From the Department of Plant Genetic Engineering,
Centro de Investigacion y de Estudios Avanzados del Instituto
Politécnico Nacional, Unidad Irapuato, Apartado Posta 629, Irapuato, Guanajuato 36500, México and the ¶ Department
of Biology, Technion-Israel Institute of Technology,
Haifa 32000, Israel
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Photolyases and blue light receptors belong to a
superfamily of flavoproteins that make use of blue and UVA light either
to catalyze DNA repair or to control development. We have isolated a
DNA photolyase gene (phr1) from Trichoderma
harzianum, a common soil fungus that is of interest as a
biocontrol agent against soil-borne plant pathogens and as a model for
the study of light-dependent development. The sequence of
phr1 is similar to other Class I Type I eukaryotic
photolyase genes. Low fluences of blue light rapidly induced
phr1 expression both in vegetative mycelia, which lack
photoprotective pigments, and, to a greater extent, in
conidiophores. Thus, visible light induces the development of
pigmented, resistant spores as well as the expression of
phr1, perhaps announcing in this way the imminent exposure
to the more damaging short wavelengths of sunlight. Light induction of
phr1 in non-sporulating mutants shows that a complete
sporulation pathway is not required for photoregulation. The light
requirements for photoinduction of phr1 were not altered in
dimY photoperception mutants. This suggests that
photoinduction of sporulation and of photolyase expression is distinct
in their photoreceptor system or in the transduction of the blue light signal.
Since its origin, life has been a continuous fight against the
harmful effects of UV light. Photoreactivation is an efficient repair
mechanism for cyclobutane pyrimidine dimers
(CPDs),1 the most common type
of DNA damage induced by UV light. It is mediated by photolyase (EC
4.1.99.3), an enzyme that uses blue and UVA light (320-400 nm) to
split dimers (1). Photolyase absorbs light due to the presence of
two intrinsic chromophores: a catalytic pigment, reduced flavin, and a
light-harvesting pigment that is either a deazaflavin
(8-hydroxy-5-deazaflavin) or a pterin (MTHF). CPD photolyases are
present in the three kingdoms of life, but two classes, distantly
related, can be distinguished: the Class I or microbial CPD photolyases
and the Class II or CPD photolyases from higher eukaryotes (2). Based
on the kind of light-harvesting pigment, Class I photolyases are
subdivided in two types: Type I or the MTHF class and Type II or the
8-hydroxy-5-deazaflavin class. CPD photolyase genes belong to the
emerging photolyase/blue light receptor family, which also includes
(6-4) photolyases and blue light photoreceptors. (6-4) photolyases
repair (6-4) photoproducts. Blue light receptors sense light in plant
seedlings and trigger responses such as inhibition of hypocotyl
elongation and phototropism (3, 4). All members of the family have
chromophores for light absorption, but only CPD and (6-4)
photolyases have repairing activity. These different proteins have
probably evolved by a divergent process from a common ancestor that had
a primarily CPD photolyase function (5, 6). As photoreactivation is separated from photoreception in photosynthetic organisms, it has been
proposed that fungi, non-photosynthetic bacteria, and lower eukaryotes
with blue/UVA morphogenetic responses constitute suitable candidates in
which to search for an ancestral photolyase that would also function as
a photoreceptor (5). However, a Neurospora mutant in which
the photolyase gene was disrupted (7) exhibits a full response to the
light stimulus.
In view of the DNA protective role of photolyase, it seems likely that
photoreactivation might be increased by exposure to any environmental
factors that are harmful to DNA. In this sense, it has been reported
that UVB light (280-320 nm) induces photoreactivation in
Arabidopsis (8). It has also been reported that UVC
(200-280 nm) and chemical mutagens induce photolyase expression in
Escherichia coli and yeast (9, 10). Furthermore, visible
light induces photoreactivation in goldfish cultured cells and in
Phycomyces spores (11, 12). Visible light also induces
photolyase gene expression in goldfish cell cultures (13). Thus,
visible light could be acting as a cue that is associated with the more
damaging UV regions of the spectrum; it has been proposed that blue
light responses evolved to alert organisms to impending stress
(14).
Although photoreactivation has been studied extensively, the role of
photolyase and its developmental regulation in different stages of
eukaryotic life cycles are poorly understood. According to its
function, more photolyase activity would be expected upon transitions
from dark to light or during continuous exposure to light. In fact,
high levels of photolyase transcription in Drosophila ovaries were found and related to survival in the embryonic stage (15).
To test the hypothesis that associates photolyase with photomorphogenesis and development, we decided to study the function of
a photolyase in Trichoderma harzianum. This common soil
fungus is used as a biocontrol agent of some phytopathogenic fungi (16) and as a photomorphogenetic model due to its ability to conidiate upon
exposure to light. In total darkness, T. harzianum grows indefinitely as a mycelium provided that nutrients are not limiting. However, a brief pulse of blue light given to a radially growing colony
induces synchronous sporulation. A ring of conidiophores bearing
green conidia is produced at what had been the colony perimeter at the
time of the light pulse (17). A cryptochrome-type photoreceptor is
involved in this response (18). The action spectrum of
photoreactivation in T. harzianum corresponds to a photolyase of the MTHF class (19). Here we report the cloning of a
T. harzianum photolyase gene (phr1), its
strong and rapid induction by low fluences of blue light, and a subtle
but significant developmental regulation during sporulation.
Strains and Growth Conditions--
T. harzianum
(ATCC32173) was grown at 24-25 °C on complete PDYC medium (24 g/liter potato dextrose broth, 2 g/liter yeast extract, and 1.2 g/liter
casein hydrolysate; all from Difco). Some experiments employed the
dimY mutants LS44 (lys-133W5 dimY) and RB10
(rib-Br dimY) and their corresponding parental strains, LS
(lys-133W5) and RB (rib-Br) (20). Two non-sporulating mutants (NS1 and
NS2) and their parental strains were also used. Mutants NS1 and NS2
were isolated from strain 43arg-Y1 (kindly provided by Dr. D. Aviv)
following mutagenesis with x-rays for 40 min at 270 R/min, giving a
survival rate of 23%.
Isolation of phr1--
The completely degenerate primers PL1
(18-mer, 5'-GCNTGG(C/A)GNGA(T/C)TT(T/C)TA(T/C)-3') and PL3 (20-mer,
5'-GG(A/G)TT(G/A)AANACNC(G/T)(G/A)AA(G/A)TA-3') were synthesized
(Biotechnology General, Rehovot, Israel) according to the amino acid
sequences indicated in Fig. 1. These primers were used to PCR-amplify a
fragment using a program including 2 min at 95 °C, followed by 45 cycles for 1 min at 94 °C, 45 s at 45 °C, and 2 min at
72 °C. Thermocycling was followed by an additional extension step of
10 min at 72 °C. A Southern blot (21) of the products was probed at
low stringency with the Neurospora crassa phr
cDNA clone (22). An abundant band of 438 bp, which hybridized to
N. crassa phr, was cloned in pCRscript (Stratagene), sequenced, and used to screen genomic and cDNA libraries. Six different cDNA libraries of strains IMI206040 and ATCC32173
previously described (23, 24) were screened with the 438-bp probe, and two partial cDNA clones were obtained. 10,000 plaques of
recombinant phage of the Photoinduction--
Colonies were grown on filter paper
soaked with PDYC medium: an 8-cm Whatman No. 50 disc overlaying a 7-cm
Whatman No. 1 disc in a 9-cm plastic Petri dish. Experiments were begun
after 36 h of growth in total darkness, when colony diameter
reached 40-50 mm. For the non-sporulating mutants NS1 and NS2, the
upper filter paper was replaced with a layer of dialysis tubing, and
the colonies were grown for 4 days in the dark. The cultures were
photoinduced by exposure to a standard blue source consisting of light
from a cool-white fluorescent tube filtered through a blue acrylic filter (maximum transmission = 442 nm; fluence rate = 2 µmol m RNA Extraction and Northern Analysis--
Total RNA was isolated
using a modified phenol-SDS method (27) and was used for Northern
analysis using standard procedures (21). A 1.35-kb EcoRV
fragment from phr1 was used in all Northern analyses.
Primer Extension Analysis--
Primer extension of the
phr1 mRNA was performed as described previously (28).
The oligonucleotide used was 5'-GGTCTCAACTCCATTGGC-3', which is
complementary to the phr1 mRNA starting at position
+160.
Isolation of a T. harzianum Photolyase Gene--
Using degenerate
primers targeted to conserved regions of photolyases, a 438-bp fragment
was PCR-amplified from Trichoderma DNA. Sequence analysis of
this fragment exhibited 82.4% identity, at the amino acid level, to
N. crassa phr, thus confirming that it indeed encodes a
member of the photolyase family.
Screening a cDNA library with the 438-bp probe resulted in the
isolation of partial clones (600 bp) containing the 3'-end. A 5.8-kb
PstI fragment, from recombinant phage isolated by screening a genomic library with the above-mentioned probe, was subcloned. Comparison between the restriction patterns from the 5.8-kb fragment and the cDNA clones allowed us to localize the coding sequence in a
3-kb region. The 3-kb fragment sequence contains an open reading frame
of 1887 bp, encoding a protein of 629 amino acids, interrupted by a
single intron of 59 bp at bp 2389. The gene was named phr1.
The 1887-bp open reading frame has three potential initiation codons.
Only the first ATG codon has a nucleotide sequence (TGTAAATGCT) in its vicinity that matches the consensus sequence (C/T)CA(A/C)(A/C)ATG(A/T)(C/T) reported around
initiation codons of T. harzianum (23-25,
28).2
The overall identity of the T. harzianum phr1 deduced amino
acid sequence to the N. crassa and Saccharomyces
cerevisiae photolyases is 62 and 30.6%, respectively. The
sequence identity in the helical domain, where most amino acids
involved in chromophore binding and substrate interaction are located,
is 73.3 and 45.1%, respectively (Fig.
1). The deduced Phr1 sequence has all the
amino acids involved in FAD binding, present in all photolyases and
blue light receptors. Like Class I CPD photolyases, it conserves the
amino acids that interact with CPD, and it shares with Class I Type I
CPD photolyases the amino acids involved in MTHF binding (Fig. 1) (6,
29). On the basis of identity to fungal photolyases and the presence of
the conserved amino acids involved in MTHF binding, phr1 can be assigned to the Class I Type I photolyase group (2).
The transcription start point was determined by primer extension, and
it is located 45 bp upstream of the first ATG codon (Fig.
2, A and B). Two
other potential transcription start points were also detected 29 and 36 bp downstream of the first ATG codon (Fig. 2A). Analysis of
the 5'-upstream region shows the presence of several putative
regulatory sequences. These are a TATA-like box sequence at position
Steady-state Levels of phr1 mRNA Increase following Exposure to
Light and during Development--
For analysis of the expression of
phr1 during photoinduced conidiation, vegetative mycelium
was grown in the dark until it reached a conidiation-competent stage
and then was exposed to a pulse of blue light of 540 µmol
m
In blue light-induced conidiating cultures, conidiophore rings were
harvested starting 12 h after the light pulse, when phialides are
emerging from branched conidiophores (Fig. 3A). At this
stage, a weak signal of phr1 mRNA was detected from
whole colonies, which persisted through 16-24 h after the light pulse,
during completion of conidiophore development and the formation of
pigmented spores.3 To test whether phr1
expression is developmentally regulated, we analyzed separately
conidiophore rings (c) and the surrounding mycelia
(m) and found that conidiophore rings have more
phr1 mRNA than mycelia at the times sampled from 12 to
24 h after the light pulse (Fig. 3B). This suggests
that phr1 is also up-regulated during conidiophore
development. A weak band above the phr1 mRNA signal
appeared in samples from 12 to 24 h (Fig. 3B) and also appeared in dark controls (Figs. 4 and
6B); this could correspond to a transcript with some
homology to phr1 and seems to be repressed by light.
Light-induced Expression of phr1 Is Due to Transcriptional
Activation and Does Not Require Protein Synthesis--
The high levels
of phr1 mRNA detected upon illumination may be the
result of transcriptional activation or increased stability of the
phr1 message. For this reason, we tested the effect of actinomycin D on the light-induced accumulation of phr1
mRNA. Inhibition of transcription initiation by actinomycin D
(AD) prevented accumulation of the message (Fig. 4),
indicating that the increase in the phr1 mRNA level is
mainly due to transcriptional activation. In addition, inhibition of
protein synthesis by cycloheximide (CH) did not block
phr1 photoinduction, indicating that protein synthesis is
not needed for transcriptional activation of phr1 (Fig.
4).
phr1 Is Regulated by Light in Conidiophores and Vegetative
Mycelia--
To test whether phr1 can be light-induced in
conidiophores, conidiating cultures at 12, 16, 24, and 36 h after
photoinduction were exposed to continuous illumination (3 µmol
m Characterization of phr1 Induction by Light in Non-sporulating and
Photoreception Mutants--
As phr1 is up-regulated both by
light and during development, we tested whether photoinduction would
occur under conditions in which morphogenesis is blocked. In two
non-sporulating mutants (NS1 and NS2), phr1 expression was
induced by a pulse of blue light (Fig.
6A). The non-sporulating
mutants had a general decrease in mRNA content as compared with
their parental strains, as shown by hybridization with an actin gene
(not regulated in the time window of the experiment) (Fig.
6A). In addition, we tested whether photoinduction would
occur under conditions in which photoreception is altered.
dimY photoresponse mutants (LS44 and RB10) were compared with the strains from which they were derived. In this case,
phr1 expression was induced by exposure to blue light that
saturated the induction of conidiation and weakly by a subsaturating
pulse (Fig. 6B). Both dimY strains showed strong
induction of phr1 mRNA by a pulse of blue light, which
was indistinguishable from the control strains (Fig. 6B).
There is therefore no increase in the threshold for induction of
phr1 mRNA. In contrast, the induction of sporulation was
shifted to higher fluences in dimY strains (Fig.
6C) (20).
The predicted sequence of phr1 contains the features of
a functional photolyase (Fig. 1). Furthermore, it does not have the C-terminal extension that is found in several blue light
photoreceptors, but not in functional CPD photolyases. The prediction
of the first ATG codon as the start codon in the deduced Phr1 protein
was consistent with primer extension analysis. It differs from the
N. crassa gene, in which the second ATG codon was reported
as the translation start site. Thus, the Trichoderma
photolyase is 38 amino acids longer than the N. crassa
photolyase at its N terminus. To gain some insight as to the possible
function of this extra region in Phr1, it was compared with the SBASE
library of protein domain sequences, indicating best similarity to the
signal peptide of the clostripain precursor. It was also compared
against the BLOCKS data base, giving hits with bovine calcitonin,
bacterial leader peptidase, and goldfish blue light-sensitive opsin
domains (36). These results suggest a translocating or interacting
function for this region of Phr1.
The presence of nuclear and mitochondrial localization signals in Phr1
(Fig. 1) suggests that it repairs dimers in both compartments, as
demonstrated in yeast (37). The Class I Type I CPD photolyase predicted
from the phr1 sequence can account for the photoreactivation of UV-inactivated spores (19). All efforts to detect additional photolyase genes, by using low stringency Southern analysis, PCR, and
complementation experiments of E. coli phr1 Visible light induction of photoreactivation seems to be a phenomenon
common to all vertebrates (14), suggesting that the induction of repair
mechanisms by long wavelengths of light may be an adaptive response to
the environment, in which visible light components are acting as a cue
for harmful effects of the UV component of sunlight. Increased
photolyase expression induced by visible light in goldfish cell
cultures was observed 4-8 h after exposure to light equivalent to 100 kJ m The levels of phr1 mRNA in dark-grown mycelia are
undetectable, consistent with the fact that Trichoderma
mycelium, which is hyaline, develops in soil, where UVC levels are
virtually zero. It has been proposed that blue light acts as a stress
signal, alerting the organism to impending photodamage (14).
According to this light-adaptive response theory, the transient and
strong phr1 mRNA increase caused by illumination can be
explained as a mechanism designed to turn on a system for protection
against any potential light-induced damage. The remaining levels of
phr1 mRNA detectable even after completion of
conidiation could be correlated with the enzyme levels able to repair
potential DNA damage.
Molecular correlations with photoinduced conidiation of
Trichoderma include a decrease in expression of
pgk (phosphoglycerate kinase) (28), gpdh
(glyceraldehyde-phosphate dehydrogenase) (24), and actin3
and an increase in a highly expressed gene
(con-1)3 and in a multidomain cell-surface
protein and its mRNA.2 All these changes occur in the
later stages of blue light-induced conidiation (12 h after the light
pulse). As phr1 induction occurs within the first minutes
after the light pulse, phr1 can be considered the first
light-regulated gene isolated in this organism.
In N. crassa, the genes al-1, al-2, and
al-3, which code for carotenogenesis enzymes, are induced
shortly after a brief illumination, and their transcript levels reach a
maximum in ~30 min. Similar patterns of induction are shared by the
conidiation genes con-5, con-6, and con-10 and
the blue light-induced genes bli-3 and bli-4. All
these genes are considered as a fast responding group (7). Deletion
analysis of the al-3 promoter indicated that the APE and
CCAAT boxes are responsible in part for the response to light. The
putative APE element was also found in al-1, con-10, and
ccg-2 (31). Expression of fast light-regulated genes in
Neurospora requires functional WC-1 and WC-2, putative
transcription factors. Recent evidence that the WC-1 zinc finger binds
to GATA boxes emphasizes the importance of these sequences in light
inducibility (40).
A sequence similar to the APE element is present in the phr1
promoter (Fig. 2B). The phr1 APE sequence
GAA-TTGGCG is 6 out of 8 bases identical to that of the al-3
gene. The APE element is flanked by CCAAT, GATA, and TATA-like boxes.
Comparison of al-3 and phr1 promoter sequences
shows a conservation between elements and their arrangement (Fig.
7). Considering the similarities between
the phr1 and al-3 promoters, we can also expect
similarities in their regulatory mechanisms. Indeed,
Trichoderma could be an additional model in which to test if
a regulatory protein binds the APE element.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EMBL3 genomic library of strain IMI206040
(25) were screened with the above-mentioned probe, and three clones showing similar restriction patterns were obtained. A 5.8-kb
PstI fragment was subcloned into the pT3T7 vector (Roche
Molecular Biochemicals). Four fragments, 1.35 kb (EcoRV),
0.5 kb (EcoRV-HindIII), 1.2 kb
(HindIII-EcoRV), and 1 kb (BglII),
from the 5.8-kb original clone were subcloned into the Pzero vector
(Stratagene). Sequencing was carried out in an Applied Biosystems Prism
377 DNA sequencer using a dye terminator cycle sequencing kit with
Amplitaq polymerase. The amino acid alignment was obtained with the
MEGALIGN program (DNASTAR, Inc., Madison, WI) according to the CLUSTAL
method (26).
2 s
1) or two cool-white fluorescent
tubes filtered with LEE filter 183 (fluence rate = 3 µmol
m
2 s
1). Wide-band green and red light were
obtained using LEE filters 124 and 106, respectively. At the indicated
times, the mycelia or conidiophore rings were scraped from the surface
of the filter paper under weak yellow or red safelight (0.1 µmol
m
2 s
1). Samples for RNA extraction were
immediately frozen in liquid nitrogen. In assays with cycloheximide (50 µM) and actinomycin D (160 µg/ml), colonies were
transferred to fresh medium containing the inhibitor by gently lifting
the upper filter paper, 1 h and 5 min before illumination, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (59K):
[in a new window]
Fig. 1.
Comparison of fungal CPD photolyases.
Alignment of T. harzianum (Th), N. crassa (Nc)
(22), and S. cerevisiae (Sc) (43) photolyases.
Boxed amino acids show identities between phr1
and other photolyases. Putative nuclear and mitochondrial localization
signals are indicated by lines above the sequence.
Dots indicate amino acids involved in MTHF binding;
plus signs those involved in CPD binding; and vertical
bars those involved in FAD binding. The bent arrow
indicates the beginning of the predicted helical domain. Straight
arrows indicate the positions of the PCR primers.
16; CCAAT boxes at positions
127 and
241; a DNA damage-responsive
element (called a DRE box) (30) at position
365; an APE element,
involved in blue light regulation of the N. crassa al-3 gene
(31), at position
86; and binding sites for Stunted (StuA), an
Aspergillus nidulans protein involved in conidiophore
development (32), at positions
41 and
822. Other putative control
elements found include binding sites for CreA (33) at positions
136
and
215; two GATA boxes (34) at positions
73 and +19; and heat
shock factor boxes at positions
28,
388,
823, and
839. Some of
these boxes are indicated in Fig. 2B. The sequence
CTTGCCTCTT that resembles the con-10 dark repression-related
sequence (35) is present twice in the phr1 promoter at
positions
25 and
536. The 3'-untranslated region contains a
polyadenylation site at position +2071, obtained by alignment with the
cDNA sequences. The calculated size of the transcript is 2 kb, in
agreement with the 2.2 kb estimated by Northern blotting. Phr1, like
other eukaryotic CPD photolyases, has a protruding N-terminal domain
with putative nuclear localization signals (KGSK and KRVK) and the
motif RRFYPH, which is a putative signal for mitochondrial localization
(Fig. 1) (22).
View larger version (41K):
[in a new window]
Fig. 2.
Organization of the phr1
promoter and primer extension. A, primer
extension analysis. The dot indicates the transcription
start point. B, sequence of the phr1 promoter
region showing putative regulatory boxes. CCAAT and TATA-like boxes are
thickly underlined; other boxes are thinly
underlined (see "Results" for details). The bent
arrow indicates the transcription start point.
2. Under these conditions, conidiation is triggered at
the edge of the colony, and a ring of conidiophores and mature spores
is visible 24 h later (17). No phr1 mRNA was
detected in the dark, becoming detectable immediately after the light
pulse (Fig. 3B). The message
reached its maximum accumulation between 15 and 30 min and started to
decrease 60 min after the pulse. From 2 to 8 h after the light
pulse, phr1 mRNA continued decaying. Similar induction
kinetics were observed when the cultures were exposed to continuous
illumination.3 An additional,
weaker signal was detected, of smaller size than the phr1
mRNA. This band may be a degradation product that is trapped by
ribosomal RNA, although we cannot exclude the possibility that it could
correspond to another transcript. The rapid and transient accumulation
of the phr1 transcript after the light pulse (Fig.
3B) strongly suggests that it is directly triggered by
light. The time window when phr1 expression is strongly
induced can be correlated with other physiological events that occur in photoinduced sporulation. The peak of induced phr1
expression occurs prior to the earliest sign of visible morphogenesis,
represented by stabilization of aerial hyphae, which occurs between 2 and 4 h after the light pulse (Fig. 3A). Thus,
phr1 expression is an early marker for photoinduction.
Previous experiments indicated that a blue light exposure of 125 ± 16 µmol m
2 was sufficient for half-saturation of
phr1 induction, whereas 90% saturation was obtained with
240 µmol m-2, as determined from nonlinear fits to
fluence-response curves. Experiments carried out with wide-band filters
to determine the range of wavelengths active in phr1
induction showed that blue light was more efficient. phr1
induction levels by 420 µmol m
2 of green and red light
were 65 and 0% relative to blue light induction,
respectively.3
View larger version (42K):
[in a new window]
Fig. 3.
Accumulation of phr1
mRNA in mycelia following exposure to a pulse of blue light
and during development. A, shown is a schematic
representation of the stages of photoinduced conidiation (44).
B, total RNA was extracted from dark-grown and blue
light-induced (540 µmol m 2) cultures at the indicated
times: dark controls at time 0 and 0, 5, 15, and 30 min and 1, 2, 4, and 8 h after the light pulse. These samples consisted of the
whole colony. The 12-, 16-, and 24-h samples consisted of conidiophore
rings (c) and the surrounding mycelium (m), which
includes the peripheral and central mycelia. The resulting Northern
blots were probed with a phr1 fragment and with a 28 S rRNA
clone as a loading control. The arrow indicates the light
pulse. The upper panel shows colony morphology during
photoconidiation. The black circles represent the
conidiophore ring.
View larger version (62K):
[in a new window]
Fig. 4.
phr1 photoinduction is blocked by
actinomycin D. Total RNA was extracted from dark-grown
(D) and blue light-induced (L; 15 min, 3 µmol
m 2 s
1) cultures with cycloheximide
(CH) or actinomycin D (AD) or without inhibitors.
Samples were collected immediately after illumination. The resulting
Northern blots were probed with a phr1 fragment. A 28 S rRNA
probe was used as a loading control.
2 s
1) 15 min before harvesting of
conidiophores. Under these conditions, the phr1 transcript
accumulated strongly in conidiophore-enriched samples as compared with
the dark controls (Fig. 5B).
To compare phr1 photoinduction in conidiophores
(c) with that in the mycelium, the mycelial region
surrounding the conidiophore ring (m), including the central
(cm) and peripheral (pm) mycelia, was collected.
Both the conidiophore and mycelium showed an increase in
phr1 mRNA levels in light-exposed conidiating cultures
compared with their dark controls, but conidiophore samples showed a
stronger signal than the mycelium (Fig. 5B). As these
results suggested a different capacity for photolyase induction, we
analyzed this effect in more detail. Induction of phr1 was
compared between conidiophore rings, newly formed conidiation-competent
peripheral mycelium, and central mycelium. The different regions indeed
have a different capacity for photolyase induction, with conidiophores
being more responsive than peripheral mycelium, and the latter more
responsive than central mycelium (Fig. 5C).
View larger version (38K):
[in a new window]
Fig. 5.
Induction of phr1 mRNA
expression in conidiophores. A, shown is a diagram
indicating regions of sample collection. B, total RNA was
extracted from conidiophores (c) and surrounding mycelia
(m) at the indicated times after photoinduction;
m samples are mixtures of peripheral mycelium
(pm) and central mycelium (cm). Developing
conidiophores were exposed to light before harvesting
(Light; 15 min, 3 µmol m 2 s
1)
or kept in the dark (Dark). C, total RNA was
extracted from conidiophores, peripheral mycelium, and central mycelium
at 12 and 24 h after photoinduction. The resulting Northern blots
were probed with a phr1 fragment. A 28 S rRNA probe was used
as a loading control.
View larger version (61K):
[in a new window]
Fig. 6.
Photoinduced phr1 mRNA
expression in non-sporulating and dimY mutants.
A, total RNA was extracted from dark-grown (D)
and blue light-induced (L; 480 µmol m 2)
cultures of NS1 and NS2 strains 1 h after the light pulse.
B, total RNA was extracted from dark-grown (0) and blue
light-induced (20 and 480 µmol m
2) cultures of
dimY strains RB10 and LS44 and their parental strains (RB
and LS, respectively) 2 h after the light pulse. The resulting
Northern blots were probed with a phr1 fragment. The blots
were also hybridized with an actin fragment and a 28 S rRNA clone as a
loading control. C, shown are the results from the
photoinduction of sporulation in dimY strains (LS and LS44
(redrawn from Horwitz et al. (20)) and RB and RB10 (B. A. Horwitz, unpublished results)).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutants with several Trichoderma cDNA libraries, did not
lead to cloning of additional genes. This suggests that there are no additional photolyase genes in T. harzianum. Thus, we
propose that the phr1 gene product is responsible for the
photoreactivation of UV-inactivated Trichoderma conidia.
2 (38). Up-regulation in this system was also induced
by peroxide and by conditions of growth arrest (39). It was therefore
proposed that a subtle DNA damage caused by visible light may be the
signal for photolyase induction and that oxygen radicals may be
involved in triggering photolyase expression. In contrast, the
Trichoderma photolyase gene is induced by fluences of blue
light as low as 20 µmol m
2 (Fig. 5C).
Furthermore, phr1 mRNA is detectable even at "zero time," immediately after the light pulse (Fig. 3). Thus, the blue light-induced expression of the Trichoderma photolyase is
most likely directly regulated by light rather than by a DNA damage mechanism. Indirect evidence for the latter is that, in addition to the
existence of a blue light-regulated element, there is a DNA
damage-responsive element in the phr1 promoter, suggesting that both mechanisms work independently.
View larger version (13K):
[in a new window]
Fig. 7.
Comparison of the arrangement of putative
light regulatory elements between the al-3 and
phr1 promoters. See "Results" for
details.
phr1 mRNA levels are higher in conidiophores developed in the dark than in the surrounding mycelium (Figs. 3 and 5B, lower panel). This indicates that phr1 mRNA is synthesized de novo or that it is more stable in conidiophores. The presence of the Stunted A protein binding box in the phr1 promoter adds support to the first mechanism, suggesting a possible regulation of photolyase during conidiophore development. If this is true, this regulation should also be observed in conidiophores from cultures induced to conidiate by stimuli other than light.
Developing and mature conidiophores were able to increase the phr1 mRNA in response to light in a way similar to the mycelium. The level of phr1 mRNA, however, was more inducible in conidiophores than in the surrounding mycelium (Fig. 5B). This is consistent with the fact that conidiophore development and spore maturation occur on the soil surface, when Trichoderma is exposed to environmental light. The developmental regulation of phr1 and the high responsiveness to phr1 photoinduction in conidiophores thus suggest a role for photolyase in spore survival.
The different responsiveness to phr1 photoinduction in conidiophores, newly competent mycelium, and central mycelium suggests that the response depends on particular developmental states of the cell. The photoinduction time courses of conidiophores and peripheral mycelium are similar3; therefore, their differential responsiveness is not due to different kinetics. The fact that conidiophores and new conidiation-competent mycelium are more responsive to phr1 induction than central mycelium suggests a relationship between photomorphogenesis and phr1 induction.
phr1 photoinduction in conidiation-competent mycelium is tightly correlated with photoinductive events in blue light-induced conidiation, suggesting that phr1 photoinduction could be part of this event. Photolyase binds adducts other than CPDs, but does not repair them (41). Moreover, it has been reported that visible light causes sublethal damage to DNA, as detected by an increased mutation ratio (42). Therefore, we could extrapolate that photolyase could bind visible light-distorted DNA. Transient phr1 photoinduction drives a photolyase increase, which could result in a distorted DNA-photolyase complex accumulation, perhaps acting to amplify the light signal in the cell. Although phr1 appears to be developmentally regulated, its light inducibility seems to be independent of the capacity of the fungus to complete the conidiation process, as shown in non-sporulating mutants.
Photolyase expression might be induced by the same photoreceptor(s)
responsible for the photoinduction of conidiation. In this case, one
would expect induction of phr1 to be decreased in the same
manner as conidiation in mutants belonging to the dimY
complementation group. These mutants require increased fluences to
induce conidiation. They also have altered action spectra for the
induction of conidiation and altered in vivo absorption
spectra (20). On the other hand, photoreactivation was normal in
dimY mutants, indicating that they have a functional
photolyase (19). Comparison of induction by pulses that are saturating
(480 µmol m2) or subsaturating (20 µmol
m
2) for conidiation shows that the dimY
mutations affect sporulation, but not phr1 induction. The
data for phr1 mRNA induction do not indicate an
increased requirement for light in the mutants. Furthermore, fluence
response analysis of phr1 induction indicates that twice more light is required for half-saturation of phr1 induction
than for conidiation.3 Normal sensitivity to light for
phr1 induction in dimY strains is most easily
explained by different photoreceptors for sporulation and photolyase
expression. This conclusion, however, is still limited by our lack of
knowledge of the molecular nature of dimY. It remains
possible that the same photoreceptor is involved in both responses, but
that the transduction steps for phr1 induction are distinct
from those leading to conidiation and do not depend directly on
dimY.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to A. Yasui for the Neurospora photolyase gene and for helpful suggestions and to R. Amit for participation in the initial PCR amplification experiments. We also thank L. Herrera-Estrella for helpful discussions during the work; L. Gonzales-de la Vara, J. Aguirre, C. Scazzocchio, and J. Delano for critical reading of the manuscript; and B. Jimenez and G. Corona for helping in sequencing and primer extension analysis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States-Israel Binational Science Foundation Grant 93-00168/1 (to B. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ009960.
§ Recipient of a doctoral fellowship from the Consejo Nacional de Ciencia y Tecnologia and Consejo de Ciencia y Technología del Estado de Guanajuato.
To whom correspondence should be addressed. Tel.:
52-462-39600; Fax: 52-462-45849; E-mail:
aherrera{at}irapuato.ira.cinvestav.mx.
2 M. Puyesky, N. Benhamou, M. Van Montagu, P. Ponce-Noyola, A. Herrera-Estrella, and B. A. Horwitz, manuscript in preparation.
3 G. Berrocal-Tito, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CPDs, cyclobutane pyrimidine dimers; MTHF, 5,10-methenyltetrahydrofolate; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); APE, albino proximal element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|