Cercospora zeae-maydis causes gray leaf spot of maize, which has become one of the most widespread and destructive diseases of maize in the world. C. zeae-maydis infects leaves through stomata, which is predicated on the ability of the pathogen to perceive stomata and reorient growth accordingly. In this study, the discovery that light was required for C. zeae-maydis to perceive stomata and infect leaves led to the identification of CRP1, a gene encoding a putative blue-light photoreceptor homologous to White Collar-1 (WC-1) of Neurospora crassa. Disrupting CRP1 via homologous recombination revealed roles in multiple aspects of pathogenesis, including tropism of hyphae to stomata, the formation of appressoria, conidiation, and the biosynthesis of cercosporin. CRP1 was also required for photoreactivation after lethal doses of UV exposure. Intriguingly, putative orthologs of CRP1 are central regulators of circadian clocks in other filamentous fungi, raising the possibility that C. zeae-maydis uses light as a key environmental input to coordinate pathogenesis with maize photoperiodic responses. This study identified a novel molecular mechanism underlying stomatal tropism in a foliar fungal pathogen, provides specific insight into how light regulates pathogenesis in C. zeae-maydis, and establishes a genetic framework for the molecular dissection of infection via stomata and the integration of host and pathogen responses to photoperiod.

Genome integrity is essential to maintain cellular function and viability. Consequently, genome instability is frequently associated with dysfunction in cells and associated with plant, animal, and human diseases. One consequence of relaxed genome maintenance that may be less appreciated is an increased potential for rapid adaptation to changing environments in all organisms. Here, we discuss evidence for the control and function of facultative heterochromatin, which is delineated by methylation of histone H3 lysine 27 (H3K27me) in many fungi. Aside from its relatively well understood role in transcriptional repression, accumulating evidence suggests that H3K27 methylation has an important role in controlling the balance between maintenance and generation of novelty in fungal genomes. We present a working model for a minimal repressive network mediated by H3K27 methylation in fungi and outline challenges for future research.

In Fusarium verticillioides, a ubiquitous pathogen of maize, virulence and mycotoxigenesis are regulated in response to the types and amounts of carbohydrates present in maize kernels. In this study, we investigated the role of a putative hexokinase-encoding gene (HXK1) in growth, development and pathogenesis. A deletion mutant (Dhxk1) of HXK1 was not able to grow when supplied with fructose as the sole carbon source, and growth was impaired when glucose, sucrose or maltotriose was provided. Additionally, the Dhxk1 mutant produced unusual swollen hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the Dhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On maize kernels, the Dhxk1 mutant was substantially less virulent than the wild-type, but virulence on maize stalks was not impaired, possibly indicating a metabolic response to tissue-specific differences in plant carbohydrate content. Finally, disruption of HXK1 had a pronounced effect on fungal metabolites produced during colonization of maize kernels; the Dhxk1 mutant produced approximately 50 % less trehalose and 80 % less fumonisin B 1 (FB 1 ) than the wild-type. The reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic stress in the Dhxk1 mutant. In summary, this study links early events in carbohydrate sensing and glycolysis to virulence and secondary metabolism in F. verticillioides, and thus provides a new foothold from which the genetic regulatory networks that underlie pathogenesis and mycotoxigenesis can be unravelled and defined.

Gray leaf spot (GLS), caused by the sibling species Cercospora zeina orCercospora zeae-maydis, is cited as one of the most important diseases threatening global maize production. C. zeina fails to produce cercosporin in vitro, and in most cases causes large coalescing lesions during maize infection; a symptom generally absent from cercosporin-deficient mutants in other Cercospora spp. Here we describe the C. zeina cercosporin toxin biosynthetic gene cluster. The oxidoreductase gene CTB7 contained several insertions and deletions as compared to the C. zeae-maydis ortholog. We set out to determine whether complementing the defective CTB7 gene with the full-length gene from C. zeae-maydis could confer in vitro cercosporin production. C. zeina transformants containing C. zeae-maydis CTB7 were generated by Gray leaf spot continues to be a devastating maize foliar disease of global importance that has resulted in extensive yield losses over the past few decades (Ward et al. 1999; Crous and Braun 2003). Previously classified as Cercospora zeae-maydis Group I and Group II, the causative agents of GLS, C. zeae-maydis and Cercospora zeina are differentiated by both genetic distance and phenotypic characteristics, such as their ability to produce the phytotoxin cercosporin (Goodwin et al. 2001;. Cercospora zeina predominates throughout Africa, while C. zeae-maydis is most prevalent in the majority of the USA and Mexico (Wang et al. 1998;Dunkle and Levy 2000; Goodwin et al. 2001;).The genus Cercospora is part of the class Dothideomycetes and consists of more than 600 recognized species of plant pathogens (Crous and Braun 2003). Although Cercospora species generally exhibit relatively narrow host ranges, many produce cercosporin, a photosensitizing perylenequinone that functions as a non-specific toxin that has been identified as a major pathogenicity factor (Daub and Ehrenshaft 2000;Weiland et al. 2010).Cercosporin production has been demonstrated for several Cercospora species, with isolates of Cercospora kikuchii, Cercospora beticola, C. zeae-maydis, Cercospora asparagi and Cercospora nicotianae all shown to accumulate cercosporin in vitro (Jenns et al. 1989).Isolates of C. zeina however demonstrate a lack of cercosporin production in vitro (Dunkle and Levy 2000; Goodwin et al. 2001; Koshikumo et al. 2014). These species all cause leaf spot diseases that are characterised by severe blighting of leaves (Daub and Ehrenshaft 2000). In contrast to this, isolates of the peanut pathogen Cercospora arachidicola, fail to produce cercosporin and induce only small chlorotic lesions (Fore et al. 1988). Isolates of C.arachidicola have however been shown to produce other toxins which may aid in virulence (Fore et al. 1988). Cercosporin biosynthesis mainly involves a cluster of eight cercosporin toxin biosynthetic (CTB) genes, all of which are transcriptionally induced upon exposure to light in the tobacco pathogen C. nicotianae (Chen et al. 2007b). Targeted disruption of any CTB gene blocked cercosporin production and reduced viru...

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Fusarium verticillioides is a globally important pathogen of maize, capable of causing severe yield reductions and economic losses. In addition, F. verticillioides produces toxic secondary metabolites during kernel colonization that pose significant threats to human and animal health. Fusarium verticillioides and other plant-pathogenic fungi possess a large number of genes with no known or predicted function, some of which could encode novel virulence factors or antifungal targets. In this study, we identified and characterized the novel gene FUG1 (Fungal Unknown Gene 1) in F. verticillioides through functional genetics. Deletion of FUG1 impaired maize kernel colonization and fumonisin biosynthesis. In addition, deletion of FUG1 increased sensitivity to the antimicrobial compound 2-benzoxazolinone and to hydrogen peroxide, which indicates that FUG1 may play a role in mitigating stresses associated with host defence. Transcriptional profiling via RNA-sequencing (RNA-seq) identified numerous fungal genes that were differentially expressed in the kernel environment following the deletion of FUG1, including genes involved in secondary metabolism and mycelial development. Sequence analysis of the Fug1 protein provided evidence for nuclear localization, DNA binding and a domain of unknown function associated with previously characterized transcriptional regulators. This information, combined with the observed transcriptional reprogramming in the deletion mutant, suggests that FUG1 represents a novel class of fungal transcription factors or genes otherwise involved in signal transduction.

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The increasing availability of sequenced genomes for plant pathogenic fungi has revolutionized molecular plant pathology in recent years. However, the genetic regulatory networks underlying many important components of pathogenesis remain poorly defined. Although the protocols outlined in this chapter can be utilized to identify genes regulating a wide range of biological processes in many filamentous fungi, we focus on describing how to identify genes through forward and reverse genetics, using the plant pathogenic fungus Fusarium verticillioides as a model for the protocol. Specifically, this chapter explains how to create a collection of insertional mutants via Restriction Enzyme Mediated Integration (REMI) and how to screen mutants with a high-throughput method to visualize defects in amylolysis. Next, techniques are described to define the genomic lesions in REMI mutants with genome-walker PCR in order to identify candidate genes. Finally, protocols are presented describing a reverse-genetic approach to disrupt candidate genes in the wild-type strain with a split-marker strategy to confirm the phenotype observed in the REMI mutant.