Infection-Associated Nuclear Degeneration in the Rice
Blast Fungus Magnaporthe oryzae Requires Non-
Min He1,2, Michael J. Kershaw1, Darren M. Soanes1, Yuxian Xia2, Nicholas J. Talbot1*
1School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom, 2Genetic Engineering Research Center, College of Bioengineering, Chongqing University,
Chongqing, People’s Republic of China
Background: The rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium
to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle
progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by
programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to
be essential for plant infection, but the precise mechanism by which it occurs is not known.
Methodology/Principal Findings: In yeast, nuclear breakdown requires a specific form of autophagy, known as piecemeal
microautophagy of the nucleus (PMN), and we therefore investigated whether this process occurs in the rice blast fungus.
Here, we report that M. oryzae possesses two conserved components of a putative PMN pathway, MoVac8 and MoTsc13,
but that both are dispensable for nuclear breakdown during plant infection. MoVAC8 encodes a vacuolar membrane protein
and MoTSC13 a peri-nuclear and peripheral ER protein.
Conclusions/Significance: We show that MoVAC8 is necessary for caffeine resistance, but dispensable for pathogenicity of
M. oryzae, while MoTSC13 is involved in cell wall stress responses and is an important virulence determinant. By functional
analysis of DMoatg1 and DMoatg4 mutants, we demonstrate that infection-associated nuclear degeneration in M. oryzae
instead occurs by non-selective macroautophagy, which is necessary for rice blast disease.
Citation: He M, Kershaw MJ, Soanes DM, Xia Y, Talbot NJ (2012) Infection-Associated Nuclear Degeneration in the Rice Blast Fungus Magnaporthe oryzae Requires
Non-Selective Macro-Autophagy. PLoS ONE 7(3): e33270. doi:10.1371/journal.pone.0033270
Editor: Yong-Hwan Lee, Seoul National University, Republic of Korea
Received January 16, 2012; Accepted February 13, 2012; Published March 20, 2012
Copyright: ? 2012 He et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Biotechnology and Biological Sciences Research Council (BB/G013896/1) and a studentship to MH from the China
Scholarship Council (No. 2009605071). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: N.J.Talbot@exeter.ac.uk
Rice blast disease is a widespread constraint to rice production
and therefore poses a persistent threat to global food security .
Rice blast infections, caused by the ascomycete fungus Magnaporthe
oryzae, are initiated by attachment of a three-celled spore, or
conidium, to the rice leaf cuticle. The conidium sticks tightly to the
leaf surface by means of an adhesive released from the spore tip
during hydration . Once attached, the conidium quickly
germinates and forms a single polarized germ tube. Within
4 hours, the germ tube ceases apical extension and terminal
hooking of the hypha starts, which represents the initiation of
cellular differentiation to form a specialised dome-shaped cell, the
appressorium, that is necessary for successful plant infection . A
narrow penetration hypha is formed at the base of the
appressorium and enters the underlying epidermis, rupturing the
cell wall and invaginating the plant plasma membrane .
Development of the M. oryzae appressorium requires external
cues including a hard, hydrophobic surface and the absence of
exogenous nutrients . Multiple cellular signal transduction
cascades, such as the cyclic AMP and Pmk1 MAPK signaling
pathways, are initiated in response to these external triggers and
bring about the terminal differentiation of the germ tube apex into
an appressorium [3,5]. The appressorium of M. oryzae ruptures the
plant cuticle by application of mechanical force through
accumulation of very high concentrations of glycerol, which draws
water into the appressorium to create enormous hydrostatic turgor
. Autophagic re-cycling of the contents of the conidium is
necessary for formation of a functional appressorium .
Consistent with this, lipid and glycogen mobilization, under
control of the MAPK and cAMP response pathways, have been
shown to occur during appressorium development and may
provide precursors for glycerol synthesis [8,9].
It is now clear that appressorium development by M. oryzae is
genetically controlled by cell cycle progression and that entry of a
nucleus in the germinating conidial cell into S-phase is the key step
in initiating infection structure development [7,10]. During
germination and appressorium development, one nucleus in the
conidium undergoes mitosis in the germ tube, after which one
daughter nucleus moves into the incipient appressorium and the
other returns to the conidium and degenerates . Completion of
mitosis leads to collapse and death of the conidium and is
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necessary for appressorium maturation and plant infection .
Systematic deletion of genes encoding each component of the
macroautophagy machinery renders M. oryzae non-pathogenic,
providing evidence that autophagy is essential for plant infection
Despite evidence to show the importance of autophagy in
programmed cell death of the conidium and subsequent
appressorium maturation, the molecular machinery responsible
for nuclear degeneration in the conidium of M. oryzae remains
unknown. Moreover, the factors regulating nuclear degeneration
and the destiny of degraded nuclei in the conidium have yet to be
characterised. In S. cerevisiae, it has been shown that piecemeal
microautophagy of the nucleus (PMN) is a separate process that is
necessary for recycling of non-essential portions of the nucleus
and is induced by starvation or exposure to rapamycin, an
inhibitor of the TOR signalling pathway [15,16,17,18,19,20,21].
PMN occurs constitutively at nucleus-vacuole (NV) junctions,
formed through a specific binding interaction of Vac8p on the
vacuole membrane and Nvj1p in the outer nuclear envelope
[15,16]. During PMN, small teardrop-shaped portions of the
nucleus are extruded along NV junctions into invaginations of the
vacuolar membrane, which results in formation of tethered blebs
that finally release vesicles containing non-essential nuclear
material into the vacuole lumen for degradation by resident
hydrolases [16,21]. Lipid metabolic proteins Osh1p and Tsc13p
have been shown to be recruited and enriched at NV junctions by
physical association with Nvj1p and may function in non-
vesicular lipid trafficking and biogenesis of a distinctive lipid
environment at NV junctions [19,22]. In addition, a spectrum of
core autophagy machinery genes is required for the terminal
vacuolar enclosure of the invaginated blebs and efficient
production of intravacuolar PMN vesicles .
In this study, we set out to determine whether there is an
identifiable PMN pathway in M. oryzae and to ask whether this
process drives nuclear degeneration in the conidium during rice
blast infection. Here, we report that MoVAC8 encodes a vacuolar
membrane protein, which plays a role in the caffeine response, and
that MoTSC13 is necessary for maintaining conidial morphology
and for penetration peg development during plant infection.
Importantly, we demonstrate that nuclear degeneration in the
conidium occurs even in the absence of MoVAC8 and MoTSC13
and that there is no evidence for a discernable PMN pathway in
M. oryzae. Instead M. oryzae degrades nuclei using a macroauto-
phagic mechanism, which is a necessary pre-requisite for plant
Results and Discussion
Nuclear degeneration occurs during appressorium
development in M. oryzae
To investigate nuclear behaviour during appressorium devel-
opment, we performed live-cell imaging and quantitative analysis
of nuclear number in a M. oryzae strain expressing a histone H1-
enhanced red fluorescent (H1:RFP) protein fusion . Mitosis
occurred in the germ tube emerging from the apical cell of the
conidium between 4–6 hour post inoculation (hpi) and the
daughter nucleus moved into the incipient appressorium, while
the mother nucleus returned to the conidium, as shown in Figure 1.
After the completion of mitosis and formation of the appressorium,
nuclear degeneration occurred in the conidium, during which the
nucleus in the basal cell of the conidium collapsed first, followed by
the two nuclei occupying the middle cell and apical cell,
respectively, as shown in Figure 1. Nuclear degeneration occurred
without overt nuclear fragmentation and red fluorescence
associated with nuclear material could be observed both in the
cytoplasm and in vacuoles within conidia. After 24 h, nuclear
degeneration always resulted in a single nucleus, which was
present in the mature appressorium (Figure 1).
Two components of the piecemeal autophagy of the
nucleus pathway are present in the M. oryzae genome
To identify the molecular machinery involved in nuclear
degeneration in M. oryzae, we set out to determine whether the
selective PMN pathway, described in S. cerevisiae, participates in
degradation and recycling of nuclei during appressorium devel-
opment by the rice blast fungus. In S. cerevisiae, VAC8, TSC13 and
NVJ1 are the three important components of the PMN pathway.
We interrogated the M. oryzae genome database using Blastp and
Figure 1. Nuclear degeneration occurs during appressorium
development in M. oryzae. (A). Upper panel: time course live cell
imaging experiment showing nuclear division and nuclear degenera-
tion during appressorium development in M. oryzae. Guy11 conidia
expressing H1:RFP were examined by epifluorescence microscopy at
indicated time points during appressorium development. Lower panel:
bar charts showing the percentage of spore germlings in Guy11
containing between 0 and 4 nuclei (mean 6 SD, n.100, triple
replications) during a timecourse of appressorium development. Scale
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identified putative homologues of Vac8p and Tsc13p. VAC8 is a
vacuolar membrane-associated protein, which plays important
roles in several vacuolar processes in S. cerevisiae, including
piecemeal microautophagy of
24,25,26]. VAC8 was first identified in a survey of the S. cerevisiae
genome for armadillo (ARM) repeat domain-containing proteins–
conserved modules involved in mediating protein-protein interac-
tions [23,27,28]. The gene was also identified independently by
complementation of a class I vacuole segregation mutant, vac8,
which contains multi-lobed vacuoles and arrests early in vacuole
inheritance with defects in the cytoplasm to vacuole (Cvt) targeting
pathway [29,30]. The myristoylation of glycine and palmitoylation
of three cysteine residues inside the N-terminal Src homologue 4
(SH4) domain are critical for Vac8p association with the vacuole
membrane  and, indeed, palmitoylation at the three cysteines
determines the enrichment and function of Vac8p at specific
vacuolar membrane sub-domains [25,26,31,32]. Vac8p interacts
with different proteins through its ARM repeat domains at discrete
vacuole membrane sub-domains specific to each of its distinct
functions. Interacting partners include Vac17p in vacuole
inheritance, Atg13 in the Cvt pathway, Nvj1p in NV junction
the nucleus(PMN) [15,23,
formation and Tco89p in caffeine resistance [25,26,27,31].
Homologues of S. cerevisiae VAC8 have been reported to function
in glucose-induced pexophagy in Pichia pastoris and in vacuolar
inheritance and normal hyphal branching in Candida albicans,
respectively [33,34,35,36]. In M. oryzae, MoVac8p shows 85.2%
identity to S. cerevisiae Vac8p (Figure S1 A). The predicted
MoVac8p coding region has 11 putative ARM repeats and to test
this prediction, we designed primers starting at the start codon
predicted in the genome database and performed 39 RACE.
Unexpectedly, sequencing the 39 RACE amplicon and a
subsequent 59RACE product showed that the correct start codon
was 303 bp downstream of the predicted start codon within the
first predicted intron (Genbank JN977613). The RADAR
programme was used to align the ARM repeats of MoVac8p
strated that MoVac8p contains 9 ARM repeats, with repeat 8
and repeat 9 interrupted by 53 amino acids, which contrasts
significantly with the 11 continuous ARM repeats in S. cerevisiae
Vac8p (Figure 2). MoVac8p shares similar N-terminal acylation
sites to those found in S. cerevisiae Vac8p (Figure 2), consistent with
its predicted function.
Figure 2. Bioinformatic identification of components of the S. cerevisiae PMN pathway in M. oryzae. (A) Alignment of acylation
modification amino acids in the N-terminus of Vac8p from M. oryzae and S. cerevisiae. Triangles indicate putative myristoylation modification site (Gly)
and stars indicate palmitoylation modification sites (Cys) that have been shown to be important for localisation and function of Vac8p in S. cerevisiae.
Similar acylation modification sites are present in MoVac8p. (B) ARM repeat organization in MoVac8p is distinct from that of S. cerevisiae Vac8p. Upper
panel: alignment of 9 ARM repeats from M. oryzae Vac8p. A dot indicates a space. Lower panel: Comparison of distribution of the ARM repeats in
Vac8p between M. oryzae and S. cerevisiae. Scale bar indicate 100 amino acids. (C) MoTsc13p shares the same topology of six tramsmembrane
domains as S. cerevisiae Tsc13p.
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A second major component of the PMN pathway in yeast, the
TSC13 gene, encodes enoyl reductase, an enzyme that catalyzes
the last step of long-chain fatty acid (C16and C18) elongation to
produce very-long-chain fatty acids (VLCFAs) . Tsc13p is an
integral membrane protein located in the peripheral and peri-
nuclear endoplasmic reticulum (ER), enriched at NV junctions,
and is essential for cell viability [15,22,37]. The activity of Tsc13p
in the VLCFA elongation cycle has been proposed to contribute
to the biogenesis of PMN blebs . There have, however, been
no reports of the functions of VAC8 or TSC13 orthologues in any
filamentous fungus to date. The M. oryzae MoTsc13p showed
60.1% identity to S. cerevisiae Tsc13p, with six predicted
transmembrane domains (Figure 2 C), consistent with the
topology of S. cerevisiae Tsc13p . MoTsc13p has conserved
amino acids (Figure S1 B), essential for activity of Tsc13p 
(Genbank JN977614). Importantly, we were unable to find a
homologue of S. cerevisiae NVJ1, using either nucleotide or amino
acid sequences of NVJ1, based on BLASTP or TBLASTN
analysis, in the M. oryzae genome database, or by immuno-
precipitation which we used to identify proteins interacting with
MoVac8-GFP (data not shown).
MoVac8-GFP localises to the vacuole membrane and
Tsc13-GFP to the perinuclear and peripheral ER
To investigate whether MoVac8p and MoTsc13p showed
similar sub-cellular localisation patterns to their yeast counterparts
(consistent with a PMN function) we generated MoVAC8:GFP and
MoTSC13:GFP gene fusion constructs and expressed them under
their native promoters in the wild type M. oryzae strain Guy11.
MoVac8-GFP showed a membrane-associated distribution pattern
in conidia, appressoria and invasive hyphae, as shown in Figure 3
A and B. When stained with FM4–64 during appressorium
development, MoVac8-GFP showed a similar distribution to
FM4–64 (Figure 3A), suggesting that MoVac8p in M. oryzae
localises to the vacuolar membrane and partially to the vacuolar
lumen. Vacuoles in the conidium were initially small and those
inside the apical cell moved into the germ tube and nascent
appressorium (Figure 3 A; 4 h timepoint), after which all vacuoles
in the conidium fused together to form a large central vacuole
(Figure 3 A; 4 h and 8 h timepoints). The vacuole finally
degenerated in the conidium (Figure 3 A) after 24 h and eventually
MoVac8:GFP disappeared from the collapsing conidium following
appressorium development. The mature appressorium contained a
large central vacuole after 24 h (Figure 3 B), consistent with
previous studies showing the importance of the vacuole as a key
lytic organelle in degrading lipid storage reserves during
appressorium development in M. oryzae .
In hyphae of transformants expressing MoTSC13:GFP, the
fusion protein was also membrane-associated, as shown in
Figure 3E. To stain nuclei, 2,4,-Diamidino-phenyl-indole (DAPI)
was used in hyphae of these transformants and showed that the
MoTsc13p is detected predominantly at locations consistent with
the peri-nuclear ER membrane and peripheral ER (Figure 3E).
This membrane-associated distribution pattern of MoTsc13p was
also found in the conidium, appressorium, penetration peg and
invasive hyphae during plant infection (Figure 3 C and D). During
appressorium development, MoTsc13p was detected in the germ
tube and differentiating appressorium, indicating that MoTsc13p-
anchored ER moves into the appressorium (Figure 3C). Taken
together, these data revealed that both MoVac8p and MoTsc13p
showed sub-cellular distribution patterns consistent with a role in a
variety of vacuole and ER functions.
Conservation of MoVAC8 and MoTSC13 function
To determine whether MoVAC8 and MoTSC13 are functional
equivalents of S. cerevisiae VAC8 and TSC13, respectively,
complementation experiments were performed. Heterologous
expression of a MoTSC13 cDNA in a S. cerevisiae tsc13-1 Delo
double mutant, was sufficient to restore its ability to grow at 37uC,
as shown in Figure 4 A, suggesting that MoTSC13 is the functional
homologue of yeast TSC13 enoyl-CoA reductase [40,41]. When
we expressed yeast enhanced GFP (yEGFP)-tagged MoTSC13 in
tsc13-1 Delo mutants, they also complemented the mutant
phenotype and displayed the same perinuclear and peripheral
ER membrane-anchoring distribution in yeast cells (Figure 4E).
We conclude that MoTSC13 probably serves an evolutionarily
conserved function in catalyzing the fourth reaction of fatty acid
elongation to produce VLCFAs in both fungi .
In S. cerevisiae, Dvac8 mutants show various vacuole-associated
phenotypes, including caffeine hypersensitivity, multi-lobed vacu-
oles, loss of protein transport from the cytoplasm to vacuoles, an
inability of budding daughter cells to inherit vacuoles from the
mother cell and, importantly defects in PMN [15,23,24,25,26].
When MoVAC8 cDNA was expressed in a S. cerevisiae Dvac8 mutant
BY4741 under control of the GAL1 promoter, growth of yeast was
partially restored in the presence of 0.05% or 0.1% caffeine
(Figure 4 B), suggesting that VAC8 has conserved functions
between S. cerevisiae and M. oryzae in regulating the caffeine
response. We used pulse-chase labelling with FM4–64 to track
vacuolar morphology and inheritance during budding of the S.
cerevisiae strain BY4741 expressing MoVAC8. Vacuoles remained
multi-lobed and identical to those observed in the Dvac8 mutant.
Moreover, yeast daughter cells failed to inherit vacuoles from
mother cells (Figure 4C and D). To test whether MoVac8p was
targeted to the vacuolar membrane of S. cerevisiae, a MoVAC8
cDNA:yEGFP was constructed and introduced into the S. cerevisiae
Dvac8 mutant BY4741. Interestingly, MoVac8p was mostly
distributed in the cytoplasm and failed to accumulate at the
vacuolar membrane (Figure 4F), indicating that the N-terminal
vacuole-membrane anchoring peptide found in M. oryzae is not
fully functional in S. cerevisiae. Partial complementation of the yeast
Dvac8 mutant by MoVAC8 may reflect the different structural
organization of Vac8p between S. cerevisiae and M. oryzae. Taken
together, we conclude that MoTSC13 is a direct functional
homologue of S. cerevisiae TSC13 while MoVAC8 appears to fulfil
a role in the caffeine response but may have diverged in both
structure and function in M. oryzae.
MoVAC8 and MoTSC13 are not required for conidial
nuclear degeneration during appressorium development
To determine the function of both putative PMN proteins in M.
oryzae, we generated DMovac8 and DMotsc13 mutants in Guy11
using a split marker method and confirmed targeted gene deletion
by Southern blot hybridization (Figure S2). In order to determine
whether MoVAC8 and MoTSC13 are involved in nuclear
degeneration, H1:RFP was introduced into both DMovac8 and
DMotsc13 mutants to allow live cell imaging of nuclear behaviour.
We monitored nuclear numbers during appressorium develop-
ment and, strikingly, nuclei showed the same behaviour between
DMovac8, DMotsc13 and Guy11, as shown in Figure 5A and B.
These observations suggest that nuclear degeneration occurs
independently of a PMN pathway in M. oryzae because nuclear
degeneration was unaffected in either mutant. However, com-
pared to Guy11 and DMovac8, a much higher percentage (,30%)
of conidia of the DMotsc13 mutant contained only one or two
nuclei, as a consequence of a conidial morphology phenotype that
was associated with loss of MoTSC13 (Figure 5C).
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Figure 3. Sub-cellular localisation of MoVac8p and MoTsc13p in Magnaporthe oryzae. (A) MoVac8p-GFP is localised at the vacuolar
membrane in both conidia and appressoria. FM4–64 was used to stain vacuoles and endosomes of Guy11, expressing MoVAC8:GFP. Conidia were
collected and resuspended in 50 ml of CM with 7.5 mM FM4–64. Appressorium development was observed on coverslips at indicated time points. (B)
MoVac8p-GFP is localised on the vacuolar membrane in penetration pegs and invasive hyphae. Penetration of onion epidermis was examined at
indicated time points. (C) Tsc13p-GFP is associated with of perinuclear and peripheral ER in both conidium and appressorium. (D) Tsc13p-GFP is
associated with perinuclear and peripheral ER in penetration pegs and invasive hyphae. Images were taken from onion epidermis infected with
Guy11 expressing MoTSC13:GFP at 24 hpi. (E) MoTsc13p-GFP is localised at the perinuclear and peripheral ER in hyphae grown in CM. DAPI was used
to stain nuclei of Guy11 expressing MoTSC13:GFP. Scale bar=10 mm.
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We also introduced MoVAC8:GFP gene fusion into the H1:RFP-
expressing strain of M. oryzae to investigate formation of putative
nuclear-vacuolar (NV) junctions. A time-course experiment was
carried out with the MoVAC8:GFP, H1:RFP strain to observe
appressorium development (Figure 6 A). The large vacuole in the
conidium occupied the majority of the conidial cell volume and,
Figure 4. MoTSC13 is functionally equivalent to S. cerevisiae TSC13, while MoVAC8 only partially substitutes for VAC8. (A) MoTSC13
complemented the temperature-sensitive lethality of the yeast strain TDY2058 (double mutant tsc13-1 Delo). Yeast tsc13-1 Delo mutants were
transformed either with empty vector or a plasmid expressing MoTSC13 cDNA under control of the galactose-inducible GAL1 promoter. Cells were
grown in YPDA overnight, normalized to and subjected to 10-fold serial dilutions, spotted onto SD+Gal plates and incubated at 26uC, 30uC or 37uC for
3 days prior to photographing. The experiments were carried out in triplicate, examining two independent yeast transformants. (B) MoVAC8
complemented the caffeine sensitivity of yeast strain BY4741 vac8D::KANMX4. Yeast vac8D::KANMX4 mutants were transformed with empty vector or
a plasmid expressing MoVAC8 cDNA under the control of the GAL1 promoter. Cells were grown in YPDA overnight, normalized and subjected to 10-
fold serial dilutions, spotted onto SD+Gal plates containing either 0.05% or 0.1% caffeine, and incubated at 26uC for the indicated period of time. The
experiments were carried out in triplicate, examining two independent yeast transformants. (C) MoVAC8 does not complement vacuole morphology
and vacuole inheritance defects of yeast mutant vac8D::KANMX4. FM4–64 was used to stain cells of S. cerevisiae, vac8D::KANMX4 and vac8D::KANMX4
strain, expressing MoVAC8 by pulse-chase labelling. Arrows indicates presence (b) or absence (d, f, h, j) of segregating vacuoles. (D) MoVAC8 failed to
complement vacuole inheritance defects of yeast mutant vac8D::KANMX4 during budding. Pulse-chase labelled S. cerevisiae cells by FM4–64 were
counted for the proportion of daughter cells carrying vacuoles inherited from the mother cell during budding. (N indicates the total number of cells
counted). (E) MoTSC13p localises to the perinuclear and peripheral ER membrane in S. cerevisiae. yEGFP tagged MoTSC13p was expressed in yeast
strain TDY2058 under control of the GAL1 promoter in plasmid pYES2. Cells were stained with DAPI to visualise nuclei. (F) MoVAC8p failed to
accumulate at the vacuolar membrane in S. cerevisiae. yEGFP tagged MoVAC8p was expressed in yeast strain vac8D::KANMX4 under control of GAL1p
promoter in plasmid pYES2.
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consequently, the nucleus and vacuole were often apposed to one
another, but no distinct, regulated physical interaction of vacuoles
and nuclei was observed. The typical teardrop-shaped blebs,
which in S. cerevisiae originate from NV junctions and release PMN
vesicles into the vacuole [15,16], were also absent from conidia
undergoing autophagic cell death, as shown in Figure 6A. More
importantly, Vac8p was degraded in the conidium at a time when
nuclei were still present (Figure 6 A; 4 h and 8 h timepoints),
indicating that vacuole degeneration may proceed before the onset
of nuclear degeneration. Because PMN is induced to high levels in
S. cerevisiae by starvation [15,16], we also carried out microscopy of
the MoVAC8:GFP; H1:RFP strain grown under nitrogen starvation
Figure 5. MoVAC8 and MoTSC13 are not required for nuclear degeneration in M. oryzae. (A) Nuclear degeneration during appressorium
development occurs independently of MoVAC8. Upper panel: time course live cell images showing nuclear division and nuclear degeneration during
appressorium development in M. oryzae DMovac8 deletion mutants. DMovac8 expressing H1:RFP were examined by epifluorescence microscopy at
indicated time points during appressorium development. Lower panel: time series of bar charts showing the percentage of spore germlings of
DMovac8 containing between 0 and 4 nuclei (mean 6 SD, n.100, triple replications). (B) Nuclear degeneration during appressorium development
occurs independently of MoTSC13. Upper panel: time course live cell images showing nuclear division and nuclear degeneration during appressorium
development in M. oryzae MoTSC13 deletion mutants. DMotsc13 expressing H1:RFP were examined by epifluorescence microscopy at indicated time
points during appressorium development. Lower panel: time series of bar charts showing the percentage of spores in DMotsc13 containing between
0 and 4 nuclei (mean 6 SD, n.100, triple replications). (C) Micrographs showing abnormal conidia of DMotsc13 containing two nuclei. Scale
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Figure 6. Nuclear-vacuolar (NV) junctions are not present in M. oryzae during appressorium development or starvation stress. (A)
Time course live cell imaging of appressorium development in M. oryzae expressing both MoVAC:GFP and H1:RFP. (B) Micrographs of hyphae of M. oryzae
expressing both MoVAC:GFP and H1:RFP. M. oryzae was grown in nutrient-rich medium (CM) or nitrogen stress minimal medium (MM-N) for the indicated
period of times, and hyphae collected for epifluorescence microscopy. Hyphae cultured in CM for 40 h were washed with distilled water, before being
transferred into MM-N. (C) MoTsc13p is associated with perinuclear and peripheral ER membrane in invasive hyphae. M. oryzae expressing both
MoTSC13:GFP and H1:RFP was inoculated onto rice sheath epidermis for 36 h before epifluorescence microscopy. (D) Distribution of MoTsc13p-GFP is not
microscopy. Pictures shown are representatives of at least 30 different hyphae cell compartments analyzed for each time point. Scale bar=10 mm.
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conditions to determine whether NV junctions were apparent after
starvation of M. oryzae. No obvious NV junctions or teardrop-
shaped blebs were detected in M. oryzae following starvation stress,
as shown in Figure 6B.
In comparison to the accumulation of S. cerevisiae Tsc13p at NV
junctions from peripheral and nuclear ER pools during starvation
stress [22,37], we found that MoTsc13-GFP in the conidium of M.
oryzae was equally distributed at perinuclear and peripheral ER
membranes during appressorium development (Figure 3C). When
the MoTSC13:GFP gene fusion was introduced into Guy11
carrying H1:RFP, MoTsc13p also showed a nuclear and
(Figure 6 C), and no enrichment of the MoTsc13p at the nuclear
membrane was observed in hyphae grown under starvation
conditions (Figure 6 D). Taken together, we conclude that there is
no formation of NV junctions, the typical structures of PMN, in M.
oryzae either during appressorium development or following
The N-terminal SH4 domain of MoVac8p is required for
association of MoVac8p with the vacuolar membrane
The N-terminus of Vac8p in S. cerevisae contains a SH4 domain,
which serves as a membrane anchoring signal peptide . SH4
domains are normally composed of 18 amino acids and
characterised by a myristoylation motif (MGxxxS/Tx) and a
palmitoylation site (a cysteine residue) or several basic amino acids
. The SH4 domain within Vac8p of S. cerevisae for instance,
possesses three palmitoylation sites, which play roles in the
localisation of Vac8p in the vacuolar membrane [26,31]. Analysis
of the N-terminal sequences of MoVac8p revealed the presence of
a myristoylation motif and three potential palmitoylation sites, as
shown in Figure 2 A.
To investigate whether MoVac8p contains a functional SH4
domain, we used the first 21 amino acids of MoVac8p to generate
a putative SH4 domain:GFP fusion protein. Localisation of the
SH4 domain:GFP fusion protein was examined in conidia,
appressoria, invasive hyphae and vegetative hyphae by epifluor-
escence microscopy (Figure 7). The distribution of the SH4
domain:GFP fusion protein in each cell type was coincident with
FM4–64 stained membranes and vacuoles, and also overlapped
with the CFW stained cell wall and septa, suggesting that
SH4:GFP is membrane-associated.
To address whether the myristoylation and palmitoylation sites
of the putative SH4 domain are involved in localisation of
MoVac8p at the vacuolar membrane, we performed site-directed
mutagenesis to generate constructs expressing variants of
MoVac8p-GFP, in which glycine and cysteine residues within
the SH4 domain were replaced by alanine residues (Figure S3A).
Single point mutations of MoVAC8:GFP, including G2A, C4A, C8A
and C9A, did not abolish association of MoVac8p-GFP with the
vacuolar membrane (See Figure S3B and Figure S4). However,
these single point mutations did result in mislocalisation of
MoVac8p-GFP into the septal pore region in vegetative hyphae
(Figure S3B and C), but not in conidia (Figure S4, at least 50
conidia were examined for each variants). When two of the
palmitoylation sites were mutated, including C4A/C8A, C4A/C9A
and C8A/C9A, mislocalisation of MoVac8p-GFP in the mycelia
septa pore area was further increased (Figure S3B and C), and
MoVac8p-GFP in the C4A/C8A, C4A/C9A variants showed
strong cytosolic localisation and loss of association with the
vacuolar membrane. The C8A/C9A substitution resulted in an
increase in the mislocalisation into the septa pore (Figure S3 B
and C). Similar results were obtained in mutants expressing the
double point mutations, C4A/C8A, C4A/C9A and C8A/C9A when
conidia were examined (Figure S4). Moreover, in vegetative
hyphae and conidia of the MoVac8p-GFP strain expressing a
triple point mutation C4A/C8A/C9A, the association of fusion
proteins with the vacuolar membrane was completely disrupted
resulting in completely cytosolic localisation and enrichment at
the septal pore (Figure S3B and C, Figure S4). We conclude that
both myristoylation and palmitoylation are involved in localisa-
tion of MoVac8p.
In S. cerevisae palmitoylation of Vac8 is required for caffeine
resistance . To examine the relationship between acylation
of the SH4 domain and MoVac8p function, we therefore
measured sensitivity of strains expressing mutant alleles of
MoVAC8:GFP to caffeine. DMovac8 mutants showed hypersen-
sitivity to 0.1% caffeine, while expression of MoVAC:GFP
restored normal growth (Figure S5). In DMovac8 mutants
expressing MoVAC:GFP variants C4A, C8A, C9A, C4A/C8A,
C4A/C9A and C8A/C9A, growth on CM containing 0.1%
caffeine was restored, but variant G2A only partially restored
growth and the triple mutant C4A/C8A/C9A failed to restore
full growth (Figure S5). These results indicate that myristoyla-
tion, in particular, and complete palmitoylation of MoVac8p
plays a role in caffeine resistance in M. oryzae.
MoVAC8 is necessary for the caffeine response, while
MoTSC13 is required for full virulence and cell wall
We investigated the functions of MoVAC8 and MoTSC13 by
analysis of the phenotypes of each deletion mutant. In view of the
role of Vac8 in vacuole inheritance and movement, we
investigated movement of vacuoles and endosomes during
appressorium development in both Guy11 and DMovac8 mutants
by staining with FM4–64. Both DMovac8 mutants and Guy11
showed a similar pattern of vacuole and endosome movement, in
which vacuoles in the conidium moved into the germ tube during
germination and into the appressorium, during cellular differen-
tiation (Figure 8A). Moreover, the fusion of vacuoles was not
impaired in DMovac8 mutants (Figure 8A). These results indicate
that MoVac8p does not serve roles in vacuole inheritance or
vacuole fusion during conidium germination or appressorium
development. DMovac8 mutants did, however, show enhanced
caffeine sensitivity and slightly increased sensitivity to calcofluor
white and high concentrations of Congo red, consistent with a role
in cell wall integrity (Figure S7). Plant infection assays also
suggested that MoVAC8 is dispensable for pathogenicity of M.
oryzae (Figure 9B), because DMovac8 mutants caused similar
numbers of disease lesions to the isogenic wild type strain Guy11
and appressoria formed normally.
In contrast to the essential function of TSC13 in S. cerevisiae,
MoTSC13 is not essential for viability in M. oryzae, but loss of
MoTSC13 did reduce vegetative growth and conidiation of M.
oryzae and increased sensitivity to osmotic stress and Calcofluor
White (Figure S8A, B and C). Importantly, DMotsc13 mutants
were only able to produce very small disease lesions on rice leaves
as shown in Figure 9A. DMotsc13 mutants formed appressoria
normally (Figure S8D), implying that neither MoVAC8 nor
MoTSC13 serve essential functions in appressorium development.
To determine which stage of infection was impaired in DMotsc13
mutants, we measured appressorium turgor and the frequency of
penetration peg formation (Figure 9B, Figure S8 E). Turgor was
unaltered in DMotsc13 mutants (Figure S8 E). However,
penetration peg formation was severly impaired with only 20%
of appressoria able to elaborate a penetration peg after 24 h
(Figure 9B). By 36 hpi, invasive hyphae of Guy11 had moved into
the second or third rice epidermal cell adjacent to the invasion site,
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but most DMotsc13 mutant appressoria failed to penetrate, and
invasive hyphae were limited to the initial cell at the invasion site
(Figure 9B). Re-introduction of the MoTSC13:GFP fusion construct
into a DMotsc13 mutant restored normal vegetative growth,
penetration peg formation and pathogenicity on rice leaves
(Figure 9A and B; Figure S8 A). We conclude that MoTsc13 is
involved in penetration hypha development during plant infection.
Macroautophagy is required for nuclear degeneration
during appressorium development
Given the absence of a discernable PMN pathway, we decided
to investigate alternative means by which nuclei might be
degraded in M. oryzae. We first expressed the H1:RFP gene fusion
in a DMoatg1 mutant to allow in vivo observation of nuclei during
Figure 7. The N-terminus of MoVAC8p contains a SH4 domain. (A) SH4:GFP fusion proteins are anchored at the vacuole, septa and the
plasma membrane in both conidia and appressoria. The first 21 amino acid residues of MoVAC8p were fused into the N-terminus of GFP to produce
SH4:GFP and subcellular localisation of the GFP fusion analysed by epifluorescence microscopy. Guy11 conidia expressing SH4:GFP fusion proteins
were stained with FM4–64 and allowed to germinate and undergo appressorium development. SH4:GFP fusions colocalise with the FM4–64-stained
vacuole, septa and plasma membrane. (B) SH4:GFP fusion proteins associate with the vacuole, septa and the plasma membrane in penetration pegs
and invasive hyphae. Penetration of onion epidermis was examined at indicated time points and Calcofluor White (CFW) used to stain the cell wall.
SH4:GFP fusion proteins colocalise with the vacuole (as seen in the bright field image) and CFW-stained cell wall. (C) SH4:GFP fusion proteins were
targeted to the vacuole, septa and the plasma membrane in vegetative hyphae. Vegetative hyphae expressing SH4:GFP fusion proteins were grown
in CM for 24 h, and 200 ml of the cultures incubated with 7.5 mM FM4–64 at 26uC for 1 h, followed by CFW staining. In vegetative hyphae, SH4:GFP
fusion proteins also colocalise with FM4–64- stained vacuoles and CFW-stained septa and cell walls. Scale bar=10 mm.
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appressorium development in a macroautophagy-deficient mu-
tant. Live cell imaging showed that nuclei in the conidium were
misshapen and failed to degenerate even after 24 hpi, as shown in
Figure 10A. We also examined whether MoATG4 was required for
nuclear degeneration in M. oryzae. To achieve this, we performed
targeted gene deletion of MoATG4 in a M. oryzae strain expressing
both MoVAC8:GFP and H1:RFP (Figure S2 C). We found that
MoATG4 was required for conidial collapse and nuclear degen-
eration during appressorium development (Figure 10B). We went
on to examine nuclear degradation in targeted deletion mutants
affecting both macro-autophagy and selected autophagy. We
found that mutants in genes associated macro-autophay all showed
defects in nuclear degeneration as observed in DMoatg1 and
DMoatg4 mutants . By contrast mutants in genes associated
exclusively with selective autophagy (ATG11, ATG24, ATG26,
ATG27, ATG28, ATG29) did not show any defect in nuclear
degeneration (data not shown).
We also investigated the localisation of MoTSC13:GFP and
MoVAC8:GFP gene fusion constructs in a DMoatg1 mutant in order
to see the effect of arresting autophagy on protein localisation
during infection relateddevelopment
MoTsc13p-GFP accumulated in the conidium until 24 hpi (Figure
S6), in contrast to the gradual disappearance of MoTsc13p in
Guy11 after 4–6 h (Figure 3C). In Guy11 expressing Mo-
VAC8:GFP, vacuole degeneration started after completion of
mitosis, and vacuoles were absent from the conidium after 24
hpi (Figure 3A). While in an DMoatg1 mutant expressing
MoVAC8:GFP, vacuoles failed to degenerate even after 24 hpi
(Figure S6), suggesting a crucial role for macroautophagy in
mediating vacuole degeneration or trafficking from the conidium
during appressorium development in M. oryzae. Consistent with
this idea, vacuoles also accumulated in the conidium of DMoatg4
mutants, as shown in Figure 10B. When considered together these
data suggest that macroautophagy is important for nuclear
degeneration, ER degeneration and vacuole degeneration within
conidia during plant infection by M. oryzae.
In this study we set out to determine the mechanism by which
nuclei are broken down in conidia of the rice blast fungus prior to
appressorium formation. Appressorium-mediated plant infection
by the rice blast fungus is tightly linked to cell cycle control and
conidial cell death and degeneration of nuclei within the spore is
an essential pre-requisite to successful plant infection [7,10,11]. In
yeast, it is apparent that nuclei are degraded by a selective
autophagic process, PMN, in which nuclei bind to vacuoles via
nucleus-vacuole (NV) junctions. These NV junctions invaginate
and release PMN vesicles containing nuclear material into the
lumen of vacuoles for hydrolysis [16,17]. We have demonstrated
that M. oryzae possesses two strong candidate PMN genes, MoVAC8
and MoTSC13, but does not possess a NVJ1 homologue and,
importantly, does not appear to form NV junctions associated with
PMN-mediated nuclear breakdown. Furthermore, we have shown
that mutants lacking either MoVAC8 and MoTSC13 still undergo
nuclear breakdown and appressorium differentiation, indicating
that PMN does not mediate nuclear degeneration in M. oryzae.
Based on yeast complementation experiments, we observed that
MoVAC8 fulfils only a sub-set of the functions of its yeast
counterpart and was unable to localize correctly when expressed
in a yeast Dvac8 mutant. This is likely to be a consequence of its
distinct structure with only 9 ARM repeats present in the protein,
compared to 11 in Vac8p. It is clear, however, that MoVac8 is a
vacuolar membrane protein, which is both myristoylated and
palmitoylated  in M. oryzae and is involved in the response to
caffeine, because DMovac8 mutants show hypersensitivity to
caffeine (1,3,7-trimethyl xanthine). This function is also conserved
when MoVAC8 was expressed in a yeast Dvac8 mutant. Caffeine
sensitivity in S.cerevisiae appears to be associated with the Pkc1/cell
integrity pathway because caffeine treatment induces rapid
phosphorylation of the Mpk1 MAP kinase and leads to large
scale changes in gene expression associated with cell wall stress
. However, the similarity in transcriptional response to
rapamycin treatment, coupled with the hypersensitivity of Tor1
kinase mutants to caffeine, also point to an effect on the Ras/
cAMP response pathway, and the control of cellular viability,
which is coupled with the regulation of autophagy. The
hypersensitivity of DMovac8 mutants to caffeine may therefore be
associated with an impairment in vacuole transport function,
which is consistent with the requirement for myristoylation and
palmitoylation for vacuolar membrane localization. Interestingly,
the wider reported roles for Vac8p in vacuolar inheritance were
Figure 8. Phenotypic analysis of DMovac8 mutants. (A) MoVAC8 is
not involved in vacuole movement from the conidium into the
appressorium. FM4–64 was used to stain endosomes and vacuoles in
conidia to visualize movement of vacuoles during appressorium
development. (B) Rice blast infection assay of DMovac8 mutants on
rice leaves. The virulence of DMovac8 is similar to that of strain Guy11.
Lesion density represents the lesion number per 5 cm of infected leaf
area on rice CO-39 seedlings (n.40).
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not conserved in M. oryzae, suggesting significant divergence in
function, consistent with the distinct structural organisation of the
protein. The MoVac8-GFP gene fusion did allow visualization of
vacuole behaviour during infection-related development in M.
oryzae in live cell imaging experiments and highlighted the
importance of formation of a large central appressorial vacuole
during during appressorium turgor generation, which had been
suggested in earlier cytochemical studies . We can therefore
conclude that MoVac8 is a vacuolar protein that is unlikely to
serve a role in PMN in the rice blast fungus, but instead plays a
role in vacuolar function which may be vital for contending with
abiotic stresses such as exposure to caffeine.
In contrast to MoVAC8, MoTSC13 appears to have a highly
conserved function as an enoyl reductase that catalyzes the fourth
reaction of fatty acid elongation to produce very long chain fatty
acids. This function appears to be completely conserved with the
role of TSC13p in yeast, but strikingly, MoTSC13 is not essential
for cellular viability in M. oryzae and DMotsc13 mutants instead
grow well in culture. Furthermore, we found no evidence for a role
for MoTsc13p in PMN and there was no distinct localization of
the protein at specific NV junctions. Instead, we found that
MoTsc13-GFP localized to the perinuclear and peripheral ER.
Importantly, we did observe that DMotsc13 mutants are signifi-
cantly impaired in their ability to cause rice blast disease and that
this results as a consequence of a reduced ability to colonize rice
epidermal cells following appressorium-mediated penetration of
the cuticle. We can conclude that very long chain fatty acid
biosynthesis is therefore likely to be important in invasive hyphae
development, perhaps pointing to the membrane components
of invasive hyphae possessing a distinct lipidic characteristic
compared to those of vegetative hyphae– a feature worthy of
The final conclusion that can be made from this study is that
nuclear degeneration during appressorium formation, which is
known to be essential for plant infection [7,10,11], occurs via non-
selective macroautophagy. In contrast to yeast, there is no
evidence for a separate selective PMN process in M. oryzae. We
found that macro-autophagy-associated genes such as MoATG1 or
MoATG4 were necessary for nuclear degeneration and their
absence rendered the fungus non-pathogenic , whereas
mutations in genes affecting selective forms of autophagy did not
show any difference from the wild type Guy11. Macroautophagy
has very recently been reported to mediate nuclear degeneration
in Aspergillus oryzae , but in that case involved formation of large
ring-like autophagosomal structures (1–2 mm) that encircled and
mediated degradation of whole nuclei in A. oryzae basal cells. In
this study we were only able to detect punctate autophagosomes in
both conidia and appressoria of M. oryzae (consistent with
[11,12,13]), rather than the much larger, ring-like autophagosome
structures reported in A. oryzae , suggesting that nuclear
breakdown may be performed by distinct macroautophagy-
dependent processes in filamentous fungi. Furthermore, nuclei
did not appear to be degraded in their entirety, but rather there
was dissolution of nuclear material, which could be observed both
sytoplasmically and ithin vacuoles during autophagy. When
considered together, we can conclude that conidial cell death
and nuclear degeneration, which occur as part of the essential
programme for appressorium-mediated plant infection by M.
oryzae, both require non-selective autophagy, which re-cycles the
contents of these cells, including nuclei, ER and other organelles
Figure 9. MoTSC13 is required for full virulence of M. oryzae. (A) Targeted deletion of MoTSC13 results in reduced pathogenicity on rice CO-39.
Lesion density and size was severely reduced in DMotsc13 rice infections compared to Guy11. Full pathogenicity of DMotsc13 mutants was restored
by introduction of MoTSC13:GFP (Compl.). Lesion density represents the lesion number per 5 cm of infected leaf area (n.40). (B) MoTSC13 is
necessary for penetration peg formation and invasive hyphae expansion during in planta growth. Scale bar=10 mm.
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into the specialized infection structure, prior to plant cuticle
rupture and tissue colonization.
Materials and Methods
Fungal strains, growth conditions, and DNA analysis
The fertile rice pathogenic M. oryzae strain, Guy11, was used in
all studies . Culture, maintenance, and storage of M. oryzae
isolates, media composition, nucleic acid extraction, and fungal
transformation were all as previously described . Yeast strains
were manipulated using standard methods. All primers used in this
study are described in Table S1. S. cerevisiae strain BY4741
vac8D::KANMX4 (MATa his3D1 leu2D0 met15D0 ura3D0 vac8D::-
KANMX4) used for expression of MoVAC8 cDNA was obtained
from EUROSCARF. S. cerevisiae strain TDY2058 (MATa
elo3::TRP1 tsc13-1 ade2-101 ura3-52 trp1D leu2D) used for
expression of MoTSC13 cDNA was kindly provided by Dr. Teresa
M. Dunn (Department of Biochemistry, Uniformed Services
University of the Health Sciences, Bethesda, Maryland). Gel
electrophoresis, restriction enzyme digestion, gel blots, PCR and
sequencing were performed using standard procedures .
Targeted deletion of MoVAC8, MoVAC8:GFP and
MoVAC8SH4:GFP fusion plasmid construction, site-
directed mutagenesis of MoVAC8:GFP and DMovac8
The split-marker recombination method was used for efficient
targeted deletion of M. oryzae genes [11,48]. The hph gene, which
confers resistance to HygromycinB (HYG) was used as the split
marker. The two split hph templates were amplified by primers
M13F with HYsplit and M13R with YGsplit, as previously
described . A 1 kb sequence flanking either side of the MoVAC8
coding sequence was amplified, with left flanking (LF) sequences
amplified by primers vac850.1 and vac8m13f, right flanking (RF)
sequences amplified with primers vac830.1 and vac8m13r. The LF
sequences were fused with split HY, using primers vac850.1 and
HYsplit to form LF-HY, while the RF sequences fused with the split
YG fragment using primers YGsplit and vac830.1, to form YG-RF.
The resulting amplicons LF-HY and YG-RF were gel-purified and
co-transformed into protoplasts of Guy11. The DMovac8 mutants
were confirmed by DNA gel blot analysis and two independent
mutants selected for further phenotypic analysis.
Figure 10. Macroautophagy is necessary for nuclear degeneration during appressorium development in M. oryzae. (A) The
Macroautophagy gene MoATG1 is required for nuclear degeneration. Upper panel: time course live cell images showing nuclear division and nuclear
degeneration during appressorium development in a M. oryzae DMoatg1 mutant. DMoatg1 conidia expressing H1:RFP were examined by
epifluorescence microscopy at indicated time points. Lower panel: time series of bar charts showing the percentage of spores in DMoatg1 containing
0 to 4 nuclei (mean 6 SD, n.100, triple replications). (B) The Macroautophagy core gene MoATG4 is required for nuclear degeneration. Upper panel:
time course live cell images showing nuclear division and nuclear degeneration during appressorium development in M. oryzae DMoatg4 mutant.
DMoatg4 conidia expressing H1:RFP were examined by epifluorescence microscopy at indicated time points during appressorium development.
Lower panel: time series of bar charts showing the percentage of spores in DMoatg4 containing 0 to 4 nuclei (mean6SD, n.100, triple replications).
Scale bar=10 mm.
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The MoVAC8:GFP construct was made by fusion PCR and
standard restriction enzyme-mediated cloning. The MoVAC8 gene
(1.7 kbp promoter and 2.1 kbp CDS) was amplified with primers
Vac8fusionFor and Vac8GFPRev. Primer Vac8GFPRev con-
tained overhanging sequences at its 59 end, which were
complementary to the sGFP sequence. The 1.5 kb sGFP coding
region, together with TrpC terminator, was amplified with primers
GFPTrpCFor and GFPTrpCRev. The MoVAC8:GFP fusion
cassette was then generated with primers Vac8fusionFor and
GFPTrpCRev. An XhoI restriction enzyme recognition site was
introduced into the 59 end of both primers, Vac8fusionFor and
GFPTrpCRev, to facilitate cloning of the MoVAC8:GFP fusion
cassette into pCB1532 vector for fungal transformation . The
first 21 amino acid section of the N-terminus of MoVac8p,
covering the predicted SH4 domain, was fused into the N-
terminus of GFP and the yeast recombination method employed to
generate a MoVAC8SH4:GFP fusion . The 1.7 kb MoVAC8
promoter sequence was amplified with primer pair, Vac8P-For
and Vac8P-Rev, while SH4 domain-coding sequences were
amplified with primer pair VAC8SH4-For and VAC8SH4-Rev
from cDNA prepared from conidial total RNA. The amplicons
were gel-purified and co-transformed into the relevant yeast strain
together with plasmid pAGL1:GFP, which was linearized with
HindIII and contains the selectable marker gene SUR conferring
resistance to chlorimuron ethyl.
Site-directed mutageneses was performed on plasmid pCB1532-
MoVAC8:GFP to generate alleles containing replacement of the
predicted myristoylation (glycine) or palmitoylation (cysteine)
modification sites by alanine residues, including G2A, C4A, C8A,
C9A, C4A/C8A, C4A/C9A, C8A/C9A and C4A/C8A/C9A. In brief,
a 714 bp region was amplified initially in two fragments, and
nucleotide substitutions introduced into the primers, located at the
overlapping region of the two fragments that were joined together
by fusion PCR using primer pair Vac8mut-For and Vac8mut-Rev.
The 714 bp fragment carrying the respective nucleotide substitu-
tions was then digested with FseI and PmlI to release a 320 bp
fragment that was used to replace the region spanning FseI and PmlI
in plasmid pCB1532-MoVAC8:GFP. DNA sequencing was utilised
to confirm successful introduction of each nucleotide substitution.
Finally, the plasmid variants of pCB1532-MoVAC8:GFP were
transformed into DMovac8 mutants, and at least two independent
transformants selected for phenotypic analysis. For complementa-
tion of DMovac8 mutants, the pCB1532 vector carrying Mo-
VAC8:GFP was transformed into a DMovac8 mutant and at least
two independent transformants tested for complementation.
Targeted deletion of MoTSC13, MoTSC13:GFP fusion
plasmid construction and DMotsc13 complementation
Targeted gene deletion of MoTSC13 was performed with the
split-marker recombination method, as described above. The
1.0 kbp LF sequences were amplified with primers tsc1350.1 and
tsc13m13f, while the 1.0 kbp RF sequences were amplified with
primers tsc1330.1 and tsc13m13r. Generation of DMotsc13
mutants was confirmed by DNA gel blot analysis and two
independent mutants selected for further analysis . The
MoTSC13:GFP fusion was made using the yeast recombination
method, as described above . Briefly, the bialaphos resistance
selectable marker gene BAR was amplified using primers BarF and
BarR, and a 3.0 kb fragment of the MoTSC13 gene amplified with
primers Tsc13GFPFor and Tsc13GFPRev, GFP-TrpC terminator
cassette amplified with primers GFPTrpCFor and GFPTrpR .
The amplicons were gel-purified and co-transformed into the
relevant yeast strain together with vector pNEB-Nat which had
been linearized with HindIII and SacI.
Expression of MoVAC8 and MoTSC13 in S. cerevisiae
Full-length double-stranded cDNAs of MoVAC8 and MoTSC13
were amplified from 1st strand cDNA using primer pair
Vac8yeast50.1 and Vac8yeast30.1 and primer pair Tsc13yeast50.1
and Tsc13yeast30.1, respectively. MoVAC8 cDNA was cloned into
KpnI and XbaI sites of yeast expression vector pYES2 (Invitrogen)
and introduced into S. cerevisiae strain BY4741 vac8D::KANMX4,
while the MoTSC13 cDNA was cloned between HindIII and XbaI
sites of pYES2 and introduced into S. cerevisiae strain TDY2058. For
expression of yeast-enhanced GFP (yEGFP) tagged MoVAC8 and
MoTSC13 in S. cerevisiae, the cDNA of MoVAC8 was amplified by
primer pair Vac8yEGFPFor and Vac8yEGFPRev, MoTSC13
amplified by Tsc13yEGFPFor and Tsc13yEGFPRev, and yEGFP
amplified by primer pair yEGFPFor and yEGFPRev from plasmid
pKT127 (obtained from EUROSCARF). The MoVAC8:yEGFP and
MoTSC13:yEGFP fusion cassettes were generated by primer pair
Vac8yEGFPFor and yEGFPRev, and primer pair Tsc13yEGFPFor
and yEGFPRev respectively, both cloned between EcoRI and SphI
sites in pYES2 . The MoVAC8:yEGFP construct was introduced
into S. cerevisiae strain BY4741 vac8D::KANMX4, while MoTS-
C13:yEGFP was expressed into TDY2058. The cDNA sequences of
both MoVAC8 and MoTSC13 in pYES2 were confirmed by DNA
sequencing. All yeast transformants were confirmed by PCR and at
least two independent yeast transformants chosen for analysis.Sen-
sitivity to caffeine was assessed by spotting a dilution series of yeast
cells (1072104cells ml21) on synthetic drop-out (SD) medium
containing 0.05% or 0.1% caffeine in the presence of galactose.
Vacuole morphology and inheritance in S. cerevisiae were observed
by staining with FM4–64 (Molecular Probes, Invitrogen) according
to . For assessing vacuolar inheritance, pulse-chase labelling
with FM4–64  was performed by washing FM4–64 stained cells
twice with fresh medium and incubating for an additional 4 h at
30uC. For testing temperature-sensitive lethality of yeast strains
TDY2058, a dilution series of yeast cells (1062104cells ml21)
expressing MoTSC13 were spotted onto synthetic drop-out (SD)
medium in the presence of galactose at 37uC, 30uC or 25uC.
Generation of M. oryzae macroautophagy deficient
strains carrying either H1:RFP (tdTomato), MoVAC8:GFP or
MoTSC13:GFP – gene fusions
A H1:RFP fusion construct was introduced into DMovac8 and
DMotsc13 mutants for live cell imaging of nuclei. MoVAC8:GFP and
MoTSC13:GFP gene fusion constructs were introduced into Guy11
carrying H1:RFP. H1:RFP, MoVAC8-GFP. MoTSC13:GFP gene
fusions were also introduced into a DMoatg1 mutant to investigate
behaviour of these fusion proteins in macroautophagy-deficient
mutants. Transformants were selected by DNA gel blot, and at
least two independent transformants investigated for all experi-
ments. For targeted deletion of MoATG4 in Guy11 expressing both
H1:RFP and MoVAC8:GFP gene fusions, the split-marker BAR was
used. Briefly, the two split BAR templates were amplified by
primers M13F with BAsplit and M13R with ARsplit, and
MoATG4 LF amplified by primers Atg450.1 and Atg4m13f,
MoATG4 RF amplified by primers Atg430.1 and Atg4m13r, as
previously described . The LF-BA was obtained with primers
Atg450.1 and BAsplit, while AR-RF obtained with primers
ARsplit and Atg430.1.
FM4–64 staining of conidia or mycelia in M. oryzae
The lipophilic styryl dye, FM4–64 (N-(3-triethylammoniumpro-
pyl-)-4-(6(4-(diethylamino)phenyl) hexatrienyl) pyridinium dibro-
mide) was used to stain vacuoles and endosomes of conidia or
mycelia in M. oryzae (Molecular Probes, Invitrogen). Conidia
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grown in CM agar plate culture were collected with 4 ml of sterile
distilled water, filtered through miracloth (Calbiochem). Approx-
imately 200 ml of conidial suspension, at 16106ml21was
centrifuged at 6, 000 g for 5 min to precipitate conidia. After
washing with 1 ml of sterile distilled water and centrifugation at 6,
000 g for 5 min, the conidial pellet was resuspended in 50 ml of
liquid CM with 7.5 mM FM4–64. The suspension was incubated
at 26uC for 20 min, and then conidia were recovered by
centrifugation. The supernatant was discarded to remove excess
FM4–64 and pellet washed twice with 1 ml of sterile distilled
water. Conidia were finally resuspended in sterile distilled water at
a concentration of 56104ml21. Appressorium development was
observed on coverslips at indicated time points with epifluores-
Plant pathogenicity and infection structure development
Cuticle penetration was assessed by recording the frequency of
penetration peg formation from appressoria on onion epidermis.
A 50 ml drop of conidial suspension at a concentration of 56104
conidia ml21was placed on the surface of onion epidermis and
incubated in a humid environment at 24uC for 24 h or 48 h.
The frequency of cuticle penetration was determined micro-
scopically by counting formation of penetration pegs from at
least 100 appressoria in triplicate replications of the experiment.
Turgor generation in mature appressoria was measured by a
cytorrhysis assay in a series of glycerol solutions of varying
molarity, as previously described [6,51]. Rice infections were
performed using cultivar CO-39, a dwarf rice cultivar which is
very susceptible to M. oryzae . A conidial suspension
(56104mL21) was produced by flooding 10-day-old M. oryzae
culture plates with 0.2% (v/v) gelatine solution and the
suspension spray-inoculated onto 14-day-old rice plants. Plants
were placed in plastic bags for 24 h to maintain high humidity
and then transferred to controlled environment chambers at
24uC and 90% relative humidity with illumination and 14 h light
periods. Plants were incubated until disease symptoms were
apparent 96–144 h later.
Conidial germination and development of appressoria were
both monitored over time on hydrophobic borosilicate glass cover
slips (Fisher Scientific) using a method adapted from [2,46].
Conidial suspensions at 56104conidia mL21were inoculated onto
cover slips, incubated at 24uC, and all images of conidial
germination and appressorium development were recorded using
a Zeiss Axioskop 2 microscope (Zeiss).
Light and epifluorescence microscopy
For epifluorescence microscopy of GFP or RFP expressing
transformants, conidia were inoculated onto coverslips, incu-
bated at 24uC and collected at indicated time points for
observation using an IX81 motorized inverted microscope
(Olympus) equipped with an UPlanSApo 1006/1.40 Oil
objective (Olympus). Excitation of fluorescently-labeled proteins
was carried out using a VS-LMS4 Laser-Merge-System with
solid state lasers. The laser intensity was controlled by a VS-
AOTF100 System and coupled into the light path using a VS-20
Laser-Lens-System (Visitron System). Images were captured
using a Charged-Coupled Device camera (Photometric Cool-
SNAP HQ2, Roper Scientific). All parts of the system were
under the control of the software package MetaMorph
(Molecular Devices) and offline images were analyzed with
MetaMorph software and Adobe Photoshop CS2 (Adobe
between M.oryzae and S. cerevisiae. (A) Vac8p ClustalW
alignment. (B) Tsc13p ClustalW alignment. Star indicates con-
served amino acids shown to be important for function of Tsc13p of
ClustalW alignment of Vac8p and Tsc13p
MoATG4 genes in M. oryzae. A) Southern blot analysis was
used to confirm targeted deletion in DMovac8 mutants. MoVAC8
left flanking region, MoVAC8 ORF, and Hygromycin resistance
marker gene fragment HY were used as probes. DMovac8.3, 10, 11,
18 and 21 were defined as five independent deletion mutants, and
strains 8.6 and 8.15 were detected as ectopic insertion mutants.
Two independent deletion mutants DMovac8.10 and DMovac8.21
were chosen for further phenotypic analysis. (B) Southern blot
analysis was used to confirm targeted deletion in DMotsc13
mutants. MoTSC13 left flanking region, MoTsc13 ORF, and
Hygromycin resistance marker gene fragment HY were used as
probes. DMotsc13.4 and DMotsc13.8 were two independent
knockout mutants, and DMotsc13.1 and DMotsc13.5 were ectopic
insertion mutants. DMotsc13.2 and DMotsc13.3 were DMotsc13
mutants in the Dku70 background strain . Two independent
deletion mutants, DMotsc13.4 and DMotsc13.8, were chosen for
further analysis. (C) Southern blot analysis was used to confirm
putative DMoatg4 mutants. MoATG4 left flanking region, ORF,
and BAR marker gene were used as probes. DMoatg4.5, 8, 10, 14
and 16 and 18 were defined as six independent deletion mutants,
and strains 13, 15 and 19 were detected as ectopic insertion
mutants. Two independent deletion mutant DMoatg4.8 and
DMoatg4.18 were chosen for further analysis.
Targeted deletion of MoVAC8, MoTSC13 and
Vac8p are required for association of MoVac8p with
vacuolar membranes in vegetative hyphae. (A) N-terminal
sequences of MoVAC8p-GFP variants used in this study. Alanine
mutations within the N-terminal SH4 domain are indicated in
bold. Constructs were named according to their mutated glycine
or cysteine residues and numbers indicate the amino acid positions
within the SH4 domain. (B) Localization of MoVac8p-GFP
variant proteins in vegetative hyphae. DMovac8 mutant was
transformed with constructs expressing the indicated GFP fusion
proteins. Vegetative hyphae of transformants expressing Mo-
Vac8p-GFP variant fusion proteins were prepared and visualised
by epifluorescence microscopy, as indicated in Figure 7. Arrows
indicate the position of mis-localised MoVac8p-GFP in the septal
pore area. (C) Effects of SH4 domain mutations in localisation of
MoVac8p-GFP. MoVac8p-GFP fusion proteins were associated
with the vacuolar membrane, while mutations within the SH4
domain resulted in mislocalisation of fusion proteins in the septal
pore area. Vegetative hyphae expressing each respective Mo-
Vac8p-GFP allele were grown in CM for 24 h, followed by CFW
staining of cell wall and septa before fluorescence microscopy. The
number in parentheses indicates the total number of septa counted
and examined by CFW staining in epifluorescence microscopy
experiments, and the numbers above the grey bars represent the
percentage of septa enriched with mislocalised MoVac8p-GFP
fusion proteins. Scale bar=10 mm.
Myristoylation and palmitoylation of Mo-
association of Vac8p with the vacuolar membrane in
conidia and appressoria. Conidia of DMovac8 mutants
Palmitoylation of MoVac8p is required for
Nuclear Breakdown in Magnaporthe
PLoS ONE | www.plosone.org15March 2012 | Volume 7 | Issue 3 | e33270
expressing the variant MoVac8p-GFP fusion proteins were stained
with FM4–64, as described above and localisation of the respective
GFP fusion proteins in conidia and appressoria analysed by
epifluorescence microscopy. Arrows indicate the position of mis-
localised MoVac8p-GFP in the septal pore area. Scale bar=10 mm.
tion and palmitoylation mutants in caffeine resistance.
The MoVAC8:GFP fusion construct and each mutant allele were
transformed into the DMovac8 mutant, and three independent
transformants for each construct grown on CM plates in the
presence of 0.1% caffeine for 15 days.
Functional analysis of MoVac8p myristoyla-
essary for vacuole degeneration and ER degeneration
during appressorium development in M. oryzae. Left
Panels. Degradation of perinuclear and peripheral ER membrane-
associated protein, MoTsc13p-GFP, was blocked in DMoatg1
mutants during appressorium development. Right Panels. Degra-
dation of vacuolar membrane protein MoVac8p-GFP was blocked
in DMoatg1 mutants during appressorium development. Scale
Macroautophagy core gene MoATG1 is nec-
on different stress medium except caffeine. Uniformly
sized mycelial plugs were used to inoculate agar plate cultures
supplemented with Congo Red, Calcofluor white (CFW), Sodium
dodecyl sulfate (SDS), hydrogen peroxide or caffeine, as shown,
and incubated for 12 days at 24uC.
MoVAC8 is not required for vegetative growth
integrity and hyper osmotic stress adaptation, but not
appressorium development or turgor generation. (A)
DisruptionofMoTSC13 reduced hyphalgrowthofM.oryzaeon CM,
and made M. oryzae sensitive to hyper osmotic stress and cell wall
stress. (B) Vegetative growth was impaired by deletion of MoTSC13.
The diameter of colonies of both Guy11 and DMotsc13 mutants
grown on CM plates was recorded at indicated times in the line
graph presented. (C) Conidiation was reduced by deletion of
MoTSC13. A 3 mm mycelium plug was inoculated in triplicate and
incubated at 24uC for 12 days. Conidia generated were collected in
3 ml of distilled water, and 20 ml of conidial suspension used for
counting on a hemacytometer. Bar chart shows conidia per cm2 of
plate cultures. (D) MoTSC13 is dispensable for appressorium
development. Bar charts showing the percentage of conidia
formaing an appressorium after 6 h or 24 h (E) MoTSC13 is
dispensable for turgor generation in the appressorium. Bar charts
showing the percentage of cell collapse upon incubation in
increasing concentrations of glycerol .
MoTSC13 is involved in maintaining cell wall
in this study.
Detailed information of oligonucleotide primers used
Conceived and designed the experiments: MH NJT. Performed the
experiments: MH MJK DMS. Analyzed the data: MH MJK DMS YX
NJT. Contributed reagents/materials/analysis tools: DMS. Wrote the
paper: MH NJT.
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