Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease.
ABSTRACT To cause rice blast disease, the fungus Magnaporthe oryzae elaborates specialized infection structures called appressoria, which use enormous turgor to rupture the tough outer cuticle of a rice leaf. Here, we report the generation of a set of 22 isogenic M. oryzae mutants each differing by a single component of the predicted autophagic machinery of the fungus. Analysis of this set of targeted deletion mutants demonstrated that loss of any of the 16 genes necessary for nonselective macroautophagy renders the fungus unable to cause rice blast disease, due to impairment of both conidial programmed cell death and appressorium maturation. In contrast, genes necessary only for selective forms of autophagy, such as pexophagy and mitophagy, are dispensable for appressorium-mediated plant infection. A genome-wide analysis therefore demonstrates the importance of infection-associated, nonselective autophagy for the establishment of rice blast disease.
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ABSTRACT: Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn2Cys6 transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn2Cys6TF genes. A large variation in biological functions of Zn2Cys6TF genes was observed under the conditions tested. Sixty-one of 104 Zn2Cys6 TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae.PLoS Pathogens 10/2014; 10(10):e1004432. · 8.14 Impact Factor
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ABSTRACT: The rice blast fungus Magnaporthe oryzae is a model for studying fungal-plant interactions. Although it produces two types of spores (microconidia and macroconidia), previous infection studies have exclusively dealt with macroconidia. Germination of microconidia has not been reported, and their role in plant infection is not defined. Here we show that approximately 10% of microconidia germinate on plant surfaces, and that colonies derived from germinated microconidia are normal in growth and pathogenesis. In infection assays with rice and barley seedlings, microconidia fail to infect intact plants, but they can colonize and develop necrotic lesions on wounded leaves and stems. Microconidia also cause disease symptoms on inoculated spikelets in infection assays with barley and Brachypodium heads. Furthermore, microconidia are detected inside rice plants that developed blast lesions under laboratory or field conditions. Therefore, microconidia can germinate and are infectious, and may be an important factor in the rice blast cycle.Nature Communications 01/2014; 5:4518. · 10.74 Impact Factor
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ABSTRACT: Plant pathogenic fungi are the most diverse and drastic causal agents of crop diseases threatening stable food production all over the world. Plant have evolved efficient innate immune system to scout and counterattack fungal invasion and pathogenic fungi also developed virulence system to nullify plant resistance machinery or signaling pathways and to propagate and dominate within their niche. A growing body of evidences suggests that post translational modifications (PTMs) and selective/nonselective degradations of proteins involved in virulence expression of plant pathogenic fungi and plant defense machinery should play pivotal roles during the compatible and incompatible interactions. This review elucidates recent investigations about the effects of PTMs and protein degradations on host defense and fungal pathogens' invasions.The Korean Journal of Mycology. 01/2010; 38(2).
Genome-wide functional analysis reveals that
infection-associated fungal autophagy is
necessary for rice blast disease
Michael J. Kershaw and Nicholas J. Talbot1
School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, United Kingdom
Edited by Steven P. Briggs, University of California at San Diego, La Jolla, CA, and approved July 16, 2009 (received for review February 12, 2009)
To cause rice blast disease, the fungus Magnaporthe oryzae elab-
orates specialized infection structures called appressoria, which
use enormous turgor to rupture the tough outer cuticle of a rice
leaf. Here, we report the generation of a set of 22 isogenic M.
oryzae mutants each differing by a single component of the
predicted autophagic machinery of the fungus. Analysis of this set
of targeted deletion mutants demonstrated that loss of any of the
16 genes necessary for nonselective macroautophagy renders the
fungus unable to cause rice blast disease, due to impairment of
both conidial programmed cell death and appressorium matura-
tion. In contrast, genes necessary only for selective forms of
appressorium-mediated plant infection. A genome-wide analysis
therefore demonstrates the importance of infection-associated,
nonselective autophagy for the establishment of rice blast disease.
appressorium ? fungus ? plant pathogen
as appressoria, are a feature of some of the most important cereal
pathogens, including the devastating rice blast disease-causing
fungus, Magnaporthe oryzae (2, 3). Appressoria of the rice blast
fungus are dome-shaped, single-celled structures that generate
enormous turgor pressure through accumulation of very high
concentrations of glycerol (4). Hydrostatic turgor is generated by
rapid influx of water into the appressorium, where a layer of
allowing turgor to increase to a level sufficient to rupture the plant
and differentiates into bulbous, branched invasive hyphae, which
are bounded by the invaginated plant cell membrane, allowing the
fungus to proliferate within living plant cells (2, 3, 4).
Appressoria are formed following germination of a 3-celled
fungal spore, called a conidium, which attaches tightly to the
hydrophobic rice leaf surface (2). The conidium germinates and
to form the appressorium. Development of these cells requires
activation of the Pmk1 mitogen-activated protein kinase pathway
(5, 6) and is regulated genetically by control of the cell cycle (7).
During germination of the M. oryzae conidium, a single nucleus
migrates into the germ tube and undergoes mitosis. After this, one
of the resulting daughter nuclei migrates into the incipient appres-
and the subsequent movement of nuclei are necessary for appres-
soria to develop and for plant infection to occur. Completion of
mitosis also, however, leads to collapse and death of the fungal
conidium, the contents of which are delivered to the maturing
appressorium. Functional analysis of the M. oryzae (Mo) ATG8
gene, has suggested that type II autophagic cell death is necessary
for appressorium maturation and plant infection (7, 8).
In this study, we set out to determine whether infection-related
autophagy is necessary for rice blast disease solely as a result of its
role in conidial cell death or whether appressoria also undergo
o cause plant disease, many plant pathogenic fungi elaborate
autophagy during their maturation. We also aimed to define
whether autophagy carried out by M. oryzae during plant infection
is a selective or a nonselective form of autophagy (9, 10, 11).
Genetic analysis in the budding yeast Saccharomyces cerevisiae has
identified a family of 30 ATG genes, which encode proteins
necessary for autophagy (11, 12). TOR kinase regulates initiation
of autophagy (13, 14) leading to formation of a single membrane
ical, double-membrane autophagosome. The autophagosome ex-
pands and then fuses with a vacuole, the lytic compartment
(lysosome equivalent) of fungal cells, sequestering its contents and
inner membrane for degradation by hydrolases (10, 11). Selective
forms of autophagy degrade peroxisomes (pexophagy), mitochon-
dria (mitophagy), and endoplasmic reticulum (reticulophagy) or
can occur during the biosynthetic cytoplasm-to-vacuole-targeting
(Cvt) pathway, described in S. cerevisiae, which is used to transport
the inactive precursor of the vacuolar hydrolase aminopeptidase I
to the vacuole (15). Selective forms of autophagy require a distinct
set of proteins, such as Atg11, which encodes a peripheral mem-
brane protein that is the adaptor required for cargo loading in
pexophagy and for delivery of aminopeptidase I to the vacuole in
the Cvt pathway (15–18).
To determine why fungal autophagy is necessary for rice blast
disease and to define which type of autophagy takes place during
plant infection, we decided to adopt a genome-wide approach in
which we would systematically analyze the autophagic machinery
of M. oryzae and define the role of each of the associated gene
products. To do this, we first developed a rapid method for gene
functional analysis in M. oryzae and deployed this method to
characterize the 22 fungal genes involved in autophagy. Here, we
provide comprehensive evidence that infection-related autoph-
agy is nonselective and takes place in both conidia and appres-
soria of M. oryzae leading to death of the conidium and devel-
opment of a functional appressorium essential for plant disease.
Infection-Associated Autophagy Occurs in both Conidia and Appres-
soria of M. oryzae. We set out first to visualize infection-associated
autophagy in M. oryzae and determine the spatial and temporal
dynamics of autophagosomes during appressorium development.
To do this, we constructed a GFP-MoATG8 gene fusion, which was
introduced into a wild-type strain of M. oryzae Guy11 and also the
?Moatg8 mutant. Analysis of the cellular localization pattern and
flux of Atg8 has been shown to be a reliable marker for autophagy
(19). In yeast, ATG8 encodes an ubiquitin-like protein that can be
Author contributions: N.J.T. designed research; M.J.K. performed research; M.J.K. and
N.J.T. analyzed data; and M.J.K. and N.J.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
www.pnas.org?cgi?doi?10.1073?pnas.0901477106 PNAS ?
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modified at its C terminus by addition of phosphatidylethano-
lamine, tethering it to the autophagosome membrane where it is
necessary for phagophore expansion during autophagosome for-
mation (20). Expression of the GFP-MoATG8 fusion was sufficient
to complement the ?Moatg8 mutant phenotypes (7; supporting
information (SI) Fig. S1), providing evidence that it was functional
within M. oryzae and therefore a reliable marker for analysis of the
microscopy showed that GFP-MoATG8-labeled autophagosomes
accumulated in conidia during germination and then steadily
decreased in number during the onset of conidial cell death and
appressorium maturation (Fig. 1). Autophagosome number within
developing appressoria increased during appressorium maturation
and intense autophagic activity and vacuole expansion was associ-
ated with mature appressoria (Fig. 1). To investigate whether
in M. oryzae, we expressed GFP-MoATG8 in a ?pmk1 MAP kinase
mutant that does not elaborate appressoria and is consequently
autophagosomes were present in the conidium during germination
but in significantly smaller numbers (see Fig. S2; t test, P ? 0.05).
Conidia of the ?pmk1 mutant remained intact throughout germi-
nation and germ tube elongation, indicating that conidial pro-
grammed cell death does not occur in the absence of appressorium
formation. We conclude that infection-associated autophagy re-
quires the Pmk1 MAP kinase and occurs during the onset of
appressorium-mediated plant infection, initially within conidia,
allowing the recycling of some of their contents to the developing
appressorium where further autophagic activity occurs.
Development of a Rapid Method for Gene Functional Analysis in M.
oryzae. To investigate the molecular control of autophagy in M.
throughput method for gene functional analysis. Recently, it has
been shown that deletion of genes encoding components of the
fungal strains with enhanced frequencies of homologous recombi-
nation (for review see ref. 21). We therefore deleted the ku70-
encoding gene of M. oryzae and tested the resulting strain for the
frequency of targeted gene replacement. We used deletion of the
BUF1 gene, which encodes tri-hydroxy-naphthalene reductase, an
enzyme required for melanin biosynthesis (22), as a visual test for
have a buff color compared to the olive green/gray color of
wild-type M. oryzae cultures (22), identifying mutants was straight-
forward. We found that homologous gene replacement occurred at
a frequency of 80% (n ? 100) in the ?ku70 mutant background.
in M. oryzae, which is locus-dependent and ranges from 1% to 25%
(21). Growth rate, sporulation, and pathogenicity of the ?ku70
mutant were found to be unaltered by the mutation and therefore
function of new genes in M. oryzae (Fig. S3).
Functional Analysis of Genes Necessary for Nonselective Autophagy in
M. oryzae. Analysis of the M. oryzae genome sequence (3) provided
evidence for the presence of 23 autophagy-related genes, which are
into those that putatively play a role in the initiation of autophagy
(MoATG1, MoATG13, MoATG17), nucleation (MoATG6), phago-
phore, and autophagosome expansion (MoATG3, MoATG4,
MoATG5, MoATG7, MoATG8, MoATG10, MoATG12, and
MoATG16), and recycling (MoATG2, MoATG9, MoATG15, and
MoATG18). The most significant differences compared to the S.
cerevisiae autophagy gene family was the absence of clear ortho-
and pexophagy pathways in yeast (10) but which are not found in
development of M. oryzae. (A) Conidia were harvested from a Guy-11 trans-
formant expressing a GFP:MoATG8 gene fusion, inoculated onto glass cover-
inoculation (error bars indicate ? 2 SE).
Cellular localization of autophagosomes during infection-related
www.pnas.org?cgi?doi?10.1073?pnas.0901477106Kershaw and Talbot
the genome sequences of filamentous fungi or other multicellular
eukaryotes, and ATG19, which is a Cvt pathway-specific receptor
protein confined to S. cerevisiae (18). Other S. cerevisiae genes
absent from the M. oryzae gene set were ATG14, which in yeast
encodes the autophagy-specific subunit of phosphatidylinositol
3-kinase complex (23), and ATG31, which encodes a protein that
interacts with Atg17p and Atg29p forming a complex involved in
localizing other Atg proteins to the phagophore assembly site (24).
We classified the M. oryzae autophagy gene set into those
predicted to be required for nonselective autophagy and those
necessary for pexophagy, mitophagy, or the Cvt pathway (Table
S2). To test the role of each gene in plant infection by M. oryzae, we
carried out targeted gene replacements using the ?ku70 mutant as
recipient strain. In this way, we were able to generate a set of 22
isogenic mutants differing with respect to a single ATG gene. Two
putative orthologues of ATG22 (25) were encoded in the M. oryzae
genome, precluding analysis of this gene function by a single gene
deletion. To check the efficacy of using the ?ku70 mutant, we also
that comparative phenotype analysis could be carried out (Fig. S1).
Phenotypic analysis of mutants generated in both genetic back-
grounds gave identical results.
To confirm the role of these genes in autophagy, the GFP-
deletion set and the distribution of autophagosomes was assessed
(19). In a M. oryzae ?Moatg4 mutant expressing GFP-MoATG8, we
found that autophagosomes did not accumulate in fungal spores or
appressoria and were very significantly reduced in number (Fig. 2;
and were excluded from vacuoles. MoATG4 encodes a cysteine
protease necessary for processing of Atg8 (26) and therefore
vacuole fusion (10, 11, 26). Consistent with this predicted role,
GFP-MoAtg8 puncta were significantly reduced in number (P ?
0.0001) in both appressoria and conidia of ?Moatg4 when com-
pared to Guy11 and excluded from vacuoles. Furthermore, conidia
did not collapse and die during appressorium formation, and there
was no intense burst of autophagic activity in appressoria. When
considered together, these results indicate that autophagy is ar-
rested by the absence of MoATG4-encoded cysteine protease,
disrupting appressorium maturation.
disease, spores were collected from ?Moatg1, ?Moatg2, ?Moatg3,
?Moatg4, ?Moatg5, ?Moatg6, ?Moatg7, ?Moatg8, ?Moatg9,
?Moatg10, ?Moatg12, ?Moatg13, ?Moatg15, ?Moatg16, ?Moatg17,
and ?Moatg18 mutants and used to inoculate 21-day-old seedlings
of the blast-susceptible rice cultivar CO-39 (Fig. 3). The isogenic
?ku70 mutant and the wild-type Guy11 strain were both able to
virulence. Only the ?Moatg13 and ?Moatg18 mutants were able to
cause any disease symptoms, and lesion numbers were significantly
0.01 respectively). Furthermore, cytological analysis of prepenetra-
tion structures revealed that conidial cell collapse was prevented in
all cases by inhibition of nonselective autophagy (Fig. 3). The loss
of rice blast symptoms was found to be due to impaired appresso-
rium function, although inoculation of wounded seedlings with
tissue, in addition to its requirement for cuticle penetration. The
?Moatg6 mutant showed reduced viability upon storage and a
severe impairment in sporulation. Complementation analysis was
performed on a subset of the ?atg mutants and in all cases tested
led to restoration of the wild-type phenotype (Fig. S1). We con-
clude that each individual gene product that is necessary for
nonselective fungal autophagy in M. oryzae is also required for rice
development of a ?Moatg4 mutant of M. oryzae. (A) Conidia were harvested
from a ?Moatg4 transformant expressing a GFP:MoATG8 gene fusion, inoc-
ulated onto glass coverslips and observed by epifluorescence microscopy at
the times indicated (Scale bars, 10 ?m.). (B) Bar chart showing mean autopha-
gosome numbers present in conidium, germ tube, and appressorium 0 h, 2 h,
4 h, 6 h, 10 h and 12 h after inoculation with ?Moatg4 mutant expressing a
GFP:MoATG8 (error bars indicate ? 2 SE).
Cellular localization of autophagosomes during infection-related
Kershaw and TalbotPNAS ?
September 15, 2009 ?
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Functional Analysis of Genes Associated with Selective Autophagy.
Our analysis of the M. oryzae genome sequence revealed the
presence of 6 genes specifically associated with pexophagy or
Cvt pathway in M. oryzae based on the absence of orthologues of
ATG19, ATG20, ATG21 and ATG23 genes (Tables S1 and S2). We
therefore carried out targeted gene deletions to generate
?Moatg11, ?Moatg24, ?Moatg26, ?Moatg27, ?Moatg28 and
disease symptoms were evaluated and quantified. We found that
?Moatg11, ?Moatg24, ?Moatg26, ?Moatg27, ?Moatg28 and
not affect conidial or appressorial autophagy (Fig. 4). We conclude
that selective autophagy (16, 17) is dispensable for appressorium-
mediated plant infection.
Autophagy is a cell survival response that is triggered normally by
starvation stress and used to recycle cytoplasm, organelles, and
proteins within cells (9–11). It is becoming increasingly clear,
however, that in addition to its homeostatic functions, autophagy
may be necessary for cellular differentiation, defense from infec-
tion, and many aspects of development in multicellular organisms.
In fungi, there are relatively few reports of autophagy (27), but the
capacity to invade heterogeneous, nutrient poor substrates strongly
suggests that autophagy may be fundamental to the fungal lifestyle.
incompatibility in Podospora anserina (28) and for metal ion
homeostasis in Aspergillus fumigatus (29).
in autophagic cell death of conidia during appressorium develop-
ment (7). ?Moatg8 mutants were unable to undergo conidial cell
collapse, and although they could form appressoria, these were
nonfunctional and unable to cause plant disease. MoATG8 has also
been shown to be involved in regulation of glycogen metabolism
during conidiogenesis (30), and ?Moatg8 mutants produce less
conidia than an isogenic wild-type strain of M. oryzae (7, 30). The
MoATG1 gene was independently identified as a differentially
expressed gene during appressorium formation and shown to be
necessary for pathogenicity (31). These previous studies did not,
however, investigate the precise onset or spatial pattern of auto-
able to demonstrate that autophagosomes are enriched in conidia
as soon as they begin to germinate, less than an hour after landing
on a rice leaf surface. The large burst of autophagic activity
continues in the conidium until it collapses and undergoes cell
death. Significantly, we also observed that autophagy also occurs in
the appressorium during its development and maturation. Auto-
phagosomes and are highly enriched in appressoria and a large
central autophagic vacuole acts as the lytic compartment in matur-
ing appressoria, consistent with previous studies of lipolysis during
appressorium maturation (32). During infection-related morpho-
nonselective autophagy renders M. oryzae unable to
cause rice blast disease. (A) Seedlings of rice cultivar
CO-39 were inoculated with uniform conidial suspen-
sions (1 ? 105ml?1) of ?ku70, Guy11 (wt) and autoph-
?Moatg5, ?Moatg6, ?Moatg7 ?Moatg8, ?Moatg9,
?Moatg15, ?Moatg16, ?Moatg17, and ?Moatg18.
Seedlings were incubated for 5 days to allow develop-
ment of disease symptoms. Very reduced symptom
development was observed on some plants sprayed
with ?Moatg18 conidia, but lesion density was signif-
icantly reduced compared to Guy-11 (P ? 0.001). All
pathogenic. (B) Conidia were germinated on hydro-
phobic glass coverslips and incubated for 24 h to form
appressoria. Micrograph shows conidial collapse in
Guy11. Conidia of macroautophagy mutants such as
?Mgatg4 and ?Mgatg8 did not show conidial cell
death (Scale bars, 10 ?m.)
Deletion of any of the 16 genes required for
uniform conidial suspensions of Guy-11, ?ku70, and autophagy mutants
?Moatg8, ?Moatg11, ?Moatg24, ?Moatg26, ?Moatg27, ?Moatg28, and
?Moatg29. Seedlings were incubated for 5 days to allow development of
disease symptoms. ?Moatg24, ?Mgatg26, ?Moatg27, ?Moatg28, ?Mgatg11,
and ?Moatg29 produced similar disease lesion density on rice seedlings to
and incubated for 24 h to form appressoria. Micrograph shows conidial
collapse in Guy11. Conidia of selective autophagy mutants such as ?Mgatg11
showed conidial cell death in contrast to ?Mgatg8 (Scale bars, 10 ?m.).
Genes involved in selective autophagy in M. oryzae are not required
www.pnas.org?cgi?doi?10.1073?pnas.0901477106Kershaw and Talbot
genesis in M. oryzae, autophagy is therefore necessary for pro-
grammed cell death in the conidium and for differentiation and
active growth in the appressorium. How autophagy occurs in cells
with such different fates in M. oryzae is an intriguing question.
Autophagy is normally considered a pro-survival response that is
essential for cells to contend with nutrient shortage in the extra-
cellular environment. Autophagy is therefore up-regulated when
the nutrient supply is insufficient to meet cellular energy demands
and when cells are exposed to different forms of stress (8–10).
Under these conditions, several studies have shown that autophagy
acts to protect cells from death in a variety of eukaryotic organisms
(8, 33). However, autophagy has also been shown to be a contrib-
uting factor in cell death (34–36), indicating that a nonapoptotic
programmed cell death pathway exists in eukaryotes that is depen-
dent on autophagy genes (8, 36). It is likely that these dual roles for
autophagy are highly context-dependent in most cases, dependent
on the prevailing extracellular environmental conditions. It is
such a way that it is necessary both for conidial cell death and also
for maturation and differentiation of functional appressoria. In-
duction of autophagy during infection-related development is de-
velopmentally regulated and requires the Pmk1 MAP kinase
pathway (6), but is also a consequence of starvation stress. The
purpose of autophagy is therefore to fuel infection-related devel-
opment in the absence of exogenous nutrients before entry into the
plant; for this reason it has to be tightly coupled to genetic control
of development of the appressorium, which occurs via the cell cycle
since completion of mitosis is a necessary prerequisite both for
appressorium maturation and conidial cell death.
We cannot, however, preclude the possibility that conidial cell
death also involves apoptosis and M. oryzae possesses 2 metacap-
sase-encoding genes that require functional analysis. It is clear,
however, that the absence of any component of the autophagic
machinery is sufficient to prevent both conidial collapse and
appressorium-mediated plant infection. Understanding the role of
TOR kinase in the initiation of infection-associated autophagy and
the potential interplay with cAMP-dependent protein kinase A
signaling, which has been shown to be necessary for appressorium
S. cerevisiae, protein kinase A and the Sch9 kinase, for instance,
cooperatively regulate induction of autophagy (14).
Our other major aim in this study was to test whether infection-
associated autophagy is a selective or nonselective process, which
could not be determined by analysis of MoATG8 (7). We decided
this correctly. The analysis of gene function in plant pathogenic
fungi has generally proceeded by the analysis of single genes using
targeted gene replacement normally to validate the role of a gene
product in pathogenesis (31). This process has been a powerful
means of identifying new virulence factors but has been less
successful in defining cellular processes critical for plant diseases to
occur. Furthermore, gene replacement is time-consuming, has
rather poor levels of efficiency, and has been carried out on an ad
hoc basis, with little further validation of predictions made from
initial studies. This means that while a large number of discrete
fungal genes are known to be necessary for plant disease, there has
been less associated new insight into the molecular basis of plant
in M. oryzae, we therefore generated a ?ku70 mutant and showed
that it significantly enhanced the frequency of homologous recom-
bination to 80%. A recent study has demonstrated that deletion of
the Ku80-encoding gene in M. oryzae has a similar effect (38), and
we were able to validate use of the ?ku70 mutant in this systematic
analysis of autophagy in M. oryzae.
Our results comprehensively demonstrated that nonselective
autophagy is necessary for rice blast appressoria to form. De-
letion of any of the 16 gene products necessary for macroauto-
of an impairment in appressorium function. The only mutants
that were able to cause any disease symptoms, ?Moatg13 and
?Moatg18, were severely reduced in virulence. The phospho-
protein Atg13 is part of a multiprotein regulatory protein
Atg1 protein kinase in S. cerevisiae and is necessary for induction
of autophagy (9). Atg13 mutants, however, show a reduction, but
not elimination of autophagy, and this may also be the case for
M. oryzae ?Moatg13 mutants, explaining the phenotype. Simi-
larly, Atg18 is involved in recycling of Atg9, together with a
number of other proteins (9) and may have a partially redundant
function in M. oryzae. Conidial collapse was impaired in all
macroautophagy mutants and cytological analysis of ?Moatg4
carrying the GFP-MoATG8 reporter confirmed disruption of
autophagosome generation and autophagy in both cellular com-
partments—conidia or appressoria. In contrast, deletion of any
of the 6 specific genes associated with pexophagy or mitophagy
did not affect fungal pathogenicity. We can conclude that these
selective forms of autophagy are therefore not necessary for
plant infection. The significance of peroxisome biogenesis to
appressorium function and fatty acid ?-oxidation to appresso-
rium physiology (39–41) predicts that pexophagy may play a role
in subsequent stages of plant tissue colonization following
breach of the cuticle. A recent study in the pathogenic fungus
Colletotrichum orbiculare, for example, suggests that pexophagy
is important in plant infection (42). Investigating the efficiency
of growth in plant tissue of ?Moatg11, ?Moatg24, ?Moatg26,
?Moatg27, ?Moatg28, and ?Moatg29 will therefore be impor-
tant, although they are clearly not severely impaired due to the
severe disease symptoms observed at 72–96 h. Our study also
11) is absent from M. oryzae, providing further evidence that it
is restricted to yeast (10, 11) and not present in multicellular
filamentous fungi (27).
In conclusion, we have provided the first genome-wide anal-
ysis of autophagy in a filamentous fungus and have validated the
importance of nonselective autophagy in the establishment of
plant disease by M. oryzae. Controlling the initiation of fungal
autophagy may provide an effective target for development of
new and novel antifungal strategies, given the fact that the plant
infection process is so sensitive to perturbation of this process.
Materials and Methods
Fungal Strains, Growth Conditions, and DNA Analysis. Growth, maintenance,
and storage of M. oryzae isolate, media composition, nucleic acid extraction,
and transformation were all as previously described (43). Gel electrophoresis,
restriction enzyme digestion, gel blots, and sequencing were performed by
using standard procedures (44).
were Ku70Ff and Ku70LFr to amplify a 1.0 kb region upstream from the start
codon and introduce a NdeI site at the 3? end and primers Ku7-RFf and
Ku70RFr to amplify a 1.0 kb region downstream of the gene and introducing
a NdeI site at the 5? end. The 2 flanking DNA fragments were cloned into
pGEM-T (Promega) and then the left flank excised with NdeI/NotI and cloned
resistance to sulfonylurea (44) was amplified as a 2.8 kb fragment using
primers SurF and SurR introducing an NdeI site to both ends of the amplicon.
This fragment was cloned into the NdeI site of pMG1 to create pMJG2. NotI
and ApaI restriction sites within the pGEM-T polylinker were used to liberate
presence of chlorimuron ethyl (100 ?gml?1). Two independent ?Moku70
deletion mutants were obtained as assessed by Southern blot.
Generation of ?Moatg Mutants. Targeted gene replacement of the M. oryzae
MoATG genes was performed using the split marker strategy (45). Vectors were
constructed using a hygromycinB resistance selectable marker, hph (46), for
transformation of ?Moku70. The hph gene cassette was cloned into pBluescript
Kershaw and Talbot PNAS ?
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vol. 106 ?
no. 37 ?
the primers used were M13 F with HY and M13R with YG, as described in ref. 45
and shown in Fig. S4. M. oryzae autophagy genes were identified by homology
to S. cerevisiae ATG genes obtained from the Saccharomyces genome database
(Department of Genetics at the School of Medicine, Stanford) (Table S1). The
sequence data for each M. oryzae autophagy gene was retrieved from the M.
nology, Cambridge, MA) (www.broad.mit.edu/annotation/fungi/magnaporthe)
and used to design specific primers (see Table S4). The M. oryzae ?ku70 mutant
was transformed with each Mgatg:hph deletion cassette (2 ?g of DNA of each
flank). Transformants were selected in the presence of hygromycin B (200 ?g.
mL?1). Two independent deletion mutants were obtained for each autophagy
gene as assessed by Southern blot. Complementation analysis was performed as
described in Fig. S1.
GFP:MoATG8 Gene Fusion Construction. The MoATG8 gene was amplified as a
at the ends of the fragment (See Table S2). The amplicon was digested and
cloned into pCB1532 (5), which carries a selectable marker bestowing resis-
tance to sulfonyurea. The promoter region of the MoATG8 gene was ampli-
NcoI sites. The fragment was sub cloned into pMJK142.2 in frame with sGFP
gene as a SpeI - NcoI fragment. MoATG8p:GFP was obtained by PCR using
primers ATG8p5 and GFPrev, and the 2.15bp amplicon was digested with
SpeI-ClaI and subcloned in-frame with the MoATG8 ORF. The resulting vector
pCB GFP-MoATG8 was transformed into Guy-11, ?pmk1 and ?Moatg4. Trans-
formants were selected in the presence of chlorimuron ethyl (100 ?g mL-1).
Light and Epifluorescence Microscopy. Epifluorescence microscopy to visualize
eGFP and MDC-stained samples was routinely carried out using a Zeiss Axioskop
2 microscope (Zeiss) with differential interference microscopy (DIC) used for
bright field images. For epifluorescence examination of the GFP:MoATG8 trans-
formants, conidia were incubated onto coverslips and placed onto a 2% agar
cushion, then observed using a IX81 motorized inverted microscope (Olympus)
equipped with a UPlanSApo 100X/1.40 Oil objective (Olympus). Excitation of
fluorescently-labeled proteins was carried out using a VS-LMS4 Laser-Merge-
System with solid state lasers (488 nm/50mW). 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 CoolSNAP HQ2, Roper Scientific). All parts of the
system were under the control of the software package MetaMorph (Molecular
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