The Cercospora nicotianae SOR1 (singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygengenerating phototoxins. Although homologues to SOR1 occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show that SOR1 is essential in pyridoxine (vitamin B6) synthesis in C. nicotianae and Aspergillus flavus, although it shows no homology to previously identified pyridoxine synthesis genes identified in Escherichia coli. Sequence database analysis demonstrated that organisms encode either SOR1 or E. coli pyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways for de novo pyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.The filamentous, phytopathogenic fungus Cercospora nicotianae exhibits a uniquely effective, broad-spectrum resistance to potent photosensitizers of diverse chemical structure and solubility (1, 2). C. nicotianae is resistant to cercosporin, a light-activated, singlet oxygen ( 1 O 2 )-generating toxin it produces in culture and during plant parasitism, and also to other potent photosensitizers including porphyrins and xanthine and thiazine dyes. Photosensitizers are highly toxic compounds that produce their deleterious effects only after activation by light. Absorbed light energy converts the photosensitizer to an excited (triplet) state molecule that may transfer an electron to oxygen to generate superoxide and͞or transfer energy directly to oxygen, yielding 1 O 2 (3). Exposure of cells to photosensitizers plus light leads to the destruction of critical cellular components including proteins, membranes, and DNA and often results in cell death. Studies on the mechanisms by which organisms protect themselves against reactive oxygen species have focused primarily on reduced and radical forms of oxygen, including hydrogen peroxide (H 2 O 2 ), superoxide (O 2 . ), and the hydroxyl radical (OH⅐). These active oxygen species are byproducts of normal cellular metabolism, and cells contain numerous and conserved defenses against them. By contrast, the highly reactive, but nonradical 1 O 2 is produced primarily via light activation of photosensitizing compounds. Most organisms do not tolerate 1 O 2 , and few biological defenses have been identified (2). The broad-spectrum resistance expressed by Cercospora species against cercosporin and other photosensitizers of diverse structure make these organisms an excellent model for understanding the cellular basis of 1 O 2 resistance. To study specific genes and proteins involved in photosensitizer and 1 O 2 resistance...

Plant pathogenic fungi in eight genera produce light-activated perylenequinone toxins that are toxic to plants via the generation of activated oxygen species, particularly singlet oxygen. Studies on the cercosporin toxin produced by Cercospora species have documented an important role for this toxin in pathogenesis of host plants. Cercosporin-generated active oxygen species destroy the membranes of host plants, providing nutrients to support the growth of these intercellular pathogens. Resistance of Cercospora species to the toxic effects of their own toxin has allowed these organisms to be used as a model for understanding the cellular basis of resistance to singlet oxygen and to general oxidative stress. In particular, the recent discovery that pyridoxine (vitamin B6) quenches singlet oxygen has led to the understanding of a novel role for this vitamin in cells as well as the discovery of a novel pathway of biosynthesis.

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.

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