John B. Ridenour

Regulation of Stomatal Tropism and Infection by Light in Cercospora zeae-maydis: Evidence for Coordinated Host/ Pathogen Responses to Photoperiod?

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.

HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production and pathogenesis in Fusarium verticillioides

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 (Δhxk1) 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 Δhxk1 mutant produced unusual swollen hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the Δhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On maize kernels, the Δhxk1 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 Δhxk1 mutant produced approximately 50 % less trehalose and 80 % less fumonisin B₁ (FB₁) than the wild-type. The reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic stress in the Δhxk1 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.

UBL1 of Fusarium verticillioides links the N‐end rule pathway to extracellular sensing and plant pathogenesis

Fusarium verticillioides produces fumonisin mycotoxins during colonization of maize. Currently, molecular mechanisms underlying responsiveness of F.verticillioides to extracellular cues during pathogenesis are poorly understood. In this study, insertional mutants were created and screened to identify genes involved in responses to extracellular starch. In one mutant, the restriction enzyme-mediated integration cassette disrupted a gene (UBL1) encoding a UBR-Box/RING domain E3 ubiquitin ligase involved in the N-end rule pathway. Disruption of UBL1 in F.verticillioides (Δubl1) influenced conidiation, hyphal morphology, pigmentation and amylolysis. Disruption of UBL1 also impaired kernel colonization, but the ratio of fumonisin B1 per unit growth was not significantly reduced. The inability of a Δubl1 mutant to recognize an N-end rule degron confirmed involvement of UBL1 in the N-end rule pathway. Additionally, Ubl1 physically interacted with two G protein α subunits of F.verticillioides, thus implicating UBL1 in G protein-mediated sensing of the external environment. Furthermore, deletion of the UBL1 orthologue in F.graminearum reduced virulence on wheat and maize, thus indicating that UBL1 has a broader role in virulence among Fusarium species. This study provides the first linkage between the N-end rule pathway and fungal pathogenesis, and illustrates a new mechanism through which fungi respond to the external environment.

The HAP complex in Fusarium verticillioides is a key regulator of growth, morphogenesis, secondary metabolism, and pathogenesis.

Among eukaryotic organisms, the HAP complex is a conserved, multimeric transcription factor that regulates gene expression by binding to the consensus sequence CCAAT. In filamentous fungi, the HAP complex has been linked to primary and secondary metabolism, but its role in pathogenesis has not been investigated extensively. The overarching goal of this study was to elucidate the role of the HAP complex in Fusariumverticillioides, a ubiquitous and damaging pathogen of maize. To this end, orthologs of core HAP complex genes (FvHAP2, FvHAP3, and FvHAP5) were identified and deleted in F. verticillioides via a reverse genetics approach. Deletion of FvHAP2, FvHAP3, or FvHAP5 resulted in an indistinguishable phenotype among the deletion strains, including reduced radial growth and conidiation, altered colony morphology, and derepression of pigmentation. Additionally, disruption of the HAP complex impaired infection and colonization of maize stalks. Deletion strains were hypersensitive to osmotic and oxidative stress, which suggests the HAP complex of F. verticillioides may mediate responses to environmental stress during pathogenesis. This study directly implicates the HAP complex in primary and secondary metabolism in F. verticillioides and provides one of the first links between the HAP complex and virulence in a plant pathogenic fungus.

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla.

To facilitate functional genomics in the soybean pathogen Phomopsis longicolla, we developed a robust Agrobacterium tumefaciens-mediated transformation system that yielded 150-250 transformants per 1×10(6) conidia of P. longicolla. This first report of P. longicolla transformation provides a useful tool for insertional mutagenesis in an increasingly important pathogen of soybean.

The novel fungal-specific gene FUG1 has a role in pathogenicity and fumonisin biosynthesis in Fusarium verticillioides.

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.

Identifying Genes in Fusarium verticillioides Through Forward and Reverse Genetics

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.

Regulation of Pathogenesis by Light in Cercospora zeae-maydis: An Updated Perspective

The fungal genus Cercospora is one of the most ubiquitous groups of plant pathogenic fungi, and gray leaf spot caused by C. zeae-maydis is one of the most widespread and damaging foliar diseases of maize in the world. While light has been implicated as a critical environmental regulator of pathogenesis in C. zeae-maydis, the relationship between light and the development of disease is not fully understood. Recent discoveries have provided new insights into how light influences pathogenesis and morphogenesis in C. zeae-maydis, particularly at the molecular level. This review is focused on integrating old and new information to provide an updated perspective of how light influences pathogenesis, and provides a working model to explain some of the underlying molecular mechanisms. Ultimately, a thorough molecular-level understanding of how light regulates pathogenesis will augment efforts to manage gray leaf spot by improving host resistance and disease management strategies.

The HAP Complex Governs Fumonisin Biosynthesis and Maize Kernel Pathogenesis in Fusarium verticillioides

Contamination of maize ( Zea mays ) with fumonisins produced by the fungus Fusarium verticillioides is a global concern for food safety. Fumonisins are a group of polyketide-derived secondary metabolites linked to esophageal cancer and neural tube birth defects in humans and numerous toxicoses in livestock. Despite the importance of fumonisins in global maize production, the regulation of fumonisin biosynthesis during kernel pathogenesis is poorly understood. The HAP complex is a conserved, heterotrimeric transcriptional regulator that binds the consensus sequence CCAAT to modulate gene expression. Recently, functional characterization of the Hap3 subunit linked the HAP complex to the regulation of secondary metabolism and stalk rot pathogenesis in F. verticillioides . Here, we determine the involvement of HAP3 in fumonisin biosynthesis and kernel pathogenesis. Deletion of HAP3 suppressed fumonisin biosynthesis on both nonviable and live maize kernels and impaired pathogenesis in living kernels. Transcriptional profiling via RNA sequencing indicated that the HAP complex regulates at least 1,223 genes in F. verticillioides , representing nearly 10% of all predicted genes. Disruption of the HAP complex caused the misregulation of biosynthetic gene clusters underlying the production of secondary metabolites, including fusarins. Taken together, these results reveal that the HAP complex is a central regulator of fumonisin biosynthesis and kernel pathogenesis and works as both a positive and negative regulator of secondary metabolism in F. verticillioides .

Draft genome sequence of Xylaria sp., the causal agent of taproot decline of soybean in the southern United States

The draft genome of Xylaria sp. isolate MSU_SB201401, causal agent of taproot decline of soybean in the southern U.S., is presented here. The genome assembly was 56.7 Mb in size with an L50 of 246. A total of 10,880 putative protein-encoding genes were predicted, including 647 genes encoding carbohydrate-active enzymes and 1053 genes encoding secreted proteins. This is the first draft genome of a plant-pathogenic Xylaria sp. associated with soybean. The draft genome of Xylaria sp. isolate MSU_SB201401 will provide an important resource for future experiments to determine the molecular basis of pathogenesis.