David C. Straney

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

BackgroundGenes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.ResultsPutative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.ConclusionETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.

Tissue-Specific Localization of Pea Root Infection by Nectria haematococca. Mechanisms and Consequences

Root infection in susceptible host species is initiated predominantly in the zone of elongation, whereas the remainder of the root is resistant. Nectria haematococca infection of pea (Pisum sativum) was used as a model to explore possible mechanisms influencing the localization of root infection. The failure to infect the root tip was not due to a failure to induce spore germination at this site, suppression of pathogenicity genes in the fungus, or increased expression of plant defense genes. Instead, exudates from the root tip induce rapid spore germination by a pathway that is independent of nutrient-induced germination. Subsequently, a factor produced during fungal infection and death of border cells at the root apex appears to selectively suppress fungal growth and prevent sporulation. Host-specific mantle formation in response to border cells appears to represent a previously unrecognized form of host-parasite relationship common to diverse species. The dynamics of signal exchange leading to mantle development may play a key role in fostering plant health, by protecting root meristems from pathogenic invasion.

Regulatory signals influencing expression of the PDA1 gene of Nectria haematococca MPVI in culture and during pathogenesis of pea

The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes a cytochrome P450 monooxygenase that detoxifies pisatin, the isoflavonoid phytoalexin produced by its host, garden pea (Pisum sativum). PDA1 is regulated by several signals in culture that may control its expression during pathogenesis of pea. It is induced by pisatin and repressed by glucose and amino acids. Deletion analysis was performed on the PDA1 promoter to define regulatory regions, using a β-glucuronidase (GUS) reporter gene fusion. The results identified a region between -287 and -429, relative to the start of transcription, that mediated repression by either glucose or amino acids in culture, independent from pisatin induction. Transformants bearing PDA1 promoter constructs displaying altered regulation in response to the different signals were used to infect pea epicotyls in order to correlate regulation in culture with that observed during pathogenesis of the host. Removal of the nutritional ...

A binuclear zinc transcription factor binds the host isoflavonoid‐responsive element in a fungal cytochrome p450 gene responsible for detoxification

The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes pisatin demethylase, a cytochrome P450. Pisatin is a fungistatic isoflavonoid produced by garden pea (Pisum sativum), a host for this fungus. Pisatin demethylase detoxifies pisatin and functions as a virulence factor for this fungus. Pisatin induces PDA1 expression both in cultured mycelia as well as during pathogenesis on pea. The regulatory element within PDA1 that provides pisatin‐responsive expression was identified using a combination of in vivo functional analysis and in vitro binding analysis. The 40 bp pisatin‐responsive element is located 635 bp upstream of the PDA1 transcription start site. This element was sufficient to provide strong pisatin‐induced expression to a minimal promoter in vivo and was required for pisatin regulation of the PDA1 promoter. A gene encoding a DNA‐binding protein specific to this 40 bp element was isolated from a N. haematococca cDNA library using the yeast one‐hybrid screen. The cloned gene possesses sequence motifs found in the binuclear zinc (Cys 6‐Zn 2) family of transcription factors unique to fungi. The results suggest that it is a regulator of this fungal cytochrome P450 gene and may provide pisatin‐responsive regulation.

Host Recognition by Pathogenic Fungi Through Plant Flavonoids

A common characteristic among fungal pathogens of plants is that each specializes on a narrow range of specific plants as hosts. One adaptation to a specific host plant is the recognition of the hosts chemicals which can be used to trigger genes or developmental pathways needed for pathogenesis. The production of characteristic flavonoids by plants, particularly those exuded from roots by legumes, appear to be used as signals for various microbes, including symbionts as well as pathogens. Nectria haematococca MPVI (anamorph: Fusarium solani) is a soil-borne pathogen of garden pea (Pisum sativum) which serves as a useful model in studying host flavonoid recognition. This fungus displays flavonoid induction of specific pathogenicity genes as well as stimulation of development needed for pathogenesis. Here, we summarize the study of flavonoid-inducible signal pathways which regulate these trait, through identification of transcription factors and regulatory components which control these responses. The characterization of the components a pathogen uses to specifically recognize its host provides insights into the host adaptation process at the molecular level.

Identification of elements in thePDA1 promoter ofNectria haematococca necessary for a high level of transcription in vitro

Expression of thePDA1 gene in the ascomyceteNectria haematococca MPVI (anamorph:Fusarium solani) is induced by exposure of mycelium to pisatin, an isoflavonoid phytoalexin produced by its host plant, garden pea. ThePDA1 gene encodes a cytochrome P-450 monooxygenase which detoxifies pisatin. Regulatory elements controlling transcription from thePDA1 promoter were identified using a homologousNectria in vitro transcription system through analysis of 5′ deletions, specific oligonucleotide competition, and fusion of upstream segments to a heterologous promoter. A promoter-distal element which provided transcriptional activation was localized to a 35-bp region positioned − 514 to − 483 upstream of the transcriptional start site. This 35-bp region binds a previously characterized pisatin-responsive DNA-binding factor (PRF) and thus may provide pisatin-responsive control of transcription. A second promoter-proximal positive-acting region was found to be necessary for promoter transcription in both homologous and heterologous extracts, and so is likely to bind less gene-specific transcription activator(s). A negative-acting element located between these two positive regions may act to make the positive-acting elements interdependent. The identification of an activator responding to pisatin provides a model for the control of a number of genes and processes controlled by host-specific signals, particularly the flavonoids.

Analysis of determinants of binding and transcriptional activation of the pisatin-responsive DNA binding factor of Nectria haematococca.

Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to pisatin. A pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.

In vitro transcription from the Nectria haematococca PDA1 promoter in an homologous extract reflects in vivo pisatin-responsive regulation

The PDA1 gene of Nectria haematococca MP VI (anamorph: Fusarium solani) encodes pisatin demethylase. This enzyme detoxifies the isoflavanoid phytoalexin pisatin produced by the plant on which this fungus is pathogenic. Expression of pisatin demethylase activity is induced in a mycelium by pretreatment with pisatin. We have developed homologous in vitro system which accurately initiates transcription from the PDA1 promoter. Transcription levels in vitro reflect the same pisatin-responsive stimulation as measured for PDA1 mRNA in vivo, and are dependent upon sequences in the 5′ upstream region of PDA1. Pisatin-responsive transcription from the PDA1 promoter indicates that initiation of transcription is a major regulatory step in the pisatin induction of pisatin demethylase expression.