Abstracts from Dr. Straney's Publications:

Khan, R. and D. C. Straney 1999. Regulatory Signals Influencing Expression of the PDA1 Gene of Nectria haematococca MP VI in Culture and During Pathogenesis of Pea. Molecular Plant-Microbe Interactions. 12: 733-742The PDA1 gene of the filamentous fungus Nectria haematococca MPVI (anamorph: Fusarium solani) encodes a cytochrome P450 monooxygenase which detoxifies pisatin, the isoflavonoid phytoalexin produced by its host, garden pea (Pisum sativum L.). PDA1 is regulated by several signals in culture which 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 GUS reporter gene fusion. The results identified a region between -287 and -429, relative to the start of transcription, which 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 response region did not have a major effect on the induction of the promoter observed during growth in pea. However, induced expression in planta was lacking in a PDA1::GUS construct which lacked pisatin-response in culture. These results suggest that the host-specific stimulus, pisatin, is a primary stimulatory signal for PDA1 regulation during pea pathogenesis.

He, Y., Y. Ruan & D.C. Straney. 1996. Analysis of determinants of binding and transcriptional activation of the pisatin-responsive DNA-binding factor of Nectria haematococca. Molec. Plant-Microbe Interact. 9: 171-179.
Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca MPVI (anamorph: Fusarium solani) which are highly virulent on pea have been found to posses 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 which reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts which 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 blotting, UV-crosslinking and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter which is responsive to a host-specific compound and provides insight into the regulation of fungal genes in response to plant flavonoids.

Ruan, Y. & D.C Straney. 1996. Identification of elements in the PDA1 promoter of Nectria haematococca necessary for a high level of transcription in vitro. Molec. Gen. Genetics 250: 29-38.
Expression of the PDA1 gene in the ascomycete Nectria haematococca MP VI (anamorph: Fusarium solani) is induced by exposure of mycelium to pisatin, an isoflavonoid phytoalexin produced by its host plant, garden pea. The PDA1 gene encodes a cytochrome P450 monooxygenase which detoxifies pisatin. Regulatory elements controlling transcription from the PDA1 promoter were identified by the use of a homologous Nectria 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 located in 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.

Suleman, P., A.M. Tohamy, A. Saleh, M. Madkour & D.C. Straney. 1996. Variation in sensitivity to tomatine and rishitin among isolates of Fusarium oxysporum f.sp. lycopersici and strains not pathogenic on tomato. Physiol. Molec. Plant Pathol. 48: 131-144.
Studies of several fungal plant pathogens have found an association between greater virulence and increased tolerance to the host's defense compounds among different isolates of that pathogen species. This study examined 17 Fusarium oxysporum isolates to determine if tolerance to either of two fungitoxic compounds produced by tomato, rishitin and tomatine, would correlate with virulence or pathogenicity on tomato. Among the 12 pathogenic isolates (forma specialis lycopersici), quantitative levels of virulence were significantly correlated with rishitin tolerance and, in more limited circumstances, with tomatine tolerance. A group of four highly virulent isolates displayed a relatively high tolerance to both tomatine and rishitin compared to the other isolates. When these pathogenic isolates were compared to five F. oxysporum isolates nonpathogenic on tomato, the nonpathogens generally displayed the highest sensitivity to tomatine, but not to rishitin. Although these results do not prove a role for rishitin or tomatine tolerance in virulence or pathogenicity, they do indicate that sufficient natural variation in these traits exists for them to contribute to an isolate's disease potential on tomato.

Wilhite, S.E. & D.C. Straney. 1996. Timing of gliotoxin biosynthesis in the fungal biological control agent Gliocladium virens (Trichoderma virens) Appl. Microbiol. Biotechnol. 45: 513-518.
Gliocladium virens is a filamentous fungus formulated for the biological control of damping-off diseases of plants. Part of its antagonistic activity is due to its production of an epidithiodiketopiperazine antibiotic, gliotoxin. A relatively short period of biocontrol activity limits the use of this biocontrol agent in certain applications. This report examines the apparent transient accumulation of gliotoxin, a potential limitation in biocontrol activity. 35S pulse labelling of gliotoxin indicated that G. virens strain G20-4VIB synthesizes gliotoxin only within a short 16 h period during replicative growth. An apparent lack of gliotoxin production in later growth phases was due to the cessation of synthesis rather than an increase in catabolism of gliotoxin. Media transfer experiments indicated that cessation of gliotoxin synthesis could not be explained by gliotoxin feedback inhibition, a diffusible inhibitor, or changing the nutritional status of the medium over a two-hour response time. These results demonstrate that the regulation of gliotoxin biosynthesis is a major determinant in the kinetics of gliotoxin appearance and focuses the need for further study on the regulation of gene expression.

Ruan, Y., V. Kotraiah & D.C. Straney. 1995. Flavonoids stimulate spore germination in Fusarium solani pathogenic on legumes in a manner sensitive to inhibitors of cAMP-dependent kinase. Molec. Plant-Microbe Interactions 8: 929-938.
Many soilborne fungal plant pathogens remain as resting propagules until the appearance of a potential host stimulates their germination. The plant-derived stimulus for germination has generally been assumed to be nutrients exuded from roots. We show that certain flavonoids, including defense-related isoflavonoid phytoalexins, stimulate spore germination of Fusarium solani formae speciales pathogenic on pea or bean. The stimulatory action of specific flavonoids are consistent with the flavonoids previously identified in root exudates of these two hosts and with the levels of flavonoids reported to be exuded by bean roots. Inhibitors of cAMP-dependent protein kinase (PKA) prevented flavonoid-responsive germination, but not nutrient-responsive germination. Thus these two stimuli, flavonoids and nutrients, appear to utilize separate signal pathways to initiate germination. Germination of macroconidia in root exudates was significantly inhibited by a PKA inhibitor, indicating that flavonoids present in root exudates may be at least as active as nutrients in stimulating germination. These results suggest that flavonoids in legume root exudate may be perceived as a signal in a number of plant-microbe interactions, not only for initiating symbiotic rhizobial interactions but also for initiating pathogenic fungal interactions.

Ruan, Y. & D.C. Straney. 1994a. PCR-based construction of promoter/Gfree templates for in vitro transcription analysis allows selection of plasmids with optimal activity in homologous extracts. Gene 146: 227- 232.
In vitro transcription has been used for dissecting transcriptional controls in many eukaryotic systems. One modification which greatly reduces background non-specific transcription is the placement of a guanosine-free (G-free) region of DNA immediately downstream of a promoter (Sawadogo and Roeder, 1985, Proc. Natl. Acad. Sci. USA 82:4394); transcription in the presence of RNAase T1 and 3' O-Me-GTP eliminates non-specific transcripts but produces the G-free transcripts initiated at the promoter. Restriction site-based fusion of a G-free cassette downstream of promoters is complicated by the requirement for G nucleotides to be excluded from the coding strand downstream of the site(s) of transcription initiation. We present an approach to add a G-free template onto a eukaryotic promoter by combining PCR-based termini construction and terminal deoxynucleotidyl transferase extension. The pisatin demethylase (PDA1) promoter of the filamentous fungus Nectria haematococca was used as the test promoter. Three PDA1 promoter/G-free constructs were tested in heterologous Drosophila and HeLa and homologous N. haematococca transcription extracts. Each extract produced a PDA1 promoter-specific transcript from each construct, but the relative level of transcription between constructs varied with extract, particularly in the homologous extract. Since the choice of G-free sequence influences transcription differently in among systems, this method for producing multiple G-free constructs should be useful for constructing and selecting optimal promoter/G-free templates for in vitro transcription in other homologous systems.

Ruan, Y. & D.C. Straney. 1994b. In vitro transcription from the Nectria haematococca PDA1 promoter in a homologous extract reflects in vivo pisatin-responsive regulation. Current Genetics 27: 46-53.
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 mycelium by pretreatment with pisatin. We have developed a homologous in vitro transcription 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 pisatin induction of pisatin demethylase expression.

Straney, D., Y. Ruan & J. He. 1994. In vitro transcription and binding analysis of promoter regulation by a host-specific signal in a phytopathogenic fungus. Antonie van Leeuwenhoek 65: 183-189.
The PDA1 promoter of the phytopathogen Nectria haematococca MPVI (anamorph Fusarium solani) offers a model for regulation of a fungal virulence gene in response to plant host-specific signals. Expression of the PDA1 gene, encoding pisatin demethylase, is induced in culture by pisatin, the isoflavanoid phytoalexin of pea. This pisatin induction is suppressed by nutritional factors. We have been studying the mechanism of pisatin induction through in vitro identification of regulatory factors and regulatory elements of the PDA1 promoter. We have developed an in vitro transcription system for N. haematococca which accurately initiates at the PDA1 promoter and reflects the pisatin induction of PDA1 mRNA observed in vivo. This in vitro activity allowed a functional test of a limited set of 5' upstream deletions in the PDA1 promoter. In vitro binding studies have identified a DNA binding factor which is appears in mycelial extract after treatment of the mycelium with pisatin. This pisatin-responsive factor binds to a minimum size region of 35 bp approximately 500 bp upstream of the transcription initiation site. Tests using the in vitro transcription assay and in vivo competition both indicate a role for this binding region in the high expression of PDA1 under pisatin-induced conditions. Southwestern blotting has identified one component of this binding activity to be a ~35 kDa protein. The availability of these functional and structural tests of function, in conjunction with complementary in vivo tests, allow the detailed dissection of the signal pathway leading from exposure of the cell to pisatin towards the activation of PDA1 transcription.

Straney, D. & H.D. Van Etten. 1994. Characterization of the PDA1 promoter of Nectria haematococca and identification of a region which binds a pisatin-responsive DNA binding factor. Molec. Plant-Microbe Interact. 7: 256-266.
Isolates of Nectria haematococca (anamorph: Fusarium solani) are able to detoxify the pea phytoalexin pisatin through expression of pisatin demethylase (pda). This enzyme is a substrate inducible cytochrome P450 monooxygenase that is encoded by the PDA gene family. In the current study, PDA1, a highly inducible PDA gene, was cloned and the 5' untranslated region was sequenced. The PDA mRNA levels were measured in pisatin-treated mycelium and found to increase by 20 fold over untreated control. Gel shift assays identified a 35 bp region, -514 to -480 bp relative to the first mRNA start site, that binds a factor found in extracts of pisatin-treated mycelium and absent in untreated mycelium. The function of the binding site in pisatin regulation of the PDA1 gene was tested in an in vivo competition assay by introduction of multiple ectopic copies of the binding site into N. haematococca through transformation. In such transformants, induction of pda activity by pisatin was delayed and reduced, consistent with the titration of a trans-acting factor which responds to pisatin. Together, these results suggest the 35 bp region in PDA1 is functioning in binding a pisatin-responsive activator. Additional regulatory signals were characterized which act on PDA1 expression. Induction of pda by pisatin was suppressed by the addition of 0.8% casamino acids or 5% glucose to the suspended mycelium. A unique DNA binding factor was detected only in extracts from mycelia treated with the casamino acids which binds to the same 35 bp region of the PDA1 gene as the pisatin-responsive factor.

Wilhite, S.E., R.D. Lumsden & D.C. Straney. 1994. Mutational analysis of gliotoxin production by the biocontrol fungus Gliocladium virens in relation to suppression of Pythium damping-off. Phytopathology 84: 816-821.
The fungus Gliocladium virens is an important biocontrol agent against plant pathogenic fungi, such as Pythium ultimum and Rhizoctonia solani, that cause damping-off disease. Gliocladium virens strain G20 (syn GL21) has been commercially formulated into the disease-suppressing product GliogardTM. One possible mechanism of G. virens biocontrol may be through the production of the fungistatic metabolite gliotoxin. The presence of this metabolite has previously been associated with disease suppressive activity towards P. ultimum. The purpose of this study was to critically test, using mutational analysis, the importance of gliotoxin production in the disease-suppressiveness effected against P. ultimum. Seven mutants lacking gliotoxin production (glx- phenotype) were isolated using selection-based enrichment and screening procedures following UV-treatment of parental strain G20-4VIB (WT). On average, these glx- mutants displayed only 54 % of the disease-suppressive activity of the wild-type isolate in vivo, and experienced a near-total loss of antagonistic activity in vitro, towards P. ultimum.