view article - American Phytopathological Society

The nit-2 gene of N. crassa encodes an activator protein that
governs overall nitrogen metabolism in the cell. We have used
the cloned gene as a heterologous probe to survey various
fungal phytopathogens for homologues. A wide variety of genera
showed homology as determined by Southern blotting. To further
study nitrogen regulation, we have chosen Fusarium app.
Genomic libraries of Fusarium moniliforme and Fusarium sacchari
have been constructed. Following screening, we have isolated a
6kb fragment from both fungi with homology to the nit-2 gene
from N. crassa. We also used the nit-2 gene to complement a
mutant of Fusarium graminearum that is defective in overall
nitrogen metabolism. Following transformation colonies were
obtained which now could utilize nitrogen sources similar to
the wildtype. This suggests that this heterologous regulatory
gene is expressed in Fusarium and functions to activate
expression of nitrogen regulated genes in this organism.
297
STRUCTURAL AND FUNCTIONAL ANALYSES OF TWO Q-TUBULIN GENES IN
COLLETOTRICHUN GRAMINICOLA. D. G. Panaccione and R. M. Hanau.
Dept. of Botany and Plant Pathology, Pudue University, West
Lafayette, IN 47907.
In organisms with multiple tubulin genes, expression of
individual tubulin genes is often developmentally regulated.
Two S-tubulin genes, TUBI and TUB2, were cloned from
Colletotrichum graminicola with the interest of studying their
involvement in conidial development. Southern hybridization
and DNA sequencing demonstrated that although the two genes
are considerably divergent, they both have the capacity to
encode 3-tubulin. RNA blots indicated that the level of TUB2
message relative to TUBI message increased in conidiating
cultures. To study the functional significance of this, the
TUB2 gene was replaced with a truncated copy of the gene by
site-specific integrative transformation. Transformants
carrying the truncated TUB2 allele did not display any
294 abnormalities in conidial development.
IDENTIFICATION OF SEQUENCES WITH PROMOTER ACTIVITY FROM 298
GIBBERE1IXA PULICARIS (FUSARIUM
Yangkyo
SAMBUCINUM)
P. Salch and
TRANSFORMANTS.
Marian N. Beremand, USDA, Agricultural
Research Service,
TRANSFORMATION
Northern Regional
OF TRICHODERMR
Research
SPP.
Center,
TO HYGROMYCIN
Peoria,
B RESISTANCE.
A. Siva, TE. Stasz, G.E. Harman and M. Hemmat,
IL 61604
Dept. of Horticult
Sciences, Cornell Univ., Geneva, NY, 14456.
G. pulicaris (GP) is a heterothallic
rot on potato
ascomycete
tubers.
and
GP
causes
protoplasts
dry
were transformed with a
cosmid
r
cosHygl containing
protoplasts
hygromycin
were obtained
B phosphotransferase
by digesting mycelium with
Novozyme
(hygB) fused
234.
to
Protoplasts
promoter
were
1 from
treated
Cochliobolous
with the plasmid
heterostrophus.
pH11B (obtained
Transformation
from O.C.
occurred
Yoder) containing
by random an
integration
Escherichia
of the
col
cosmid
hygromycin B
phosphotransferase gene, which was used
into
with
the GP
the
genome.
permission
Based on restriction of
digestion
Eli
and
Lilly &
Southern
Co., fused to a Cochliobulus
hybridization
heterostrophus promoter. Treated
analyses,
protopla
one of the transformants with
a single
Co.,
copy
fused
insertion,
to a m
63C3, had the recombination promoter
event
Tred prtopasts
were
occurring
plated in
between
a molten
the
agar
5'
medium
end of
which
hygB
later
coding
was
sequence
covered with
and
a second
the
end
3'
of promoter 1.
medium
Expression
layer
of
containing
hygB resistance
the antibiotic.
most
The
likely
application time
antibiotic
of the
resulted from endogenous GP
was
promoter-like
critical and differed
sequences.
between
Cloning
strains. Southern analysis
putative
of
and analysis of
transformants
the GP promoter-like
showed integration
sequences
into
will
the
be
genomic DNA. The
frequency of transformed nuclei in hygromycin B resistant isolates was
discussed.
usually lower than 1%. Therefore,
were
nuclei
allowed
from
to segregate
all putative
through conidiation.
transformants
Single spore isolates were
295 obtained that were mitotically stable on selective and non selective media.
CAROTENOID-OVERPRODUCING TRANSFORMANTS OF =]RQýBRQFAQM5 ARE
NOT RESISTANT TO CERCOSPORIN. _ C og_Qpperman, M. E. Daub, R 299
G. Carolina Upchurch, State and University, G. A. Payne. Raleigh, Dept. of NC Plant 27695-7616. Pathology, North
HEAT SHOCK-INDUCED DEVELOPMENT OF INFECTION STRUCTURES BY THE
The photoactivated toxin cercosporin
RUST FUNGI:
produces
EXPRESSION
singlet
OF
oxygen
THE INF GENES.
Staples,
S. Bhairi
Boyce
and R.C.__
Thompson Institute,
and
Cornell
superoxide. University,
Resistance Tower
of roso~~ spp. to cercosporin Road, Ithaca, NY 14853.
appears to act
singlet
at several
oxygen
levels.
quenchers,
Carotenoids,
are associated
which
with
are
resistance
potent
to
cercosporin.
Germlings of
A
Uromvces
Cercospora
aoDoendiculatus
nicoti__anae genomic
induced
library
by exposure to
28.5°C
constructed
heat for
in
1.5
the
hr
cosmid
developed
vector
infection
pSV50 was
structures
used to transform
similar
cercosporin-sensitive
to those induced thigmotropically
Ljjosoo Capa.
and
A
at
clone
about
(B5)
the
was
same rate.
identified
After heat
that
shock,
conferred
appressorium
increased
development
carotenoid
was
production
accompanied
to
by
the
the
transforsants.
appearance of at
Eight
least
additional
six heat-shock
B5 transformants
proteins,
also
but
synthesis of
overproduced
the thigmotropic-specific
carotenoids. Southern
proteins
blot
which
analysis
occurs
indicated
insertion
the
of
during
9 ico__tia.nae
contact-induced
DNA into the
development
genome. The
was
carotenoidnot
observed.
Thigmotropically-induced
overproducing transformants
appressorium
did not
development
show increased
is
resistance
to cercosporin.
accompanied by
These
an
results
upshift
support
in the expression
the previous
of a
hypothesis
small group
(Phytopathology
of INF genes,
79:180)
and we
that
have
carotenoids
identified
are
six
not
of
the
these
sole
by now.
Here
mechanism
we have
of
examined
cercosporin
the expression
resistance,
of four ds-genes after
inducing infection structure
'
development by heat shock. Genes
IN___FFli 56 and INF24 were induced only moderately. I__Fl F64 was
induced in germlings heat shocked for 4 hr but not when heat
shock was applied for only 2 hr. INF88 was not induced.
296
REGULATION OF NITROGEN METABOLISM IN~ FUSARIUM BY A HOMOJLOGOUS 300
NEUOSOR GEE.Mati B.Dikmn~ an
of
JhnF.Leslie,
Plant Path., Univ.
Dept.
of Nebraska,
RESTRICTION
Lincoln
FRAGMENT
68583,
LENGTH
and
POLYMORPHISMS
Dept. of
BETWEEN
AND ADZUKIBEAN
SOYBEAN
Plant Path., Kansas
PHIALOPHoRA
State Univ.,
GREGATA
Manhattan
ISOLATES.
66506.
L. E. Gray,
and A. Hepburn, USDA, Agricultural Research Service, and
1172 PHYTO PATH OLOGY