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Applied and Environmental Microbiology, December 2001, p. 5377-5383, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5377-5383.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Planta Regulation of Extension of an Endophytic
Fungus and Maintenance of High Metabolic Rates in Its Mycelium in the
Absence of Apical Extension
Yong Y.
Tan,1
Martin J.
Spiering,1,
Vicki
Scott,1
Geoffrey A.
Lane,2
Michael J.
Christensen,2 and
Jan
Schmid1,*
Institute of Molecular BioSciences, Massey
University,1 and AgResearch Grasslands
Research Centre,2 Palmerston North, New
Zealand
Received 15 May 2001/Accepted 12 September 2001
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ABSTRACT |
The fungus Neotyphodium lolii is an endophytic
symbiont. It grows in the intercellular spaces of the perennial
ryegrass Lolium perenne, producing secondary metabolites
which enhance the fitness of the association over that of uninfected
L. perenne. We report that the average number of hyphal
strands in a given section of a leaf remains constant during the life
of a leaf, indicating synchrony of leaf and hyphal extension, including
cessation of hyphal extension when leaf extension ceases. We used a
constitutively expressed reporter gene as an indicator of the
mycelium's metabolic activity during and after hyphal extension.
Reporter gene activity decreased when the mycelium stopped extending in
liquid culture but not in planta. This indicates that in planta
endophyte hyphae remain metabolically highly active when extension has
ceased and throughout the life of the leaf they are colonizing. The
behavior of the fungus in planta indicates the existence of signaling
pathways which (i) synchronize the extension of leaf and hypha by
regulating hyphal extension, (ii) suppress hyphal branching, and (iii)
stop apical extension of fungal hyphae, without reducing the
mycelium's metabolic activity. These signals may be crucial for the
symbiosis, by allowing the endophyte to switch the focus of its
metabolic activity from extension to the production of secondary metabolites.
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INTRODUCTION |
Neotyphodium endophytes
are mutualistic symbionts of grasses, living in their intercellular
spaces. Their presence confers upon the grass a number of physiological
characteristics such as drought resistance, enhanced growth, and
protection from herbivores, through the synthesis of feeding deterrents
and toxins. In return, the plant provides nutrients and propagates the
endophyte via its seeds (17, 28, 34, 35, 46, 47).
We have recently used a Neotyphodium endophyte, transformed
with the Escherichia coli 
-glucuronidase gene (GUS
reporter gene system [14]), under the control of a
heterologous constitutive promoter, to estimate the in planta
distribution of endophyte metabolic activity (as GUS activity)
(12). We found that GUS activity followed well-defined
patterns, established during the growth of ryegrass leaves
(12). The endophyte metabolic activity in plant tissue
measured in such experiments depends on (i) the number of endophyte
hyphae present, (ii) the rate of gene expression in these hyphae, and
(iii) the half-life of the GUS enzyme. Thus, while the existence of
these patterns was intriguing, these experiment allowed only limited
conclusions as to how they are generated. We have now determined
directly the in planta distribution of endophyte hyphal biomass. In
parallel, we have determined the endophyte's constitutive GUS
expression in the same ryegrass tillers. We have also determined GUS
activity in exponentially growing and stationary laboratory cultures of
the same endophyte strain. Together, these data allow us to draw
conclusions regarding the endophyte's metabolic state in different
parts of the ryegrass tiller.
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MATERIALS AND METHODS |
Strains and plant growth conditions.
All experiments were
carried out with strain KS1, created by cotransformation of
Neotyphodium lolii strain Lp19 (7) with the
hygromycin resistance plasmid pAN7-1 (29) (kindly provided by D. B. Scott) and plasmid pFG-gpd, containing the GUS reporter gene under the control of a constitutive heterologous promoter, the
Aspergillus nidulans gpdA promoter. Plasmid pFG-gpd was
constructed as follows (21). Initially, the
Escherichia coli -glucuronidase gene was obtained
as a 1.9-kb fragment by sequential digestion of plasmid pRAJ275
(14)with EcoRI, the Klenow fragment of DNA polymerase I and SalI. The A. nidulans trpC
terminator was then obtained as a 0.6-kb fragment by sequential
digestion of plasmid pNOM-102 (29) with BamHI,
the Klenow fragment, and HindIII. The two fragments were
ligated into a position adjacent to the multicloning site of pGem-1
(Promega), following digestion of the vector with
HindIII and SalI, yielding the plasmid
pFunGus. The latter was digested with SmaI and
NcoI, and the A. nidulans gpdA promoter was
ligated into it; the promoter was obtained by sequential digestion of
pNom-102 with EcoRI, the Klenow fragment and
NcoI. For cotransformation, protoplasts were generated by digestion of cell walls with Novozyme 234 (Novo Industri A/S) according
to the method of Murray et al. (22) except that the MgSO4 concentration in the osmotic medium was
raised to 1.5 M, transformed by the method of Itoh et al.
(13), and plated on osmotically stabilized CM medium
(23), containing 100 µg of hygromycin B/ml. Selected
hygromycin-resistant transformants were assessed for GUS expression by
placing them in microtiter wells containing 4-methylumbelliferyl
glucuronide (MUG) in GUS extraction buffer (14) at room
temperature for 3 h and monitoring the emergence of fluorescence
brought about by the conversion of MUG into methylumbelliferone (MU) on
a transilluminator (31). The transformant chosen for further work, KS1, contains a single copy of the GUS reporter gene
(31). KS1 could not be purified by spore purification (it does not sporulate), but we have evidence that it is a homokaryon: Lp19
is a monokaryotic fungus (33), and heterokaryons are thus expected to form pellets in liquid culture with clearly defined GUS-negative sectors (38); none were observed among 104 pellets of transformant KS1 assessed by using the dye Imagene Green,
according to the methods described by Herd et al. (12).
KS1 was introduced into perennial ryegrass seedlings (cultivar Nui) 1 year prior to the beginning of the study, by the method of Latch and
Christensen (18). Its in planta behavior is
indistinguishable from that of its untransformed parent
(49).
Grass plants originating from a single infected seedling were
maintained at 15°C in a growth cabinet with "12/24 h"
illumination (296 µmol of photons per m2 per s)
in 1.4-liter pots containing potting mix to which Osmocote (Grace
Sierra, Australia) slow-release fertilizer had been added. After growth
for 1 month, the plants were fertilized weekly (Thrive, Yates, New
Zealand). Plants were watered to saturation twice weekly and
transferred to new pots once the plants contained ca. 65 tillers (roughly every 2 months).
Counting of hyphae in cross sections.
Over a period of 9 months, single tillers were harvested and dissected as shown in Fig.
1. Hyphae were counted in aniline blue-stained transverse cuts taken from tissue samples 0.5 cm in length
(marked by shading in Fig. 1). For staining, samples were incubated in
a solution containing 63% ethanol, 7% lactic acid, and 16% glycerol
for 2 h at room temperature or, in case of the leaf blades, at
100°C for 1 to 2 min. Samples were then placed in 95% ethanol for 15 min and rinsed in distilled water for 1 min. Subsequently, all tissues
except the lowest part of the emerging leaf were placed into a solution
of 2.5 mg of chloral hydrate per ml of water for 1 to 2 h (most
parts of the emerging leaf and the leaf sheath) or 20 h (blades),
followed by 15 min in 50% ethanol and a 1 min rinse in distilled
water. Subsequently, the samples were placed in an aqueous solution
containing 27% lactic acid and 33% glycerol for 15 min. Transverse
slices for microscopy were then cut by hand with a scalpel and placed
in a drop of a solution containing 0.1% aniline blue and 2.5 g of chloral hydrate per ml of water. After 15 to 20 min, excess staining solution was removed with no. 1 Whatman paper, and sections were mounted on a microscope slide in a solution containing equal parts of
80% lactic acid, glycerol, and water, covered with a coverslip, and
hyphae were counted at ×400 magnification on a Zeiss KM microscope.
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FIG. 1.
Expanded view of a ryegrass tiller showing tissues used
for hyphal counts (shaded areas) and for GUS assays (unshaded areas).
In the uppermost sections, the tissue removed for counting was 0.5 cm
below the tip of the leaf. Shown is a three-leaf tiller, i.e., a tiller
with three mature, nonextending leaves in which the ligular zone
(marked by a thick black line) is visible, separating blade and sheath.
Some of the tillers only had two mature leaves, lacking the third leaf,
and others had only one mature leaf, lacking the second and third
leaves. The dashed lines indicate the borders of sections for which
results are reported in Table 1 and Fig. 2 and 3.
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Determination of hyphal diameters in planta
Transverse plant tissue segments, 1 to 2 mm thick, were fixed in 3%
glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer (pH 7.2)
(15) and then prepared for transmission electron microscopy as described by Spiers and Hopcroft (39).
Electron micrographs (at either ×31,800 or ×48,600 magnification)
were photocopied in duplicate onto Reflex A4 paper. Images of hyphal cross sections were then cut out and weighed on an Ohaus Explorer Balance. Squares of known size of the same paper were also weighed and
used as standards to calculate the area of the cross sections from the
weight of the cut-out images. Areas of magnified hyphal cross sections
thus obtained were divided by the square of the magnification factor to
obtain the area of the original hyphal cross sections.
Endophyte GUS activity determination.
Endophyte GUS
expression in grass tissue was determined in the same tillers used for
hyphal counts. The length and the fresh weight of each piece of tissue
taken for GUS assays (unshaded areas in Fig. 1) was determined. The
material was then ground in liquid nitrogen and freeze-dried, and its
dry weight was determined. The material was then extracted by
sonication and analyzed for GUS activity by a fluorometric assay based
on the conversion of MUG to MU (12). After subtraction of
background (0.25 pmol of MU min
1 per mg of dry
weight [12]) and correction for the interference of
plant material with the GUS assay by using a calibration curve (38; data not shown), the concentration of endophyte
metabolic activity in plant material was calculated as the picomoles of MU per minute per milligram of dry weight. Because the grinding step
could lead to substantial losses of material when small pieces of
tissue were processed, a calibration curve (38; data not shown) was used to determine the expected dry weight of the entire tissue sampled, based on its fresh weight. Expected dry weights were
then used to calculate the amount of GUS activity (in picomoles of MU
per minute) in the entire tissue sampled. To calculate the GUS activity
in the sections shown in Fig. 3, corrections were made for removal of
part of the tissue for hyphal counting, based on the length of the
tissue removed for hyphal counting.
The GUS activity of endophyte mycelium in liquid cultures was
determined on filtered, washed mycelia grown with shaking at
15°C in
YEG medium (0.5% yeast extract plus 2% glucose) and at
25°C in
potato dextrose broth (Difco) by using the assay and extraction
protocol as described above with one exception: based on estimates
of
extraction efficiency carried out as described by Herd et al.
(
12; data not shown), sonication was prolonged 2.5 times
to
achieve the same extraction efficiency as in the plant
tissue.
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RESULTS |
The average number of hyphal strands in a given section of a leaf
remains constant during the life of a leaf, indicating synchrony of
leaf and hyphal extension.
To get an indication of the
distribution of endophyte biomass in ryegrass tissue, we determined the
average number of hyphal strands in different parts of ryegrass tillers
with one, two, and three mature leaves (mature leaves are leaves that
have stopped growing and have exposed the ligular zone, which separates
the blade from the leaf sheath [37]). Figure
2 gives a graphic overview of average
numbers, listed also in Table 1. In the
figure, the leaves of the different tillers are aligned so that by
following the vertical arrows, one can follow hyphal counts throughout
the developmental history of an average leaf (for example, the emerging leaf in tillers with one mature leaf will become the innermost mature
leaf in tillers with two mature leaves and then the second leaf in
tillers with three mature leaves). Note that leaves elongate by basal
addition of tissue (37); thus, the tip of the emerging leaf will become the tip of mature leaf, and the basal parts of the
mature leaf are the last to be formed.
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FIG. 2.
Numbers of hyphal strands in sections of ryegrass
tillers infected with endophyte KS1. Three tillers with one mature
leaf, three tillers with two mature leaves, and five tillers with three
mature leaves were dissected and analyzed as described in Materials and
Methods. Tillers are represented schematically in side view with age of
leaves increasing from left to right; the emerging leaves, which are
still growing, are on the very left. Shading of the sections of the
individual leaves indicates the average numbers of hyphal strands per
section, following the key provided as part of the figure (for
instance, shading of the top section of the emerging leaf in the
uppermost row indicates that the tips of emerging leaves have an
average that falls between between 0 and 20 hyphal strands; the number
of hyphae in a section of an individual tiller is the average of hyphal
counts in cross sections taken from the top and the bottom of the
section; marked by shading in Fig. 1). Shaded circles next to the
sections indicate the standard deviation, according to the same key.
The vertical arrows indicate the developmental fate of tissues as
tillers develop more leaves (see Results for details).
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The figure shows that there are basal-apical gradients of similar
steepness in all emerging and mature leaves and that the
number of
hyphae in a given section of the average leaf remained
more or less
constant throughout its development. (The statistical
significance of
basal-apical gradients was demonstrated by ranking
the hyphal counts in
sections of each individual leaf and applying
the Kruskal-Wallis test
to these ranking data for each type of
leaf in each type of tiller
shown in Fig.
1 [
P < 0.05]; no statistically
significant changes in hyphal counts in a given leaf section with
developmental age were observed by using the
t test, nor did
we
find any trends in the data indicative of changes in hyphal counts
with age.) Given the mode of extension of the leaf, the simplest
explanation of these data is that an increasing number of hyphae
enters
the leaf as it extends from a multibranched mycelium at
its base
(leading to the basal apical gradients) and that hyphae,
once they are
in the leaf, extend at the same rate as the surrounding
leaf tissue and
without forming significant numbers of branches
(leading to a constant
number of hyphae in each plant tissue section
once it has formed). This
synchrony of growth includes a more
or less simultaneous cessation of
leaf tissue and hyphal extension,
leading to constant hyphal numbers
after the leaf has stopped
extending.
Estimates of the concentration of endophyte biomass.
To get an
indication of the contribution of endophyte biomass to the biomass of
the infected leaf, we determined the area of hyphal cross sections by
using electron microscopy in various parts of a tiller with three
mature leaves, and we used this value to estimate dry weight of the
mycelium (Table 2). According to these
estimates, even in the most densely colonized areas of the tiller, the
endophyte did not constitute more than 0.2% of the biomass of the
infected tissue. The data also suggest that endophyte biomass may
increase approximately twofold after extension has ceased, due to an
increase in hyphal thickness; such postextension increases in hyphal
thickness have also been observed in other filamentous fungi
(40).
Evidence that nonextending endophyte hyphae maintain high metabolic
rates in mature leaves.
In culture, cessation or slowing of
biomass increase and hyphal extension in fungi occurs when
environmental conditions curtail metabolic activity (1,
27). We wanted to investigate whether, in planta, the cessation
of extension of N. lolii hyphae coincides with, or is
brought about by, a curtailing of metabolic activity. The endophyte we
used carries the GUS reporter gene under control of a constitutive
heterologous promoter (see Materials and Methods for details). We could
therefore assess endophyte metabolic activity as constitutive GUS
reporter gene expression (12). Figure
3 shows the average distribution of
reporter enzyme activity in the same tillers that were used for hyphal
counts (data are also presented numerically in Table 1). No decrease of
reporter gene activity after leaf maturation was apparent.
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FIG. 3.
Distribution of GUS activity throughout ryegrass tillers
infected with endophyte KS1, constitutively expressing the GUS gene.
GUS activity was analyzed in extracts of the same sections of the same
tillers whose hyphal counts are shown in Fig. 2. Tillers are
represented schematically in a side view, with age of leaves increasing
from left to right; the emerging leaves, which are still growing, are
on the very left. Shading of the sections of the individual leaves
indicates the average GUS activity in a section, according to the key
provided as part of the figure (for instance, the shading of the top
section of the emerging leaf in the uppermost row indicates that the
tips of emerging leaves contain on average a GUS activity that falls
between 61 and 150 pmol of MU per min). The shaded circles next to the
sections indicate the standard deviation, according to the same key.
The vertical arrows indicate the developmental fate of tissues as
tillers develop more leaves (see Results for details).
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Figure
3 and Table
1 show activities in whole sections. Since the sizes
of sections varied between tillers, we also calculated
GUS activity per
grass tissue dry weight and per length of mycelium
in a section (data
not shown) and likewise saw no evidence indicating
a decline with leaf
age. We verified this by
t tests, in which
we compared GUS
activity values for the same section in mature
leaves of different
ages. Only in lower leaf sheath sections did
we find statistically
significant (
P < 0.05) decreases with age,
but only
for GUS activities per dry weight. No trend of such a
decrease was
apparent when total GUS activity in sections or GUS
activities per
mycelial length were compared for these sections.
Thus, the
age-dependent differences in GUS activity per dry weight
in lower leaf
sheaths are more likely caused by age-dependent
changes in the
composition of the sheath tissue rather than by
a decline in GUS
activity of the mycelium within
them.
The levels of GUS activity in tissue depend not only on the rate of
synthesis of the
-glucuronidase enzyme but also on the
half-life of the enzyme after its synthesis. Thus, the data presented
above would not necessarily preclude that hyphae reduce their
metabolic
rate, provided that
-glucuronidase molecules, once
synthesized, do
not decay significantly over the lifetime of a
leaf. We therefore
assessed the half-life of GUS activity in stationary-phase
mycelium in
liquid culture in YEG medium. The data shown in Fig.
4 suggest that the decay is a
three-component process with an
initial half-life of 5 days, followed
by a plateau after activity
has declined to 25% of the original,
followed by another phase
of decline with a half-life of the remaining
GUS activity of 6
days. The half-life of GUS, averaged across the
entire length
of the experiment, was 15 days. Very similar data were
obtained
when the same experiment was conducted in another growth
medium,
potato dextrose broth, or when growth was inhibited in the
latter
medium by adding KCN: there was an initial decline with a
half-life
of 5 to 6 days until activity had fallen to <50% of the
original
level, followed by a phase of slower decline (in these
experiments
GUS activity was only followed for
17 days after growth
had stopped).
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FIG. 4.
GUS activity of endophyte mycelium in liquid cultures
(YEG medium). Solid symbols show growth as the mycelial dry weight per
milliliter of culture; open symbols show the GUS activity per
milliliter culture. GUS activity was determined as described in
Materials and Methods in harvested mycelium.
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To estimate from these data whether a decrease of endophyte metabolic
activity in planta, after completion of leaf extension,
would lead to
measurable differences between plant tissues of
different ages, we next
determined the age difference between
leaves as follows. The extension
rate of leaves was determined
(0.94 mm per h, or 2.26 cm per day), as
was the maximum length
of a leaf (20 cm inclusive of the leaf sheath).
We also determined
that the next emerging leaf became visible once the
youngest mature
leaf had reached a length of ca. 11 cm. Based on these
data, one
can calculate that it takes ca. 9 days for a leaf to reach
its
final size and that each new leaf finishes its elongation ca.
4 days after the previous one. Thus, the outermost leaf of a tiller
with
two fully grown mature leaves will have been in a nonextending
state
for ca. 4 days and the outermost leaf of a tiller with three
fully
grown mature leaves will have been in a nonextending state
for ca. 8 days. If the nonextending hyphae in planta enter a metabolic
state
comparable to what occurs in stationary cultures, GUS levels
should be
lower by 30% in outer leaves of tillers with two mature
leaves than in
the mature leaf of a tiller with one mature leaf.
Likewise, GUS
activity in the outermost leaf of a tiller with
three mature leaves
should be 30% lower than in the outermost
leaf of a tiller with two
mature leaves and 60 to 70% lower than
in the mature leaf of a tiller
with one mature
leaf.
These calculations indicate that a decrease in metabolic activity of
the endophyte in planta, after leaf extension has ceased,
should be
detectable as a substantial reduction of GUS activity
in the older
tissues of the tillers we observed. The absence of
such a reduction is
therefore evidence that nonextending hyphae
in planta retain high
metabolic
activity.
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DISCUSSION |
In planta regulation of hyphal extension, hyphal branching, and
metabolism of N. lolii.
Our data provide evidence
that extension of endophyte hyphae and ryegrass tissue are highly
coordinated, as indicated by constant endophyte biomass content of leaf
tissue throughout its age. This conclusion is corroborated by recent
observations by Christensen et al. (6) that endophyte
extension ceases in mature leaves not only in the association we used
but also in a variety of other Neotyphodium-grass
associations examined.
Using the GUS reporter gene as an indicator of metabolic activity of
the mycelium, we have provided evidence indicating that
it is not the
curtailing of metabolic activity of the endophyte
that stops hyphal
growth when the leaf stops extending. This conclusion
is supported by
evidence which suggests that the environment of
the endophyte in mature
leaves is conducive to its further growth.
Christensen et al.
(
6) recently showed that in associations
containing a
mutant of the strain used here, as well as in some
other
N. lolii/ryegrass associations, older nonextending leaves
can contain
significantly higher number of endophyte hyphae than
young nonextending
leaves of the same tillers. The simplest explanation
for these
differences is the continued growth of endophyte hyphae
in these leaves
after completion of leaf
extension.
The most likely reason for coordinated growth of the endophyte and
plant tissue would thus be a signaling mechanism. The mechanism
could
make use of the movement of
Neotyphodium hyphae versus
surrounding
plant tissue during extension. We have recently proposed
(
32,
33) that
N. lolii colonizes extending
leaves from a multibranched
mycelium located in the meristematic tissue
at the base of the
leaf, with the endophyte mycelium growing by adding
material to
the hyphal apices as is customary for fungal hyphal
extension
(
2,
11,
45). If the model is correct, the
apically extending
hyphae will continuously slide through the
intercellular spaces
of the extending leaf, which grows by addition of
cells at its
base.
Based on this model, one possible mechanism for synchronizing endophyte
growth with that of the plant would be perception
by the growing tip of
the apically extending endophyte of biochemical
changes of neighboring
plant cells as they are separated farther
and farther from the basally
located meristem (
25). An even
more attractive (since it
is conceptually simpler) mechanism would
rely on the endophyte
possessing mechanosensitive channels, homologous
to those shown to be
involved in apical extension in other fungi
(
19,
20,
26,
42). Located at the tip of the hypha, such
channels could
sense a differential in speed of extension of hypha
and grass as
friction as the two slide past each other. If the
extension of both
tissues occurs at identical rates, no net movement
of tip versus
surrounding tissue and no friction would occur.
Signals from such
channels could be used to regulate the speed
of incorporation of new
cellular material into the hyphal apex,
including the cessation of
hyphal elongation when leaf extension
ceases. Likewise,
mechanosensitive channels along distal parts
of the hypha could sense
continuing friction, due to movement
of plant cells past the stationary
parts of the hypha, and this
signal could suppress branching; note that
the suppression of
branching in extending areas of the leaf is not only
supported
by the constancy of the number of hyphal strands reported
here
but also by direct microscopic assessment of in planta branching
frequency in epidermal strips and leaf sheaths (
6).
In culture, the cessation of growth of
N. lolii is
accompanied by a decline in GUS expression. The decline in expression
of
the GUS gene, controlled by a heterologous constitutive promoter,
is
to be expected as the mycelium enters stationary phase. Stationary
phase, triggered through changes in the environment such as nutrient
limitation, pH changes, or the accumulation of waste products
(
27), is accompanied in fungi and other organisms by
reduced
protein synthesis, even though specific pathways, such as
secondary
metabolite pathways, might become more active (
1,
4,
8,
9,
24,
30,
36,
41,
43,
44). In other words, in
culture
N. lolii biomass increase, through hyphal apical extension,
and
metabolic activity are correlated and linked. The maintenance
of high
GUS levels in nonextending mycelium in mature leaves indicates
that in
planta apical extension can cease without inducing a decline
in protein
synthesis rates typical of the stationary phase. This
suggests that in
planta hyphal extension and metabolic activity
can apparently be
uncoupled, with hyphal extension ceasing, while
high metabolic rates
are maintained. This should benefit the symbiosis
greatly, allowing the
endophyte to utilize, in mature leaves,
the equivalent of the
biosynthetic capacity expended previously
for rapid apical extension
(or a large part thereof) for the production
of secondary metabolites
which protect its host. If undiminished
biosynthetic capacity is indeed
switched from biomass synthesis
to secondary metabolite biosynthesis,
the end of leaf extension
should see the turning on of the respective
pathways at high rates.
While no in planta gene expression studies are
yet available to
ascertain that the pathways are switched on once
extension ceases,
the distribution of at least some of the known
alkaloids in ryegrass
tissue supports the idea of their predominant
synthesis in mature,
i.e., fully expanded leaf tissues
(
38).
Implications for biotechnology.
Our data suggest (i) that it
is possible to stop the extension of a fungal mycelium without
curtailing its metabolic activity, (ii) that N. lolii can
turn on secondary metabolite pathways under favorable environmental
conditions sustaining high metabolic rates, and (iii) the existence of
plant signals that regulate mycelial morphology in terms of branching.
These observations are of potential interest for biotechnology, given
the rheological impact of mycelial morphology on biotechnological
production and given that many biotechnological productions with fungi
aim at secondary metabolites (3, 5, 10). If we could
identify the N. lolii signaling mechanisms that regulate its
in planta growth and morphology, it might be possible to apply this
knowledge to filamentous fungi used in biotechnology to control
mycelial morphology and to switch from fungal biomass synthesis to
secondary metabolite synthesis without having to resort to creating
adverse conditions to stop growth. An additional biotechnological
application of the pathways regulating the speed of extension could be
to use their homologues in human pathogenic fungi as targets for the
development of novel therapeutic fungistatic agents.
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ACKNOWLEDGMENTS |
This work was supported by the New Zealand Foundation for
Research Science and Technology. M.J.S. was supported by a Massey University Ph.D. scholarship.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: 64-6-350-4018. Fax: 64-6-350-5688. E-mail:
J.Schmid{at}massey.ac.nz.
Present address: Department of Plant Pathology, University of
Kentucky, Lexington, Ky.
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Applied and Environmental Microbiology, December 2001, p. 5377-5383, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5377-5383.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
