctccgctcgaagta-39) and efup28/hyg4 (59-cgttgcaagaactgcctgaa-39) from pTSF1128 using the Takara Ex Tar PCR system (Takara Bio).…”

The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis

“…To identify genes directly involved in cercosporin biosynthesis, C. nicotianae mutants completely deficient in cercosporin production were generated using the restriction enzyme-mediated integration (REMI) mutagenesis (Chung et al, 2003b). A CTB1 (Cercosporin Toxin Biosynthesis) gene encoding a fungal polyketide synthase was subsequently identified from one of the REMI mutants and shown by gene disruption to be required for cercosporin biosynthesis and for high levels of virulence on tobacco (Choquer et al, 2005).…”

Cellular toxicity of elsinochrome phytotoxins produced by the pathogenic fungus, Elsinoë fawcettii causing citrus scab

“…Many of the perylenequinone pigments produced by fungi have been shown to be toxic to mice, bacteria, and many fungi, to be cytotoxic to animal tumors, to be potent antiviral agents, and to inactivate protein kinase C (Tamaoki & Nakano, 1990;Hudson & Towers, 1991;Diwu, 1995;Hudson et al ., 1997). (a) Structure of elsinochromes with various side-groups (R) (redrawn based on the work of Weiss et al, 1987 Except for cercosporin produced by many members of the fungal genus Cercospora (Callahan et al ., 1999;Choquer et al ., 2005Choquer et al ., , 2007Chen et al ., 2007;Dekkers et al ., 2007), none of the perylenequinones of fungal origin have been demonstrated to act as a crucial factor in plant diseases caused by the producing pathogen.…”