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Gray leaf spot is a globally important foliar disease of maize caused by the fungus Cercospora zeae-maydis. In C. zeae-maydis, light is a critical environmental signal linked to pathogenesis and secondary metabolism. However, the mechanisms by which the fungus senses and responds to light are not fully understood. Thus, the overarching goal of this project was to unravel the connection between light and pathogenesis in C. zeae-maydis. The conserved fungal protein White Collar-1 (WC-1), designated Crp1 in C. zeae-maydis, senses light and initiates physiological responses. In fungi, WC-1 functions in the White Collar Complex via interaction with White Collar-2 (WC-2). In this study, the WC-2 ortholog of C. zeae-maydis, Crp2, was found to be involved in many of the same processes as Crp1, including pathogenesis. The light-mediated response initiated by Crp1 included transcriptional induction of the conserved circadian clock component frequency (FRQ1), which also displayed circadian rhythmicity. Deletion of FRQ1 impaired pathogenesis. Although overexpression of frequency in other fungi impairs clock function, overexpression of FRQ1 did not impact virulence in wild-type C. zeae-maydis but partially restored virulence in a Crp1-deficient strain. Characterization of the Defective in Binding DNA (DBD) region of Crp1 revealed a conserved role in the fungal circadian clock, but the domain was dispensable for pathogenesis. Additionally, Crp1 and Frq1 regulated the biosynthesis of, and self-protection against, cercosporin, a light-regulated secondary metabolite produced by C. zeae-maydis. Specifically, the stress response MAPKKK gene CZK3 and the cercosporin transporter gene CFP1 showed circadian rhythmicity, and the physiological response to cercosporin was dependent on the circadian clock. Lastly, Set3, a conserved component of histone deacetylation, was found to be important for pathogenesis, secondary metabolism, and transcriptional regulation of FRQ1. These findings suggest that (1) C. zeae-maydis has a circadian clock, (2) a Crp1/Crp2-mediated light response drives pathogenesis through regulation of FRQ1, (3) Set3 modulates FRQ1 following Crp1/Crp2 activation possibly through changes in histone acetylation, and (4) Frq1 has clock-independent role in pathogenesis and a clock-dependent role in cercosporin resistance. These phenomena have not been previously described and substantially advance the fundamental understanding of how C. zeae-maydis utilizes light to regulate pathogenesis.

Genetic markers and whole genome sequencing (WGS) were used to study the disease dynamics and population structure for three important plant pathogens; Cercospora sojina (frogeye leaf spot of soybean), Corynespora cassiicola (target spot of soybean, cotton and many other crops) and Phytophthora colocasiae (taro leaf blight). For each pathogen, genome sequencing was used to guide the development of single nucleotide polymorphism (SNP) markers and both were used to investigate diversity in field populations. Investigation of C. sojina in Tennessee included comparisons of extant populations and historical isolates, revealing a dominant, potentially long-lived, clonal lineage. Characterization of QoI fungicide resistance indicates populations have adapted rapidly and that sexual recombination and heteroplasmy may play a role. Genome comparison of isolates of C. cassiicola from cotton and soybean indicates isolates from Tennessee are very similar but significantly different from an isolate recovered from rubber and that a revision of the species may be necessary. Genotype analyses of isolates of C. cassiicola from five southern US states indicates populations are dominated by a single clonal lineage. Genome sequencing and SNP genotyping for P. colocasiae recovered from Hawaii, Vietnam, China and Nepal revealed both inter- and intra-genomic variation in ploidy and a long-lived clonal population on the islands of Hawaii. This is the first report of intra-genomic ploidy variation within Phytophthora and the implications for evolution, adaptation and research are discussed.

Gray leaf spot, caused by Cercospora zeae-maydis, is one of the most destructive foliar diseases of maize worldwide. C. zeae-maydis orients hyphal growth towards stomata (stomatal tropism) and forms infectious structures (appressoria) that are necessary for successful infection. Although some genes involved in pathogenesis in C. zeae-maydis have been identified, the molecular mechanisms are not well understood. In fungi, heterotrimeric G-proteins consist of three subunits (α, β, and γ) and mediate responses to environmental stimuli. They regulate diverse functions, including nutrient detection, virulence, fungal development, conidiation, secondary metabolism, and pathogenesis in many plant pathogenic fungi. This research explored the role of each G-protein α (Gpα) subunit of C. zeae-maydis in pathogenesis. To this end, the three Gpα subunits identified in C. zeae-maydis were functionally characterized. All three Gpα genes were required for appressorium formation and pathogenesis. Additionally, all three Gpα genes regulated cercosporin biosynthesis and sporulation. Together, these data demonstrated that the Gpα subunits in Cercospora zeae-maydis regulate pathogenesis and suggest that environmental sensing has been impaired. This study links G-protein signaling to infectious development in C. zeae-maydis and sets up a study for transcriptomic analysis of genes regulated by the Gpα genes.

The fungal photoactivated toxin cercosporin plays a key role in pathogenesis by Cercospora spp. The bacterium Xanthomonas campestris pv. zinnia (XCZ) is able to rapidly degrade this toxin. Growth of XCZ strains in cercosporin–containing medium leads to the breakdown of cercosporin and to the formation of a non–toxic breakdown product called xanosporic acid. EMS mutagenesis was used to generate five non–degrading mutants of a rapid–degradation strain (XCZ&#8211h;3). Mutants were complemented using a wild–type genomic library. All five mutants were complemented with the same fragment, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of the two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five had point mutations in the oxidoreductase sequence and no mutations in the regulator. Quantitative RT–PCR showed that expression of both of these genes in the wild–type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three cercosporin non–degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT–PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that other factors are involved in this process. In an effort to determine if the oxidoreductase gene has a role in pathogenesis, quantitative RT–PCR was used to assay for oxidoreductase regulation in XCZ–3 grown in the presence of zinnia and tobacco extracts. The oxidoreductase gene was upregulated in the presence of both plant extracts, but the response was greater with the tobacco extract. The five XCZ–3 oxidoreductase mutants were assayed for pathogenicity on zinnia. The mutants yielded disease symptoms equal to those of the wild–type. Furthermore, previously identified bacteria (Xanthomonas axonopodis pv. pruni, strains XAP–50 and XAP–75, and Pseudomonas syringae pv. tomato, PST DC3000) with oxidoreductase homologues were not able to infect zinnia, suggesting that the presence of a functional oxidoreductase is not correlated with pathogenesis of zinnia.