Fungal Genetics and Biology 56 (2013) 33–41
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Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi
Differences between appressoria formed by germ tubes and appressorium-like structures developed by hyphal tips in Magnaporthe oryzae
Ling-An Kong a,b, Guo-Tian Li c, Yun Liu a, Mei-Gang Liu a, Shi-Jie Zhang a, Jun Yang b, Xiao-Ying Zhou c,
You-Liang Peng b, Jin-Rong Xu a,c,⇑
a
b
c
NWAFU-Purdue Joint Research Center, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China
State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Plant Pathology, China Agricultural University, Beijing 100193, China
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history:
Received 1 October 2012
Accepted 31 March 2013
Available online 13 April 2013
Keywords:
Hyphal tips
Germ tubes
Plant penetration
Melanin layer
Wax
Pyricularia oryzae
a b s t r a c t
Melanized appressoria are highly specialized infection structures formed by germ tubes of the rice blast
fungus Magnaporthe oryzae for plant infection. M. oryzae also forms appressorium-like structures on
hyphal tips. Whereas appressorium formation by conidial germ tubes has been well characterized, formation of appressorium-like structures by hyphal tips is under-investigated. In a previous study, we found
that the chs7 deletion mutant failed to form appressoria on germ tubes but were normal in the development of appressorium-like structures on artificial hydrophobic surfaces. In this study, we compared the
differences between the formation of appressoria by germ tubes and appressorium-like structures by
hyphal tips in M. oryzae. Structurally, both appressoria and appressorium-like structures had a melanin
layer that was absent in the pore region. In general, the latters were 1.4-fold larger in size but had lower
turgor pressure than appressoria, which is consistent with its lower efficiency in plant penetration. Treatments with cAMP, IBMX, or a cutin monomer efficiently induced appressorium formation but not the
development of appressorium-like structures. In contrast, coating surfaces with waxes stimulated the
formation of both infection structures. Studies with various signaling mutants indicate that Osm1 and
Mps1 are dispensable but Pmk1 is essential for both appressorium formation and development of appressorium-like structures on hyphal tips. Interestingly, the cpkA mutant was reduced in the differentiation of
appressorium-like structures but not appressorium formation. We also observed that the con7 mutant
generated in our lab failed to form appressorium-like structures on hyphal tips but still produced appressoria by germ tubes on hydrophobic surfaces. Con7 is a transcription factor regulating the expression of
CHS7. Overall, these results indicate that the development of appressorium-like structures by hyphal tips
and formation of appressoria by germ tubes are not identical differentiation processes in M. oryzae and
may involve different molecular mechanisms.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
Fungal pathogens have evolved various strategies to overcome
diverse barriers they encounter during the penetration of plant
cells (Xu et al., 2006). The rice blast fungus Magnaporthe oryzae is
the most destructive pathogen of rice throughout the world and
a model for studying genetic mechanisms of fungal–plant interactions (Dean et al., 2005; Wilson and Talbot, 2009). It carries out a
series of well-defined developmental processes during foliar infection, including the formation of the penetration structure known as
appressorium at the tip of germ tubes (Xu and Hamer, 1996; Zhao
⇑ Corresponding author at: NWAFU-Purdue Joint Research Center, College of
Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China. Fax:
+86 765 496 0363.
E-mail address: [email protected] (J.-R. Xu).
1087-1845/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.fgb.2013.03.006
et al., 2007). In the past two decades, appressorium formation and
penetration have been extensively studied at the cellular and
molecular levels (Wilson and Talbot, 2009). Several well-conserved
signaling pathways are known to be important for appressorium
formation and penetration in M. oryzae, including the cAMP signaling, Pmk1, and Mps1 MAP kinase pathways (Li et al., 2012; Wilson
and Talbot, 2009; Xu and Hamer, 1996; Xu et al., 1998). A few
putative receptor genes such as Pth11 and Msb2 and downstream
transcription factors such as Mst12 that are functionally related to
appressorium morphogenesis and penetration also have been
identified (Kamakura et al., 2002; Liu et al., 2011; Park et al.,
2002, 2004).
In addition to the formation of appressoria at the tip of germ
tubes, M. oryzae also forms appressorium-like structures at hyphal
tips on hydrophobic or plant surfaces that are morphologically
similar to appressoria (Kong et al., 2012). Two transcription factor
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L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
genes in M. oryzae, COS1 and HTF1 (also known as MoHOX2), are
essential for conidiogenesis (Kim et al., 2009; Liu et al., 2010; Zhou
et al., 2009). The cos1 and htf1 deletion mutants failed to produce
conidia but formed appressorium-like structures at hyphal tips. Because Pmk1 was essential for both the formation of appressoria
and appressorium-like structures by germ tubes and hyphal tips
(Liu et al., 2010), it has been assumed that these melanized structures are similar infection structures involving the same regulatory
mechanisms. However, the differences between appressoria
formed by germ tubes and appressorium-like structures developed
on hyphal tips have not been closely examined.
In a previous study, we found that germ tubes of the chs7 deletion mutant failed to form appressoria on artificial hydrophobic
surfaces (Kong et al., 2012). However, under the same conditions,
deletion of the CHS7 chitin synthase gene had no effect on the formation of appressorium-like structures at hyphal tips, suggesting
that appressorium formation and development of appressoriumlike structures may be different in specific genetic backgrounds.
In this study, we determined the differences between the formation of appressoria and appressorium-like structures under different conditions in the wild type and various signaling mutants. On
hydrophilic surfaces, cutin monomers, cAMP, and IBMX efficiently
induced appressorium formation but not the development of
appressorium-like structures. In contrast, waxes stimulated the
formation of both appressoria and appressorium-like structures.
Whereas the roles of Pmk1 and Mps1 appear to be conserved for
development of appressorium-like structures and penetration, we
noticed that disruption of CON7 (Odenbach et al., 2007), a transcriptional regulator of CHS7, had more severe effects on development of appressorium-like structures. These results indicate that
appressorium formation and development of appressorium-like
structures are not identical differentiation processes in M. oryzae.
2.3. Development and penetration of appressorium-like structures
Development of appressorium-like structures on artificial surfaces was assayed as described (Kong et al., 2012; Liu et al.,
2010). Calcofluor white (CFW) and DAPI co-staining was performed as described (Li et al., 2004; Wang et al., 2011a). For penetration assays with appressorium-like structures on plant surfaces,
hyphal blocks from 1-week-old OTA cultures were placed on 5day-old leaves of barley cultivar Golden Promise and incubated
within a moist chamber. Penetration and invasive hyphae were
examined as described (Liu et al., 2010; Yang et al., 2010).
2.4. SEM and TEM examinations
For scanning electron microscopy (SEM) examination, appressoria and appressorium-like structures formed by the wild-type
strain Guy11 were fixed in 4% glutaraldehyde at 4 °C for 16 h.
The samples were then dehydrated and coated with gold before
being examined with a JSM-6360LV (Jeol Ltd., Tokyo) scanning
electron microscope as described (Kong et al., 2012; Liu et al.,
2010). For transmission electron microscopy (TEM) examination,
appressorium-like structures formed on barley leaves were fixed,
dehydrated, and embedded as described (Park et al., 2004; Wang
et al., 2011b). Serial thin-sections were removed onto slot grids
and examined with a Philips/FEI CM-100 transmission electron
microscope (FEI Company).
2.5. Cytorrhysis assays
Appressoria and appressorium-like structures formed by Guy11
were used for cytorrhysis assays as described (Howard et al., 1991;
Park et al., 2004; Yang et al., 2010). Turgor pressure was assayed
with 25%, 30%, 35%, and 40% (w/v) aqueous solutions of PEG-8000.
2. Materials and methods
2.6. Generation of the CON7-GFP transformant
2.1. Strains and culture conditions
The CON7 gene was amplified and cloned into the pFL2 vector
by the yeast GAP repair approach (Bruno et al., 2004). The resulting
CON7-GFP fusion construct was transformed into the protoplasts of
Guy11. GFP signals were examined with a Nikon Eclipse 800 epifluorescence microscope.
The M. oryzae wild-type stains, P131, 70-15, and Guy11, and
various mutants used in this study (Table S1) were cultured as described (Xue et al., 2012; Zhou et al., 2012). To assay the effects of
different chemical treatments on appressorium formation and
development of appressorium-like structures, 5 mM cAMP (adenosine 30 ,50 -cyclic monophosphate, Sigma), 2.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma), or 10 lM cutin monomer (1,16hexadecanediol, Sigma) was added to conidial suspensions or hyphal blocks placed on the artificial hydrophilic surface of GelBond
membranes (Lonza) as described (Liu et al., 2011; Zhou et al.,
2011). For wax treatment, paraffin waxes (Royal Oak) were applied
directly to the surface of glass slides. Conidial suspensions or fresh
hyphal blocks were placed on the wax-coated areas. Appressorium
formation and development of appressorium-like structures were
examined as described (Xue et al., 2002; Yang et al., 2010).
2.2. Appressorium formation and penetration assays
Appressorium formation on artificial surfaces was assayed as
described (Nishimura et al., 2003; Zhao et al., 2005). For appressorium formation and penetration assays on plant surfaces, 20 ll of
conidial suspension were deposited on 5-day-old leaves of the barley cultivar Golden Promise. After incubating in a moist chamber
for 24 h and 48 h, respectively, appressorium formation and penetration were examined as described (Park et al., 2006; Xue et al.,
2002; Yang et al., 2010).
3. Results
3.1. Development of appressorium-like structures by hyphal tips is
different from appressorium formation on germ tubes
In a previous study, we found that germ tubes of the chs7 chitin
synthase deletion mutant failed to form appressoria on artificial
hydrophobic surfaces (Kong et al., 2012). However, under the same
conditions, hyphal tips of the chs7 mutant were able to form
appressorium-like structures (Fig. 1A). In plant infection assays
with hyphal blocks as the inoculum, the chs7 mutant was reduced
in virulence (Fig. 1B), which was consistent with results from infection assays with conidia (Kong et al., 2012). Interestingly, the efficiency of appressorium-like structure differentiation on hyphal
tips was similar between the wild type and chs7 mutant after incubation for 24 h, indicating that germ tubes and vegetative hyphae
differ in tip differentiation on artificial surfaces.
To confirm this observation, we used a time course assay to
compare appressorium formation and differentiation of appressorium-like structures in the wild-type strain Guy11 on artificial
hydrophobic surfaces. Over 90% of germ tubes formed young
appressoria by 4 h (Fig. S1). Vegetative hyphae had no obvious
tip differentiation under the same conditions. Most of the
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L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
the hyphae by a septum with one nucleus in the center of the single-cell (Fig. 2A). However, appressorium-like structures appeared
to be larger and elongated. On average, appressorium-like structures were approximately 1.4-fold longer than appressoria (Table 1).
When examined by SEM and TEM, appressorium-like structures
and appressoria had similar structures (Fig. 2B and C). A melanin
layer was deposited between the cell wall and cytoplasmic membrane. Like appressoria, the pore area of appressorium-like structures attached to the plant surface was not melanized (Fig. 2C).
3.2. Appressorium-like structures are less efficient than appressoria in
plant penetration
In penetration assays with barley leaves, appressorium-like
structures were able to penetrate and develop typical invasive hyphae in plant cells (Fig. 3A). However, whereas over 90% of appressoria formed by germ tubes successfully penetrated the leaves, less
than 72% of appressorium-like structures formed invasive hyphae
by 48 h (Table 1). In addition, we noted that growth of invasive hyphae in plant cells penetrated by appressorium-like structures was
not as extensive as those penetrated by appressoria (Fig. 3A),
which may be related to the delay in development of appressorium-like structures and reduced penetration efficiency.
Because appressorium-like structures and appressoria had similar structures, the reduction in penetration efficiency is likely related to differences in the intracellular turgor, which is known to
be important for penetration in M. oryzae (Howard et al., 1991;
Wilson and Talbot, 2009). In cytorrhysis assays, appressorium-like
structures were more sensitive than appressoria to 30% or 40%
PEG-8000 (Fig. 3B), suggesting that the turgor pressure within
appressorium-like structures is lower than that within appressoria.
3.3. Exogenous cAMP or IBMX fail to efficiently stimulate development
of appressorium-like structures on hydrophilic surfaces
Fig. 1. Development of appressorium-like structures is different from appressorium
formation in the chs7 mutant. (A) The chs7 mutant forms appressorium-like
structures but not appressoria on artificial hydrophobic surfaces. Conidia and
hyphal blocks of the wild-type strain P131 (WT) and chs7 mutant were placed on
plastic coverslips and examined for appressorium formation and development of
appressorium-like structures at 24 h. ALS, appressorium-like structures; AP,
appressoria; CN, conidia; GT, germ tubes; HT, hyphal tips. Bar = 10 lm. (B) The
chs7 mutant is reduced in virulence. Seedlings of 8-day-old barley were inoculated
with hyphal blocks of the wild type and chs7 mutant. Inoculation with oatmeal agar
blocks (Agar) was used as the negative control. Photos were taken at 5 dpi.
appressoria formed by germ tubes were melanized by 8 h, but hyphal tips only began to swell and differentiate by 12 h (Fig. S1 and
Table 1). Approximately 25% of the hyphal tips formed melanized
appressorium-like structures by 24 h (Fig. S1 and Table 1). Therefore, development of appressorium-like structures on hyphal tips
was delayed in comparison to appressorium formation by germ
tubes.
Similar to appressoria, mature appressorium-like structures
(24 h) were melanized and delineated from the distal portion of
In M. oryzae, cAMP signaling is known to be involved in the recognition of surface hydrophobicity (Wilson and Talbot, 2009). On
hydrophilic surfaces, similar to appressorium formation by germ
tubes, hyphal tips of the wild-type strain Guy11 failed to form
appressorium-like structures. Remarkably, even in the presence
of 2.5 mM IBMX, less than 6% of hyphal tips formed appressorium-like structures on hydrophilic surfaces (Fig. 4 and Table 2).
Under the same conditions, over 90% of germ tubes formed appressoria. Similar results were obtained in experiments with 5 mM
cAMP (Table 2). These data indicated that cAMP or IBMX treatment
was not effective in inducing development of appressorium-like
structures by hyphal tips on hydrophilic surfaces in M. oryzae.
3.4. Waxes but not cutin monomers induce development of
appressorium-like structures
Cutin monomers and epicuticular waxes also are known to induce appressorium formation on hydrophilic surfaces in M. oryzae
(Lee and Dean, 1993; Liu et al., 2011). In the presence of cutin
Table 1
Characterization of appressoria and appressorium-like structures in the wild-type strain Guy11.
Lengtha (lm)
Appressoria
Appressorium-like structures
9.8 ± 0.5a
13.2 ± 0.6b
Formation (%)b
Penetration efficiency (%)c
8h
12 h
24 h
96.5 ± 1.3a
0c
97.5 ± 0.8a
0c
99.2 ± 0.6a
25.4 ± 3.1b
92.7 ± 2.4a
70.6 ± 3.1b
Data from three replicates were analyzed with two sample t-test. The same Greek letter indicated that there was no significant difference with the characteristics between the
appressoria or appressorium-like structures. Different letters were used to mark statically significant difference (P = 0.01).
a
Measured as the distance between the distal end of appressoria or appressorium-like structures and the junction site with germ tubes or hyphae.
b
Percentage of germ tubes or hyphal tips formed appressoria or appressorium-like structures.
c
Percentage of appressoria or appressorium-like structures penetrated and formed invasive hyphae on barley leaves 48 hpi.
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L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
Fig. 2. Microscopic examination of appressorium-like structures formed by the wild type. (A) Appressorium-like structures and appressoria formed by Guy11 were costained with Calcofluor white (CFW) and DAPI, and examined by differential interference contrast (DIC) and epifluorescence (UV) microscopy. Bar = 10 lm. (B) Scanning
electron microscope (SEM) examination of appressorium-like structures and appressoria formed by Guy11 on barley leaves at 24 hpi. Bar = 10 lm. (C) The ultrastructure of
appressorium-like structures formed by Guy11 on barley leaves at 24 hpi was examined by transmission electron microscopy (TEM). The right panel is a close-up view of the
rectangular section marked on the left panel. The melanin layer (arrows) is absent in the pore area (between arrowheads) of appressorium-like structures. Bar = 1 lm. ALS,
appressorium-like structures; AP, appressoria; CN, conidia; GT, germ tubes; HT, hyphal tips; ML, melanin layer; N, nucleus.
monomer 1,16-hexadecanediol, abundant appressoria were
formed by germ tubes of Guy11 on the hydrophilic surface of GelBond membranes (Fig. 4 and Table 2). However, only approximately 7% of hyphal tips developed appressorium-like structures
(Table 2), which was similar to results from cAMP and IBMX treatments (Fig. 4 and Table 2). In contrast, approximately 50% of hyphal tips differentiated appressorium-like structures on glass
slides coated with paraffin waxes (Fig. 4 and Table 2). These data
indicated that development of appressorium-like structures on hyphal tips is likely induced by epicuticular waxes rather than by cutin monomers on plant surface.
3.5. Development of appressorium-like structures also requires intact
cAMP signaling
Although cAMP signaling is known to be involved in surface recognition for appressorium formation, the efficiency of development of appressorium-like structures induced by cAMP or IBMX
treatment was relatively low (Table 2). Therefore, we assayed the
formation of appressorium-like structures by hyphal tips on hydrophobic surfaces in the cpkA and mac1 mutants, which are defective
in cAMP signaling. Hyphal tips of the cpkA mutant still formed melanized appressorium-like structures at 48 h (Fig. 5A). However, in
comparison with the wild type, the cpkA mutant was significantly
reduced in the efficiency of appressorium-like structure differentiation at 24 or 48 h (Table S2). In contrast, the cpkA mutant was delayed by approximately 4 h but not reduced in appressorium
formation by 24 h (Xu et al., 1997). Moreover, appressorium-like
structures formed by the cpkA mutant on hyphal tips failed to penetrate and develop invasive hyphae in plant cells at 48 hpi (Fig. 5A)
and the cpkA hyphal block failed to cause lesions at 5 dpi (Fig. 5B).
Unlike the cpkA mutant, hyphal tips of the mac1 mutant failed to
form appressorium-like structures (Fig. 5A), which was consistent
with its defects in appressorium formation (Adachi and Hamer,
1998; Choi and Dean, 1997).
3.6. Pmk1 but not Mps1 or Osm1 MAP kinase is required for
development of appressorium-like structures
In M. oryzae, three MAP kinase pathways have been functionally
characterized (Dixon et al., 1999; Xu and Hamer, 1996; Xu et al.,
1998). Whereas Pmk1 regulates appressorium formation, Mps1
L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
37
invasive hyphae on barley leaves as efficiently as the wild type,
indicating that Osm1 is dispensable for turgor generation in
appressorium-like structures formed by hyphal tips. In the mps1
mutant (Xu et al., 1998), hyphal tips formed appressorium-like
structures on artificial hydrophobic or plant surfaces as efficiently
as the wild type (Fig. 6A). However, most of the appressorium-like
structures formed by the mps1 mutant failed to penetrate and form
invasive hyphae on the barley leaves by 48 h (Fig. 6A). Although a
few appressorium-like structures appeared to penetrate epidermal
cells, the mps1 mutant failed to develop extensive invasive hyphae
and cause lesions on barley leaves (Fig. 6B).
3.7. Msb2 but not Sho1 is important for appressorium-like structure
development
Fig. 3. Appressorium-like structures were less efficient than appressoria in
penetration. (A) Barley leaves were inoculated with conidia (left) and hyphal
blocks (right) of the wild-type strain Guy11 and examined for invasive hyphae in
epidermal cells at 48 hpi. ALS, appressorium-like structures; AP, appressoria.
Bar = 10 lm. (B) Appressorium-like structures and appressoria formed by Guy11
were treated with different concentrations of PEG-8000 (w/v). Means and standard
errors of the percentage of intact appressorium-like structures or appressoria were
calculated from three independent repetitions.
and Osm1 are not essential for the differentiation of melanized
appressoria. Similar to germ tubes, hyphal tips of the pmk1 mutant
failed to form appressorium-like structures on artificial hydrophobic and plant surfaces (Fig. 6A). Therefore, Pmk1 is essential for
development of appressorium-like structures in M. oryzae, which
is consistent with published reports (Liu et al., 2010). The osm1
mutant had no defects in development of appressorium-like structures and penetration of plant cells (Fig. 6A). Appressorium-like
structures formed by the osm1 mutant on hyphal tips developed
Because Msb2 and Sho1 are two putative receptors for surface
recognition (Liu et al., 2011), we assayed development of appressorium-like structures in the msb2, sho1, and msb2 sho1 double mutants. Hyphal tips of the sho1 mutant formed appressorium-like
structures on artificial hydrophobic surfaces as efficiently as on
barley leaves (Fig. 7A). Similar to appressorium formation, the
msb2 mutant and msb2 sho1 double mutant were significantly reduced in development of appressorium-like structures on artificial
hydrophobic surfaces (Fig. 7A). By 48 h, less than 1% of the hyphal
tips of the msb2 and msb2 sho1 double mutant formed appressorium-like structures. Even after a prolonged incubation of 72 h,
only approximately 1.5% of the hyphal tips formed appressoriumlike structures in the msb2 mutant and msb2 sho1 double mutant
(Table S2). Therefore, Msb2 plays a critical role in development
of appressorium-like structures, which is consistent with the fact
that Msb2 functions upstream from the Pmk1 pathway. Hyphal
tips of the msb2 mutant and msb2 sho1 double mutant formed
appressorium-like structures more efficiently on barley leaf surfaces than on an artificial surface (Fig. 7A), which may be related
to the epicuticular waxes on the plant surface (Liu et al., 2011).
3.8. Mst12 is dispensable for the formation of appressorium-like
structures but essential for penetration
One putative downstream transcription factor regulated by
Pmk1 is Mst12, which is dispensable for appressorium formation
Fig. 4. Assays for the effects of IBMX, cutin monomer, and wax treatments on development of appressorium-like structures. Conidia and hyphal blocks of the wild-type strain
Guy11 were placed on the hydrophilic surface of GelBond films in the presence of 2.5 mM IBMX or 10 lM 1,16-hexadecanediol or on the surface of wax-coated glass slides.
The formation of appressoria by germ tubes and appressorium-like structures on hyphal tips was examined at 48 h. The same treatment with double distilled water was
negative control. ALS, appressorium-like structures; AP, appressoria. Bar = 10 lm.
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L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
Table 2
Effects of different treatments on appressoria and development of appressorium-like
structures (ALSs) by the wild-type strain Guy11.
Appressorium
formation
(%)
Differentiation
of ALS (%)
Water
cAMPa
IBMX
Cutin
monomer
Paraffin
waxesb
0
55.8 ± 1.6b
92.2 ± 1.7a
93.2 ± 2.7a
91.9 ± 2.2a
0
1.2 ± 0.4c
5.2 ± 0.6c
6.7 ± 1.3c
52.8 ± 0.6b
Data from three replicates were analyzed with two sample t-test. The same Greek
letter indicated that there was no significant difference between appressorium
formation from germ tubes and development of appressorium-like structures from
hyphal tips. Different letters were used to mark statically significant difference
(P = 0.01).
a
IBMX, cAMP, and 1,16-hexadecanediol were added to the final concentrations
of 2.5 mM, 5 mM, and 10 lM, respectively, to conidial suspensions or hyphal blocks
of Guy11 placed on the hydrophilic surface of GelBond membranes.
b
Glass slides were coated with paraffin waxes. Appressorium formation and
development of appressorium-like structures were examined after 48 h incubation.
Fig. 6. Pmk1 but not Mps1 or Osm1 was essential for development of appressorium-like structures. (A) Hyphal blocks of the wild-type Guy11, pmk1, mps1, and
osm1 mutants were placed on plastic coverslips and barley leaves. Development of
appressorium-like structures and invasive growth were examined at 24 h and 48 h,
respectively. Bar = 10 lm. (B) Barley leaves inoculated with hyphal blocks of Guy11
and the mps1 and osm1 mutants were photographed at 5 dpi.
3.9. CON7 is important for development of appressorium-like
structures
Fig. 5. Assays for development of appressorium-like structures and penetration in
the cAMP signaling mutants. Hyphal blocks of the wild-type strain Guy11 and the
cpkA and mac1 mutants were placed on hydrophobic coverslips and 5-day-old
barley leaves, and assayed for development of appressorium-like structures at
48 hpi (A). Appressoria formed by the cpkA mutant were smaller than those of the
wild type as previously reported (Xu et al., 1997). The lesions were visible on the
barley leaf infected by the wild type at 5 dpi, while the cpkA hyphal blocks failed to
cause lesions (B). Bar = 10 lm.
but essential for the development of penetration pegs (Park et al.,
2002, 2004). Hyphal tips of the mst12 mutant were normal in
appressorium-like structure formation on artificial hydrophobic
and plant surfaces (Fig. 7B). However, appressorium-like structures
formed by the mst12 mutant failed to penetrate epidermal cells of
barley leaves and develop invasive hyphae by 48 h (Fig. 7B), which
was similar to its defects in appressorium formation and penetration (Park et al., 2002, 2004).
Recently, the Pmk1-interaction transcription factor Sfl1 was
shown to be important for full virulence but dispensable for
appressorium formation (Li et al., 2011). The sfl1 mutant formed
melanized appressorium-like structures on both artificial hydrophobic and plant surfaces (Fig. 7B). Unlike the mst12 mutant, the
sfl1 mutant developed invasive hyphae on barley leaves. However,
appressorium-like structures formed by the sfl1 mutant were
reduced in the penetration efficiency (Table S2) and only occasionally developed invasive hyphae (Fig. 7B).
It has been reported that the expression of CHS7 is regulated by
the putative transcription factor Con7 (Odenbach et al., 2007). The
con7 mutant was significantly reduced in appressorium formation.
Only approximately 10% of germ tubes of the con7 mutant generated in our lab formed appressoria on artificial hydrophobic surfaces (Fig. 8A) as reported previously (Yang et al., 2007).
Interestingly, development of appressorium-like structures by hyphal tips was not observed in the con7 mutant on artificial hydrophobic or plant surfaces (Fig. 8B), even after a prolonged
incubation of 72 h (Table S2). Occasionally, some hyphal tips of
the con7 mutant swelled slightly but did not differentiate further
into melanized structures. Thus, the con7 mutant formed appressoria but not appressorium-like structures, suggesting that appressorium formation and differentiation of appressorium-like structures
involve different mechanisms. Even though these two processes
are similar, the regulatory mechanisms may be not identical. Because Con7 was reported to be localized to the nucleus in appressoria (Odenbach et al., 2007), in this study we generated the CON7GFP transformant (Table S1) and examined the expression and subcellular localization of the fusion proteins. As expected, Con7-GFP
was expressed and localized to the nucleus in vegetative hyphae
and appressorium-like structures (Fig. 8C).
4. Discussion
Appressorium formation plays a critical role in the infection cycle of M. oryzae. During the past decade, appressorium formation
and penetration have been extensively studied (Wilson and Talbot,
2009; Zhao et al., 2007). Under laboratory conditions, this pathogen also can infect through roots (Sesma and Osbourn, 2004).
Therefore, hyphal tips must be able to penetrate either directly
via the formation of pre-invasive hyphae or hyphopodia (Tucker
et al., 2010). Recently, formation of appressorium-like structures
by hyphal tips has been reported for a few mutants that are defective in conidiation (Liu et al., 2010; Zhou et al., 2009). Although
appressorium-like structures and appressoria appeared to have
similar morphology, in a previous study, we found that the chs7
mutant was normal in appressorium formation but defective in
development of appressorium-like structures, indicating that differences may exist between appressorium-like structures and
L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
39
Fig. 7. Development of appressorium-like structures and penetration assays with mutants related to the Pmk1 pathway. (A) The msb2, sho1, and msb2 sho1 double mutants.
(B) The mst12 and sfl1 mutants. Hyphal blocks of the wild-type (Guy11) and mutant strains were placed on hydrophobic coverslips or barley leaves and assayed for
development of appressorium-like structures (24 h) and invasive hyphae (48 h). Bar = 10 lm.
appressoria. In M. oryzae, it has been reported that germ tubes older than 8 h were defective in appressorium formation on artificial
surfaces (Beckerman and Ebbole, 1996). Although the existence
of a window time for germ tubes to form appressoria (Beckerman
and Ebbole, 1996) has not been confirmed in other publications,
development of appressorium-like structures on hyphal tips certainly will allow hyphae grown on plant surfaces to penetrate
underlying cells.
In comparison with appressorium formation by germ tubes,
development of appressorium-like structures on hyphal tips was
delayed by approximately 20 h on hydrophobic surfaces (Fig. S1).
Appressorium-like structures also contained a single nucleus but
tended to have stronger Calcofluor staining than appressoria
(Fig. 2). Differences in the intensity of Calcofluor staining indicated
that the cell wall structure or composition may be different between these melanized structures formed on the tips of hyphae
and germ tubes in M. oryzae. In general, germ tubes and hyphal tips
have similar morphology although the former tended to be narrower. However, it is known that a maturation process is involved
in the transition from germ tubes to vegetative hyphae. In Ashbya
gossypii, 8 h of incubation is necessary for hyphal maturation (Harris, 2008; Wendland and Walther, 2005). Whereas branching is not
observed in young germ tubes, hyphal branching is an integral
component of growth in filamentous fungi. Also, unlike germ tubes
emerged from three-celled conidia, hyphal tips are connected to a
large number of hyphal compartments. Most likely, differences in
physiology and cell wall structures are responsible for the observed differences between appressorium formation by germ tubes
and hyphal differentiation on hyphal tips. However, it is also possible that the mucilage produced by germ tubes and hyphal tips,
which allows them to attach to the surface, contribute to the differences we observed in this study.
We tested development of appressorium-like structures in various signaling mutants of M. oryzae available in our lab. Similar to
appressorium formation (Liu et al., 2010), Pmk1 but not Osm1 or
Mps1 is required for development of appressorium-like structures
on hyphal tips. Whereas Osm1 is dispensable, Mps1 is essential for
successful penetration of appressorium-like structures. On barley
leaves inoculated with hyphal blocks, unlike the osm1 mutant,
the mps1 mutant failed to cause lesions (Fig. 5B). These data indicate that the functions of these three MAPK signaling pathways are
conserved between appressorium formation and differentiation of
appressorium-like structures in M. oryzae. Phenotype characterization of the msb2, msb2 sho1, mst12, and sfl1 mutants further indicated that the role of the Pmk1 pathway is conserved for
regulating the differentiation of appressoria and appressorium-like
structures on germ tubes and hyphal tips.
For the mutants defective in cAMP signaling, the mac1 mutant
failed to form appressorium-like structures on artificial hydrophobic surfaces or barley leaves. Hyphal tips of the cpkA mutant
were reduced in the efficiency of appressorium-like structure
formation, indicating that cAMP signaling is involved in the
differentiation of appressorium-like structures. However, the cpkA
mutant is delayed but not reduced in appressorium formation
(Xu et al., 1997). Therefore, the role of CpkA in appressorium formation and development of appressorium-like structures may be
slightly different. The M. oryzae genome contains another gene,
CPK2, that encodes a catalytic subunit of PKA (Xu et al., 2007).
It is possible that CPK2 may be more effective in compensating
CPKA deletion in appressorium formation than in differentiation
of appressorium-like structures. CPKA is dispensable for pathogenesis on rice roots (Sesma and Osbourn, 2004), suggesting that
hyphophodia formed by the cpkA mutant may be functional in
plant infection and CPKA is dispensable for pathogenesis after
40
L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
Fig. 8. The con7 mutant failed to form appressorium-like structures. (A) Conidia of
the wild type and con7 mutant were inoculated on plastic coverslips and examined
for appressorium formation at 24 h. The con7 mutant was reduced but still formed
appressoria (AP). (B) Hyphal blocks of the wild type and con7 mutant were placed
on coverslips or barley leaves and examined for development of appressorium-like
structures at 24 h. Unlike the wild type, the con7 mutant failed to develop
appressorium-like structures (HS) on artificial hydrophobic and plant surfaces. (C)
Expression and localization of Con7-GFP fusion proteins in vegetative hyphae (VH)
and appressorium-like structures (ALSs) in the CON7-GFP transformant LA53.
Bar = 10 lm.
the initial penetration of roots. Interestingly, hyphal tips of the
mac1 mutant occasionally formed melanized appressorium-like
structures on rice roots. The rice root surface may have chemical
cues that are absent on leaves and stimulatory to the differentiation of appressorium-like structures at hyphal tips. Another
possible explanation is that formation of rare appressorium-like
structures by the mac1 mutant was due to a high rate of spontaneous suppressor mutation (Adachi and Hamer, 1998; Choi and
Dean, 1997).
IBMX, cAMP, and cutin monomers are functionally related to
cAMP signaling (Lee and Dean, 1993; Liu et al., 2011). Treatments
with these chemicals failed to induce development of appressorium-like structures as efficiently as they stimulate appressorium
differentiation on hydrophilic surfaces. It is possible that hyphal
tips may be less efficient than germ tubes in the uptake of these
chemicals. Nevertheless, the wild type and mutant strains efficiently formed appressorium-like structures on glass slides coated
with waxes. Therefore, waxes may be more effective in stimulating
development of appressorium-like structures than cAMP or cutin
monomers in M. oryzae. One explanation is that, unlike treatments
with these three chemicals, coating with waxes likely resulted in
the change of surface hydrophobicity. More recently, it was reported that epicuticular waxes have been shown to stimulate
appressorium formation as chemical cues (Liu et al., 2011). Nevertheless, artificial surfaces treated with different chemicals or
waxes may mimic but still quite different from plant surfaces
(Kamakura et al., 2002; Tucker et al., 2010).
In a previous study, we showed that the chs7 mutant was normal in development of appressorium-like structures but failed to
form appressoria (Kong et al., 2012). The CHS7 gene is regulated
by a putative transcription factor Con7 (Odenbach et al., 2007),
and the con7 mutant with insertion at 163 bp upstream of the
CON7 gene failed to form appressoria (Odenbach et al., 2007). We
obtained a con7 mutant with an insertion at 1479 bp upstream of
the CON7 gene and found that about 10% of the germ tubes formed
appressoria (Yang et al., 2007). The difference in appressorium formation between these two con7 mutants may be related to different locations of the insertions or different strain background. In
this study, we found that our con7 mutant failed to form appressorium-like structures on hyphal tips, further indicating differences
in appressorium formation and development of appressorium-like
structures. In the CON7-GFP transformant generated in this study,
Con7-GFP fusion proteins were expressed and localized to the nucleus in vegetative hyphae and appressorium-like structures
formed on hydrophobic surfaces. It will be interesting to determine
whether the con7 mutant generated by Odenbach et al. (2007) still
forms appressorium-like structures on hyphal tips.
In M. oryzae, Cos1 and Htf1 (=MoHox2) are two transcription
factors essential for conidiation (Kim et al., 2009; Liu et al., 2010;
Zhou et al., 2009). The cos1 or htf1 mutant failed to produce conidia
on conidiophores but formed appressorium-like structures on hyphal tips (Liu et al., 2010; Zhou et al., 2009). Results from this study
indicate that appressorium-like structures had lower turgor pressure and were less efficient than appressoria in plant penetration.
In M. oryzae, turgor pressure generated inside appressoria is essential for the physical penetration of the rice leaf cuticle and cell wall
(Howard et al., 1991; Wilson and Talbot, 2009). Energy reserves in
conidium compartments are mobilized and degraded to generate
appressorium turgor. Because normal vegetative hyphae have minimal carbohydrate reserves in the cytosol, lower turgor pressure in
appressorium-like structures may be related to the reduced availability of energy reserves in hyphal tips. Nevertheless, because hyphal compartments are interconnected, long-distance mobilization
from distal portions of the hyphae may compensate for limited carbohydrate storage in the apical regions. The delay in the delimitation of appressorium-like structures at hyphal tips in comparison
with appressorium formation may allow more time to mobilize
carbohydrates into developing appressorium-like structures for
turgor generation.
Acknowledgments
We thank Dr. Larry Dunkle for critical reading of this manuscript. We also thank Dawei Wang for insightful discussions. This
work was supported by a grant from the National Research Initiative of the USDA NIFA (Award Number: 2010-65110-20439) and
the National Major Project of Breeding for New Transgenic
Organisms (2012ZX08009003).
L.-A. Kong et al. / Fungal Genetics and Biology 56 (2013) 33–41
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.fgb.2013.03.006.
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