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.
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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.
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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.
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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.
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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.
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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.
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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.
Back to references
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.
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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.
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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.
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