CAMP regulation of ''pathogenic'' and ''saprophytic'' fungal spore germination
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Fungal Genetics and Biology 41 (2004) 317–326
www.elsevier.com/locate/yfgbi
cAMP regulation of ‘‘pathogenic’’ and ‘‘saprophytic’’ fungal
spore germination
Sima Barhoom and Amir Sharon*
Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Received 4 August 2003; accepted 13 November 2003
Abstract
We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum glo-
eosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external
conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two
cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract,
germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and
forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-
pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard
surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant
surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic
germination and for appressorium formation.
Ó 2003 Elsevier Inc. All rights reserved.
Keywords: cAMP; Spore germination; Colletotrichum gloeosporioides; Fungal pathogenicity
1. Introduction Conidial germination in the filamentous model spe-
cies Aspergillus nidulans is also initiated by fermentable
Spore germination is a key process common to all sugars. After the isotropic phase, the rounded, unicel-
fungi. It can be divided into four stages: (i) breaking of lular A. nidulans conidium differentiates a single germ
spore dormancy, (ii) isotropic swelling, (iii) establish- tube. Next, the germ tube is separated from the conid-
ment of cell polarity, and (iv) formation of a germ tube ium by a septum and a second germ tube develops op-
and maintenance of polar growth (dÔEnfert, 1997; posite to the first germ tube (dÔEnfert, 1997; Harris,
Wendland, 2001). In the yeasts Saccharomyces cerevisiae 1999; Wolkow et al., 1996). Similar to yeast spore ger-
and Schyzosaccharomyces pombe, glucose is sufficient to mination, conidial germination in A. nidulans and Neu-
induce spore germination. The signal is transmitted rospora crassa also involves activation of the cAMP
through Ga/Ras pathways that cause rapid increase in pathway and trehalose breakdown (dÔEnfert et al.,
cellular cAMP levels, which in turn activate cPKA cat- 1999). However, recent studies reveal substantial differ-
alytic subunits (Hatanaka and Shimoda, 2001; Hreman ences between S. cerevisiae and A. nidulans in regulation
and Rine, 1997; Xue et al., 1998). The activation of of cAMP levels and in the interaction with other signal
cPKA leads to activation of trehalose degradation and cascades (Fillinger et al., 2002).
glycerol synthesis that increase the cell osmotic potential Spores are the most common fungal infection struc-
(Pahlman et al., 2000). tures. Upon contact with a putative host they attach to
the surface and then germinate. Following spore ger-
mination, many plant pathogenic fungi differentiate
appressoria that assist in penetration of the fungus into
*
Corresponding author. Fax: +972-3-640-5498. the host tissues. Unlike A. nidulans that requires an ex-
E-mail address: amirsh@tauex.tau.ac.il (A. Sharon). ternal energy source, spores of plant pathogenic fungi
1087-1845/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2003.11.011
318 S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 usually germinate in a water droplet deficient of any by the type of germination-inducing signals already at nutrients. These spores depend on the limited endoge- the onset of spore germination. nous resources present within the resting spore for completing all the developmental stages that precede host penetration. These processes, which occur on the 2. Materials and methods host surface, must therefore be highly efficient to ensure successful penetration into the host tissues within a 2.1. Fungi and plants short period of time. Indeed, spores of plant pathogenic fungi respond to plant-specific signals, including chemo- Colletotrichum gloeosporioides f. sp. aeschynomene physical characteristics of the plant surface (thigmotro- 3.1.3 WT and transgenic strains were used. The fungus pism) and specific plant chemicals such as cuticular was maintained on EmersonÕs YpSs (EMS) medium as waxes, root exudates or plant volatiles (Allen et al., previously described (Robinson et al., 1998). For spray 1991a; Gilbert et al., 1996; Podila et al., 1993; Staples inoculation experiments we used 12-day-old Aeschy- and Hoch, 1997; Tucker and Talbot, 2001). Studies in nomene virginica plants, which are the natural host of several plant pathogens and especially in Magnaporthe the fungus. Droplet inoculation was performed on the grisea showed that spore germination and appressorium first true leaves of Pisum aestivum cv. White Sugar, formation are tightly linked and coordinated with each which is also susceptible to the fungus. Plants were other through several signal pathways. Involvement of grown in 10 cm pots in a growth chamber at 25 °C with a the cAMP pathway and of the Pmk1 and Mps1 MAP 14 h photoperiod. kinase cascades, have been demonstrated (Dean, 1997; Lee et al., 2003; Thines et al., 2000; Xu, 2000; Xu and 2.2. Transgenic fungi Hamer, 1996; Yang and Dickman, 1997). Thus, although the signals that induce spore germination Colletotrichum gloeosporioides transgenic isolates ex- are different, similar signal components govern spore pressing cytoplasmic or nuclear GFP were produced. germination in pathogenic and saprophytic fungi. For cytoplasmic GFP expression we used the gGFP Colletotrichum gloeosporioides f. sp. aeschynomene plasmid (Maor et al., 1998). For nuclear labeling (C. gloeosporioides) specifically attacks the legume weed we generated the plasmid pRS31w. The plasmid pRS31 Aeschynomene virginica. The fungus produces unicel- (Suelmann et al., 1997) was digested with KpnI and lular, oval conidia (asexual spores) that do not germi- BamHI to release the sGFP-StuA cassette. The fragment nate readily in liquid cultures due to self-inhibitory was ligated into plasmid pC8HS (Turgeon et al., 1993) compounds (Hegde and Kolattukudy, 1998). We pre- that was digested with the same enzymes. Transgenic viously showed that exposure of conidia to pea extract isolates were obtained by electroporation of germi- overcomes this inhibition and induces high rate of nated conidia according to Robinson and Sharon conidial germination in shake cultures (Robinson and (1999). The transgenic isolates had WT phenotypes Sharon, 1999). Germination in pea extract is charac- and pathogenicity. terized by rapid nuclear and cell division without swelling, and by the formation of a single germ tube 2.3. Dyes and staining procedures after 2.5 h. The other conidial cell does not germinate and it has been unclear whether it remains viable or FDA and FM4-64 were purchased from Molecular whether it loses viability and dies. Although not spe- Probes (Eugene, OR, USA). DAPI was obtained from cifically mentioned, this unidirectional spore germina- Sigma. Viable cells were stained with FDA (10 lg/ml in tion is typically seen in a range of additional plant phosphate buffer, pH 7.5) or FM4-64 (7 lM in water). pathogens. DAPI was added to cells after fixation with 0.1 N NaOH In this paper we report that different signals activate for 30 min. Excitation and emission wavelengths for two separate germination pathways in C. gloeosporio- FDA, FM4-64 and DAPI were 493/510 nm, 506/751 nm, ides. Unidirectional spore germination that leads to and 385/420, respectively. appressoria formation and plant infection occurs only in the presence of stimuli that are common to plant sur- 2.4. Microscopy faces, such as pea extract and a hard surface. In rich medium and in the absence of plant signals conidia Fluorescent and light microscopy were performed germinate from both sides as in Aspergillus, they do not with a Zeiss Axioskp 2 epifluorescence microscope, or form appressoria and are reduced in virulence. The un- with an Olympus SZX 12 fluorescent stereoscope equip- germinating conidial cell remains viable and can be in- ped with an eGFP filter. Confocal microscopy was per- duced to germinate by cAMP. Our results show that formed by a confocal laser scanning microscope (Zeiss, although germination and appressorium formation are CLSM 510). Digital images were captured with a DVC- separate processes, fungal pathogenicity is determined 1310 color digital camera (DVC, Austin Texas, USA).
S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 319
2.5. Spore germination microscope glass slide, on leaves, or on the surface of a
Petri dish.
The fungus was grown for 5 days on EMS plates
and conidia were harvested by washing the plates with 2.6. cAMP treatments
sterile distilled water. After dilution and counting, the
conidia were used to inoculate EMS or pea extract cAMP was purchased from CalBiochem. Conidia
(PE) liquid media (Robinson and Sharon, 1999). We were harvested in water, inoculated into media con-
used 106 conidia/ml unless otherwise mentioned. Coni- taining 20 or 25 mM cAMP and then placed under the
dia were germinated in shake cultures or on a solid different germination regimes.
surface. Germination in a shake culture was performed
in 250 ml Erlenmeyer flasks containing 50 ml liquid 2.7. Plant assay
medium. The experiments were conducted in a shaking
incubator operating at 190 rmp, at 28 °C in the light. Aeschynomene virginica plants were sprayed to runoff
Germination on a solid surface was performed on a with 0.1% Tween 20 conidial suspension containing
Fig. 1. Characteristics of C. gloeosporioides conidial germination. (A) Germination in pea extract. Conidia of a transgenic isolate expressing nuclear
GFP were inoculated into PE medium and incubated with shaking at 190 rpm. All images are from a confocal microscope and are a combination of
light and fluorescent spectra except for Ab, which shows only the fluorescent spectrum. Time points: a, resting conidia; b, 0.5 h; c, 1 h; d, 1.5 h; e,
2.5 h; and f, 3 h. (B) Viability stains of conidia germinated in PE. Conidia were stained with either FM4-64 (a, c, and e) or with FDA (b, d, and f).
(a,b) Negative controls of FM4-64 at 4 °C, and FDA at 55 °C, (c,d) positive staining of viable resting conidia, and (e,f) staining of conidia 4 h after
germination in PE. Both cells are stained with both dyes indicating that they are viable. (g) DAPI staining after 3 h showing that both cells contain an
intact nucleus. (C,D) Germination in EMS. Conidia were incubated in EMS with shaking at 190 rpm. (C) Differential interference contrast mi-
croscopy, (D) fluorescent microscopy after staining with DAPI. Time points: (C) a, resting conidia; b, 4 h; c, 6 h; d, 9 h; e, 12 h; and f, 15 h. (D) a,
resting conidia; b, 4 h; c, 9 h; and d, 13 h. (E) Germination of conidia on different surfaces. Conidia were harvested from plates in water and placed
on: a, A. virginica leaf; b, corn leaf; and c, microscope glass slide. Scale bars represent 10 lm in all pictures.
320 S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326
5 104 conidia/ml. Sprayed plants were closed in plastic To test how the different signals affect each other, we
cylinders and placed in a growth chamber at 25 °C. After placed conidia in PE for variable periods of time, then
24 h, the cover was removed and the plants were main- washed the conidia, moved them into EMS, and scored
tained until the end of the experiment in the growth the percent of germination 2.5 h after introduction into
chamber. Pea leaves were drop inoculated by placing PE. As little as five minutes in PE were sufficient to in-
5 ll droplets of conidial suspension containing duce 30% germination after 2.5 h, all from one side
5 105 conidia/ml on the leaf surface. The leaves were (Fig. 2). Germination rates after 2.5 h increased with
placed in a Petri dish with moisture and the plates were increasing exposure time to PE. Thus, even a short ex-
maintained under the same conditions as described for posure to PE was sufficient to induce a high rate of
the spray-inoculated plants. Pathogenicity was deter- unidirectional germination and the early PE signal was
mined by the rate of plant mortality and fresh weight superior to the EMS signal. The conidia that did not
(whole plants) or by microscopic examination and germinate by 2.5 h underwent swelling and germinated
scoring of necrosis in the drop-inoculated leaves. from both sides after longer periods of time (not shown).
When submerged in PE + EMS germination was mainly
from one side, even when the medium contained only
3. Results 10% PE. These results suggest that the PE signal is ep-
istatic to the EMS signal.
3.1. Germination in PE shake-culture
3.3. Germination on a solid surface
When submerged in pea extract (PE) conidia of
C. gloeosporioides undergo rapid nuclear and cell divi- On leaves of A. virginica, conidia germinated as in
sion (1 h), after which a single germ tube develops. We pea extract, only from one side (Fig. 1C, d). Over 30% of
further characterized this process using a transgenic the germ tubes differentiated appressoria, in contrast to
strain expressing nucleus-localized GFP (Fig. 1A), and germination in PE shake cultures in which appressoria
by staining the cells with viable dyes (Fig. 1B). Nuclear were almost never observed. Conidia germinated in the
migration was observed less than 30 min after sub- same way and produced appressoria on the leaf surface
merging conidia in PE shake cultures, and nuclear of non-host plants (Fig. 1E, b). To test if the signal was
division was noted after less than 1 h. Next, the two plant specific we germinated conidia in EMS on a glass
nuclei moved to opposite poles and a septum formed in slide. Under these conditions germination still occurred
the middle of the cell (Fig. 1A, d). A germ tube started to only from one side, but the germ tubes kept elongating
emerge from one side after about 2.5 h. The nucleus close and did not form an appressorium (Fig. 1E, c). On a
to the germ tube divided, one of the resulting nuclei Petri dish, (a hydrophobic surface), germination was
migrated into the germ tube and a septum was formed mainly from one side and approximately 90% of conidia
between the conidial cell and the elongating germ tube
(Fig. 1A, f). Viability and nuclear staining showed that
the un-germinating cell remained viable and contained 100
% Germination 2.5h after
an intact nucleus during the entire process (Fig. 1B, g).
80
incubation in PE
3.2. Germination in EMS shake culture 60
Different germination morphology was observed 40
when conidia were germinated in EMS. Conidia un-
20
derwent swelling after 5–7 h, and then formed a single
germ tube (Fig. 1C, d). After emergence of the germ 0
tube a septum formed inside the conidium and then a 0 15 30 45 60 75 90 105 120 135 150
second germ tube emerged from the other side. The In
Incubation period in PE (min)
whole process lasted 12–15 h, depending on conidial age.
Fig. 2. Short exposure of conidia to pea extract is sufficient to induce
The morphological changes, nuclear division and the
‘‘pathogenic’’ germination. Conidia were harvested from plates and
time course resembled the major events that take place then suspended in PE for various periods of time. At each time point
during A. nidulans spore germination (Momany and conidia were collected by centrifugation, washed three times with water
Taylor, 2000; Wolkow et al., 1996). Germination in and transferred to EMS. Germination rate was always scored 2.5 h
additional media, e.g., EMS without yeast extract or after suspending the conidia in PE. For example, after 5 min in PE
conidia were washed, incubated in EMS for 145 min and then the
glucose rich media had similar characteristics (not
germination rate was scored. All the conidia that germinated after 2.5 h
shown). Thus, two different signals, PE and a free car- germinated as in PE at all time points. At each time point we counted
bon source, induce two highly dissimilar styles of co- 250 conidia. Germination rate was calculated as percent of germina-
nidial germination. tion rate in PE after 2.5 h.S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 321
formed appressoria (Fig. 4A, f). Thus, a hard surface PE shake cultures amended with 25 mM cAMP and the
was sufficient to induce unidirectional germination even germination phenotypes were determined after 2.5 and
without PE, but a hard surface + PE or a hydrophobic 8 h. In PE + cAMP only 30% of the conidia germinated
surface was necessary for appressoria formation. from one side after 2.5 h as opposed to the 90% germi-
These results show that at least three different signals nation in PE without cAMP. The other 70% started to
affect conidial germination: PE, rich carbon source, and swell, similar to germination in EMS. After 8 h some
a hard surface. Each of the PE and hard surface signals conidia germinated from both sides, as in EMS, but
is sufficient to induce unidirectional germination, while there was a range of additional germination phenotypes
appressoria formation occurs only on a hard surface and as illustrated in Fig. 4B. The most common phenotypes
requires, in addition to a hard surface, hydrophobicity included germination from two cells at the same time,
or PE. formation of a germ tube on one side and an appres-
sorium on the other side, and formation of two germ
3.4. Effect of germination style on conidial pathogenicity tubes from the same cell. Only a few regular appressoria
formed normally, at the end of a short germ tube, while
The two styles of conidial germination suggested that the rest of them included a range of uncommon phe-
they may have different roles and may be important notypes (Fig. 4A). These changes occurred only in PE
under different conditions. Since on plants conidia ger- medium. Addition of cAMP to EMS did not affect the
minated from one side while in rich medium from two germination phenotype, but it enhanced germination;
sides, we hypothesized that only the unidirectional ger- conidia swelled earlier and formed a single or even two
mination may be associated with plant pathogenesis. To germ tubes after less than 8 h. Results of additional ex-
test this hypothesis we germinated conidia in PE and in periments showed that the saprophytic germination is
EMS up to the stages of germ tube formation and activated by cAMP, while different, cAMP-independent
swelling, respectively, and then used these conidia to signal pathways probably regulate pathogenic germi-
inoculate plants. When germinated in PE, conidia were nation (see Table 1 and Fig. 4 for complete phenotypes
as pathogenic to A. virginica as the ungerminated con- and effects of cAMP). cAMP was found important also
trol treatment, whereas pathogenicity of conidia that for appressoria formation. Normally appressoria were
were pre-incubated in EMS was drastically reduced formed only on a hard hydrophobic surface or when
compared to the other two treatments (Fig. 3A). We germinated on a glass slide in PE medium. Formation of
noted that some of the EMS-treated conidia produced appressoria was enhanced by cAMP under additional
new conidia on the plant surface (not shown). These conditions such as EMS on glass. This result suggested
conidia germinated only from one side and produced that cAMP might also cause a change in conidial
delayed symptoms. Indeed, the differences in pathoge- pathogenicity.
nicity of the PE and EMS-treated conidia were more
distinct four days than six days post inoculation. Mi- 3.6. Effect of cAMP on conidial pathogenicity
croscopic observations of pea inoculated leaves revealed
that the control and PE-germinated conidia penetrated We tested the effect of external cAMP on conidial
the plants and only very little mycelium could be de- pathogenicity after pre-incubation in PE and in EMS.
tected on the plant surface after 24 h. Necroses devel- As shown earlier, there was a clear difference in the re-
oped under the penetration sites after 36–48 h (Fig. 3B). sponse of conidia to cAMP in PE and in EMS. In PE,
In contrast, when conidia were pre-incubated in EMS, cAMP induced swelling after 2.5 h, and formation of
the mycelium developed on the plant surface without irregular germination and appressoria after 8 h (Table 1,
plant penetration, and the leaf tissue underneath the Fig. 4). In EMS, cAMP enhanced swelling and bi-di-
mycelium remained intact. These results confirmed the rectional germination in shake cultures, and appressoria
association of unidirectional germination with plant formation on a glass slide. Two different cAMP treat-
pathogenesis. We therefore coined the term ‘‘pathogenic ments were therefore used for PE and EMS germinated
germination’’ to describe this type of spore germination conidia. In PE, the purpose was to test pathogenicity of
while the bi-directional mode was termed ‘‘saprophytic conidia that have been induced to swell and formed
germination.’’ abnormal germination and appressoria. cAMP was
therefore added to the PE incubation medium and the
3.5. Effect of cAMP on spore germination and appressoria conidia were allowed to germinate for 2.5 h. This treat-
formation ment results in enhanced spore swelling and some 30%
of unidirectional germination (Table 1). After incuba-
In A. nidulans, cAMP is activated early on and in- tion the conidia were washed with water several times to
duces spore germination and swelling. We therefore remove the medium and any remnants of cAMP. In
decided to test if early external cAMP application will EMS, the purpose was to test pathogenicity of conidia
affect pathogenic germination. Conidia were grown in that were germinated under saprophytic conditions322 S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 Fig. 3. Plant infection by conidia that were germinated in PE or EMS. Conidia of a transgenic isolate expressing GFP were harvested from plates and incubated for 2.5 h in PE or for 5 h in EMS. (A) Conidia were sprayed on A. virginica plants and pictures were taken after 6 days. (a) Ungerminated conidia, (b) PE-germinated conidia, (c) EMS-germinated conidia, and (d) uninfected control. The average fresh weight of 6 plants SD is shown under each treatment. (B) Conidia were drop inoculated onto detached pea leaves and pictures were taken after 24 h. Lower part fluorescent, upper part visible light micrographs. Pictures (a–f) were taken with a microscope, pictures (g,h) were taken with a fluorescent stereoscope. (a,b) Ungerminated conidia, (c,d,h) PE-germinated conidia, (e,f,g) EMS-germinated conidia. Magnification is similar in panels (a–f). Scale bars are: a, 40 lm; g, 100; and h, 60 lm. Fig. 4. Germination and appressoria phenotypes induced by cAMP. (A) a–e, Various appressoria phenotypes induced by addition of cAMP to PE or EMS. f, Germination and appressorium formation on a Petri dish without cAMP. (B) Various germination phenotypes induced by addition of cAMP to PE or EMS. Bars represent 10 lm in all pictures. Fig. 5. Effect of cAMP on conidial pathogenicity. Conidia were harvested from plates and incubated for 2.5 h in PE or for 6 h in EMS. cAMP was added to PE before inoculation such that the conidia were incubated in PE + cAMP during the entire 2.5 h. In EMS cAMP was added after the incubation, before plant inoculation. Conidia were sprayed on A. virginica plants and pictures were taken after 24 h. Six plants were used for each treatment. All plants within a treatment showed identical results. Top without cAMP, bottom with 20 mM cAMP. (A) PE-germinated conidia; (B) EMS-germinated conidia; and (C) ungerminated conidia.
S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 323
Table 1
Summary of the germination and appressoria phenotypes with and without cAMP
EMS PEa
No cAMP +25 mM cAMP No cAMP +25 mM cAMP
Shake cultures
2.5 h No change Conidia start swelling >90% germination from 25–30% germination from
one side one side, the rest of
conidia swell
8h Swelling 30% germination from Hyphae formation, one 15–20% germination from
one or both sides, >1% cell does not germinate, one side after cell division,
form a single no appressoria 15–20% germination from
appressorium two sides after swelling,
60–70% of various
phenotypes.
Glass slide
2.5 h 80% germination from 80% germination from 90% germination from 90% germination from one
one side after cell one side after cell one side after cell side after cell division, 30%
division, >1% form division, 30% form division, 1% form form appressoria
appressoria appressoria appressoria
8h 80% germination from 80% germination from >90% one side 90% germination from one
one side, >10% of various one side, 5% various germination, 30% form side, 30% form normal
phenotypes. >1% form phenotypes normal appressoria appressoria, 5% abnormal
appressoria appressoria
a
The various appressoria and germination phenotypes are shown in Fig. 4.
(from both sides) and then induced to form appressoria enhanced by cAMP, other processes that are required
on the leaf by cAMP application. Therefore, the conidia for full pathogenesis may be mis-regulated in the EMS-
were first germinated for 5 h without cAMP until they germinated spores.
reached the stage of swelling, and then they were col-
lected, washed with water, and cAMP was added just
before plant inoculation. Conidia from both treatments, 4. Discussion
as well as ungerminated conidia were inoculated on
plants and symptoms were recorded. Addition of cAMP Colletotrichum gloeosporioides f. sp. aeschynomene is
to PE had no effect on conidial pathogenicity. No a facultative, hemibiotrophic plant pathogen: it can
symptoms were observed on plants inoculated with the grow as a saprotroph on dead organic matter, or as a
PE or PE + cAMP germinated conidia after 24 h (Fig. 5), pathogen by infecting the legume weed A. virginica.
and normal disease symptoms and plant mortality Similar to many other plant pathogens, conidia germi-
developed several days later (see Fig. 3 for disease nate upon contact with the plant and then form ap-
symptoms). In contrast, the addition of cAMP to EMS- pressoria that penetrate the plant cuticle. During the
germinated conidia caused abnormal disease develop- first 24–36 h following plant penetration the fungus de-
ment. Normally there are no symptoms until 48 h. After velops intramural or biotrophic hyphae. Minimal se-
48–72 h small anthracnose lesions appear on the leaves, cretion of hydrolytic enzymes is essential at this stage to
these lesions develop into larger necroses that spread to avoid provoking the plant defense system (Mendgen and
the stem and eventually kill the plants after 5–7 days. Hahn, 2002; Perfect et al., 2000). A necrotrophic phase
Addition of cAMP to conidia that were germinated in follows during which massive hydrolytic enzymes are
EMS caused early symptoms on the infected leaves secreted causing tissue maceration and cell death. The
(Fig. 5). Brown necroses developed already 24 h after situation is completely different when the fungus grows
spraying the plants and after 48 h the leaves died. The as a saprotroph. Under these conditions there is plen-
addition of cAMP to resting conidia caused a similar tiful external energy and nutrients available to the ger-
phenomenon while cAMP alone had no observable ef- minating conidium, and therefore germination under
fect on the plants. However, the disease caused by the saprophytic conditions does not depend on the conid-
EMS + cAMP treatment was restricted only to the leaves ium reserves and there is also no need for appressoria.
that were directly inoculated with conidia. These leaves Instead, the breakdown of large organic molecules may
developed the early necroses and died within 48 h, but require massive secretion of hydrolytic enzymes.
the disease was restricted to these leaves and it did not In this study we showed that C. gloeosporioides uses
spread to new leaves or to the stem (not shown). These two different germination strategies for pathogenic and
results suggest that although conidial penetration was saprophytic development. The pathogenic germination324 S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326
is triggered by chemical and physical plant surface sig- and the works on germination signal transduction
nals and is characterized by rapid cell division and for- pathways in other species suggest that activation of the
mation of a single germ tube 2.5 h after induction. The cAMP pathway is involved in the early stages of sap-
saprophytic germination has the A. nidulans character- rophytic germination. Cell wall and membrane modifi-
istics; it is induced by fermentable sugars and consists of cations that are regulated by the cell wall modifying
spore swelling before germ tube formation. Two germ Mpk1/Slt2 MAP kinase pathway might be required at a
tubes are eventually formed and the process takes be- later stage to allow the following morphogenetic chan-
tween 8 and 15 h. Only the unidirectional type of ger- ges (Wendland, 2001). In contrast, the early stages of
mination results in plant infection. pathogenic germination are not mediated by cAMP but
Although the different germination styles were by other, cAMP independent pathway(s). This is in-
demonstrated only in C. gloeosporioides, they seem to ferred from the disruption of the pathogenic germina-
be common to many other plant pathogens; examina- tion by cAMP application. Instead, we suggest that the
tion of various publications shows that many other first events of pathogenic germination are cell division
plant pathogenic fungi also germinate on the host only and cell wall modifications. This hypothesis is also
from one side without swelling. Moreover, work in supported by the fact that a C. gloeosporioides cell wall
other fungi such as M. grisea and Colletotrichum defected mutant exhibits saprophytic germination in PE
graminearum, showed that while these fungi normally (data not shown).
form a single germ tube, they are capable of germi- Two MAP kinase modules, homologues of the S.
nating from both sides under some conditions (Chaky cerevisiae Slt2 and Fus3/Kss1 pathways are involved in
et al., 2001; Thines et al., 2000). The work with M. the regulation of a series of developmental processes in
grisea signal transduction mutants is of a special in- filamentous fungi including conidiation, germination,
terest since some of these mutants have germination sexual reproduction, appressoria formation, and path-
defects such as longer germ tubes or formation of ogenicity (Tudzynski and Sharon, 2003; Xu, 2000).
multiple appressoria (Adachi and Hamer, 1998; Gilbert Some functions may be under regulation of either one of
et al., 1996; Mitchell and Dean, 1995). These data these MAP kinases. For example, conidiation and fe-
suggest that separate pathogenic and saprophytic ger- male sterility are regulated in M. grisea by the SLT2
mination pathways are a common strategy in plant
pathogenic fungi. Studies in additional plant patho-
genic species are necessary to verify the generality of
this phenomenon as well as to elucidate the unique
characteristics in each species.
PE induces pathogenic germination in liquid cultures
but without appressoria. Appressoria are formed only
on a hard surface with PE, or on a hard hydrophobic
surface. In M. grisea, appressoria formation requires
functional Pmk1 MAP kinase and is induced by cAMP;
pmk1 deletion mutants and various cAMP pathway
mutants do not form appressoria, while external addi-
tion of cAMP rescues some of the mutations and en-
hances appressoria formation in the WT (Adachi and
Hamer, 1998; Lee et al., 2003; Thines et al., 2000).
Treatment of C. gloeosporioides conidia with cAMP
changed the style of germination and appressoria for-
mation (see Fig. 4 and Table 1 for the various pheno-
types). Most significantly, cAMP induced swelling of a
large fraction of the conidia in PE (first stage of sap-
rophytic development) and enhanced swelling and ger-
mination in EMS. It also induced appressoria formation
under conditions which they do not normally form, such
as in a PE shake culture, on a hydrophilic surface, or in Fig. 6. Model for the regulation of saprophytic and pathogenic spore
EMS on plants. The germination and appressoria for- germination in Colletotrichum. Left, ‘‘Saprophytic’’ spore germination
mation in the presence of cAMP were not always regular is induced by fermentable sugars and is cAMP-dependent. Possible
involvement of a MAP kinase cascade is suggested. Right, ‘‘Patho-
and included many uncommon phenotypes (Fig. 4, Ta-
genic’’ germination is induced by plant signals as well as by a hard
ble 1). Application of cAMP to PE 2 h after the begin- surface, which activate cAMP-independent pathway(s) leading to rapid
ning of incubation resulted in much larger percentage cell division and germination without swelling. The cAMP pathway is
(40%) of regular appressoria. Taken together our results required later for appressorium formation.S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 325
homologue MPS1 (Xu et al., 1998), while in Cochlio- Harris, S.D., 1999. Morphogenesis is coordinated with nuclear division
bolus heterostrophus these processes are regulated by the in germinating Aspergillus nidulans conidiospores. Microbiology
145, 2747–2756.
FUS3/KSS1 homologue CHK1 (Lev et al., 1999). Only Hatanaka, M., Shimoda, C., 2001. The cyclic AMP/PKA signal
the FUS3/KSS1 homologues regulate appressoria for- pathway is required for initiation of spore germination in Schizo-
mation in a wide range of plant pathogens (Tudzynski saccharomyces pombe. Yeast 18, 207–217.
and Sharon, 2003; Xu, 2000). C. gloeosporioides forms Hegde, Y., Kolattukudy, P.E., 1998. Cuticular waxes relieve self-
appressoria only on a hard hydrophobic surface, or on a inhibition of germination and appressorium formation by the
conidia of Magnaporthe grisea. Physiol. Mol. Plant Pathol. 51, 75–
hard surface in the presence of PE. This suggests that 84.
pathogenic germination and appressoria formation are Hreman, P.K., Rine, J., 1997. Yeast spore germination: a requirement
under control of different signal pathways, which may for Ras protein activity during re-entry into the cell cycle. EMBO J.
involve the two MAP kinase cascades. A model illus- 16, 6171–6181.
trating our current understanding of the different ger- Lee, N., DÔSouza, C.A., Kronstad, J.W., 2003. Of smuts, blasts,
mildews, and blights: cAMP signaling in phytopathogenic fungi.
mination pathways is presented in Fig. 6. According to Annu. Rev. Phytopathol. 43, 03.1–03.29.
this model, the PE signal activates cell wall modification Lev, S., Sharon, A., Hadar, R., Ma, H., Horwitz, B.A., 1999. A
and cell division in a process that does not require mitogen-activated protein kinase of the corn leaf pathogen Coch-
cAMP. Appressoria formation is induced by surface liobolus heterostrophus is involved in conidiation, appressorium
signals and requires cAMP as well as additional path- formation, and pathogenicity: Diverse roles for mitogen-activated
protein kinase homologues in foliar pathogens. Proc. Natl. Acad.
ways. Under saprophytic conditions the order is re- Sci. USA 96, 13542–13547.
versed: the cAMP pathway is activated first and is Maor, R., Puyesky, M., Horwitz, B.A., Sharon, A., 1998. Use of the
followed by cell wall modifications. green fluorescent protein (GFP) for studying development and
fungal-plant interaction in Cochliobolus heterostrophus. Mycol.
Res. 102, 491–496.
Mendgen, K., Hahn, M., 2002. Plant infection and the establishment
Acknowledgments of fungal biotrophy. Trends Plant Sci. 7, 352–356.
Mitchell, T.K., Dean, R.A., 1995. The cAMP-dependent protein
We are thankful to Dr. Tamar Eilam for her valuable kinase catalytic subunit is required for appressorium formation and
assistance with the staining procedures, to Dr. Reinhard pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant
Cell 7, 1869–1878.
Fischer for providing the pRS31 plasmid and to Itai Momany, M., Taylor, I., 2000. Landmarks in the early duplica-
Sharon for his assistance in figures preparation. This tion cycles of Aspergillus fumigatus and Aspergillus nidulans:
research was supported by The Israel Science Founda- polarity, germ tube emergence and septation. Microbiology 146,
tion (Grant No. 722/01-17.1). 3279.
Pahlman, A.K., Granath, K., Ansell, R., Hohmann, S., Adler, L.,
2000. The yeast glecerol 3-phosphatases Gpp1p and Gpp2p are
required for glycerol biosynthesis and differentially involved in the
References cellular responses to osmotic, anaerobic and oxidative stress.
J. Biol. Chem. 31, 31.
Adachi, K., Hamer, J.E., 1998. Divergent cAMP signaling pathways Perfect, S.E., Pixton, K.L., OÔConnell, R.J., Green, J.R., 2000. The
regulate growth and pathogenesis in the rice blast fungus Magna- distribution and expression of a biotrophy-related gene, CIH1,
porthe grisea. Plant Cell 10, 1361–1373. within the genus Colletotrichum. Mol. Plant Pathol. 1, 213–
Allen, E.A., Hazen, B.E., Hoch, H.C., Kwon, Y., Leinhos, G.M.E., 221.
Staples, R.C., Stumpf, M.A., Terhune, B.T., 1991a. Appressorium Podila, G.K., Rogers, L.M., Kolattukudy, P.E., 1993. Chemical
formation in response to topographical signals by 27 Rust species. signals from avocado surface wax trigger germination and appres-
Phytopathology 81, 323–331. sorium formation in Colletotrichum gloeosporioides. Plant Physiol.
Chaky, J., Anderson, K., Moss, M., Vaillancourt, L., 2001. Surface 103, 267–272.
hydrophobicity and surface rigidity induce spore germination in Robinson, M., Riov, J., Sharon, A., 1998. Indole-3-acetic acid
Colletotrichum graminicola. Phytopathology 91, 558–564. biosynthesis in Colletotrichum gloeosporioides f. sp. aeschynomene.
dÔEnfert, C., 1997. Fungal spore germination: insights from the App. Environ. Microbiol. 64, 5030–5032.
molecular genetics of Aspergillus nidulans and Neurospora crassa. Robinson, M., Sharon, A., 1999. Transformation of the bioher-
Fungal Genet. Biol. 21, 163–172. bicide Colletotrichum gloeosporioides f. sp. aeschynomene
dÔEnfert, C., Bonini, B.M., Zapella, P.D.A., Fontaine, T., da Silva, by electroporation of germinated conidia. Cur. Genet. 36,
A.M., Terenzi, H.F., 1999. Neutral trehalases catalyse intracellular 98–104.
trehalose breakdown in the filamentous fungi Aspergillus nidulans Staples, R.C., Hoch, H.C., 1997. Physical and chemical cues for spore
and Neurospora crassa. Mol. Microbiol. 32, 471–484. germination and appressorium formation by fungal pathogens. In:
Dean, R.A., 1997. Signal pathways and appressorium morphogenesis. Carroll, G.C., Tudzynski, P. (Eds.), Plant Relationships The
Annu. Rev. Phytopathology 35, 211–234. Mycota, V part A. Springer, Berlin, pp. 27–40.
Fillinger, S., Chaveroche, M.K., Shimizu, K., Keller, N., dÔEnfert, C., Suelmann, R., Sievers, N., Fischer, R., 1997. Nuclear traffic in fungal
2002. cAMP and Ras signaling independently control spore hyphae: in vivo study of nuclear migration and positioning in
germination in the filamentous fungus Aspergillus nidulans. Mol. Aspergillus nidulans. Mol. Microbiol. 25, 757–769.
Microbiol. 44, 1001–1016. Thines, E., Weber, R.W.S., Talbot, N.J., 2000. MAP kinase and
Gilbert, R.D., Johnson, A.M., Dean, R.A., 1996. Chemical signals protein kinase A-dependent mobilization of triacylglycerol and
responsible for appressorium formation in the rice blast fungus glycogen during appressorium turgor generation by Magnaporthe
Magnaporthe grisea. Physiol. Mol. Plant Pathol. 48, 335–346. grisea. Plant Cell 12, 1703–1718.326 S. Barhoom, A. Sharon / Fungal Genetics and Biology 41 (2004) 317–326 Tucker, S.L., Talbot, N.J., 2001. Surface attachment and pre- Xu, J.R., Hamer, J.E., 1996. MAP kinase and cAMP signaling penetration stage development by plant pathogenic fungi. Ann. regulate infection structure formation and pathogenic growth in Rev. Phytopathol. 39, 385–417. the rice blast fungus Magnaporthe grisea. Genes Dev. 10, 2696– Tudzynski, P., Sharon, A., 2003. Fungal pathogenicity genes. In: 2706. Arora, D.D.K., Khachatourians, G.G. (Eds.), Fungal Genomics. Xu, J.R., Staiger, C.J., Hamer, J.E., 1998. Inactivation of the App. Microbiol. Biotechnol. Rev. 3, 178–212. mitogen-activated protein kinase Mps1 from the rice blast fun- Turgeon, B.G., Bohlmann, H., Ciuffetti, L.M., Christiansen, S.K., gus prevents penetration of host cells but allows activation of Yang, G., Sch€afer, W., Yoder, O.C., 1993. Cloning and analysis of plant defense responses. Proc. Natl. Acad. Sci. USA 95, 12713– the mating-type genes from Cochliobolus heterostrophus. Mol. Gen. 12718. Genet. 238, 270–284. Xue, Y., Batlle, M., Hirsch, J.P., 1998. GPR1 encodes a putative G Wendland, J., 2001. Comparison of morphogenetic networks of protein-coupled receptor that associates with the Gpa2p Galpha filamentous fungi and yeast. Fungal Genet. Biol. 34, 63–82. subunit and functions in a Ras independent pathway. EMBO J. 17, Wolkow, T.D., Harris, S.D., Hamer, J.E., 1996. Cytokinesis in 1996–2007. Aspergillus nidulans is controlled by cell size, nuclear positioning Yang, Z., Dickman, M.B., 1997. Regulation of cAMP and cAMP and mitosis. J. Cell Sci. 109, 2179–2188. dependent protein kinase during conidial germination and appres- Xu, J.R., 2000. MAP kinases in fungal pathogens. Fungal Genet. Biol. sorium formation in Colletotrichum trifolii. Physiol. Mol. Plant 31, 137–152. Pathol. 50, 117–127.
You can also read
