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Genome-wide functional analysis reveals that infection-associated fungal…
Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease
INTRODUCTION
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
Objectives
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 autophagy during their maturation
to define whether autophagy carried out by
M. oryzae
during plant infection is a
selective or a nonselective form of autophagy
to determine
why fungal autophagy is necessary
for rice blast disease
to define which
type of autophagy
takes place during plant infection
TOR kinase
regulates initiation of autophagy
leads to
formation of single membrane structure, the
phagophore
, which surrounds and engulfs cytoplasm, organelles, and other cellular components developing into a spherical, double-membrane autophagosome.
autophagasomes
expands
and then
fuses
with a vacuole, the lytic compartment (lysosome equivalent) of fungal cells,
sequestering
its contents and inner membrane for degradation by hydrolases
Selective forms of autophagy
degrade
peroxisomes
(pexophagy),
mitochondria
(mitophagy), and
endoplasmic reticulum
(reticulophagy)
occur during the
biosynthetic cytoplasm-to-vacuole-targeting (Cvt) pathway
S. cerevisiae
, which is used to transport the
inactive precursor of the vacuolar hydrolase aminopeptidase I
to the vacuole
require a distinct set of proteins, such as
Atg11
encodes a
peripheral membrane protein
that is the adaptor required for cargo loading in pexophagy and for delivery of
aminopeptidase I
to the vacuole in the
Cvt pathway
RESULT
Infection-Associated Autophagy Occurs in both Conidia and Appressoria of
M. oryzae
To determine the spatial and temporal dynamics of autophagosomes during appressorium development
GFP-MoATG8 gene fusion
Wild-type strain of
M. oryzae
Guy11 and also the
Moatg8
mutant
ATG8
encodes an ubiquitin-like protein that can be modified at its C terminus by (+) of phosphatidylethanolamine, linkedto the autophagosome membrane that necessary for
phagophore expansion during autophagosome formation
Autophagosome number within developing appressoria
increased
during appressorium maturation
I
ntense autophagic activity and vacuole expansion
was associated with mature appressoria
To investigate if autophagy specifically associated with appresorium development in
M. oryzae
Expressed
GFP-MoATG8 in a pmk1 MAP kinase mutant
that does not elaborate appressoria, nonpathogenic
In pmk1 mutant,
GFP-MoATG8-labeled autophagosomes
Present
in the conidium during germination but in small number
conidia of pmk1 mutant remained
intact
during germination and germ tube elongation = conidial cell death not occured when appressorium formation is not there
Infection-associated autophagy requires Pmk1 MAP kinase
& had to occurs with
appressorium-mediated plant infection within conidia
alliow for development of appressorium so that, further autophagic activity can occur.
Development of a Rapid Method for Gene Functional Analysis in
M.oryzae
.
used deletion of the BUF1 gene, which encodes tri-hydroxy-naphthalene reductase
as a visual test for the frequency of gene replacement
Buf1 mutants have a buff color compared to the olive green/gray color of wild-type M. oryzae cultures
identifying mutants was straightforward
tested the resulting strain for the frequency of targeted gene replacement
homologous gene replacement occurred at a frequency of 80% (n 100) in the ku70 mutant background
in
M. oryzae
, locus-dependent and ranges from 1% to 25%
deleted the ku70-encoding gene of M. oryzae
Growth rate, sporulation, and pathogenicity of the ku70
mutant were found to be unaltered by the mutation
mutant was selected as an entry strain for rapid evaluation of the function of new genes in
M. oryzae
Functional Analysis of Genes Necessary for Nonselective Autophagy in
M. oryzae.
Presence of 23 autophagy-related genes
could be separated into initiation of autophagy (MoATG1, MoATG13, MoATG17)
nucleation (MoATG6)
phagophore, and autophagosome expansion (MoATG3, MoATG4, MoATG5, MoATG7, MoATG8, MoATG10, MoATG12, and MoATG16)
recycling (MoATG2, MoATG9, MoATG15, and
MoATG18).
most significant differences compared to the
S.cerevisiae
autophagy gene family
:pencil2: absence clear orthologues (ATG20, ATG21, and ATG23) required for the selective Cvt and pexophagy pathways in yeast :pencil2:not found in the genome sequences of filamentous fungi or other multicellular eukaryotes, and ATG19,
:pencil2:absent from the M. oryzae gene set :pencil2:(ATG14), yeast encodes the autophagy-specific subunit of phosphatidylinositol 3-kinase complex :pencil2: 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
autophagy gene set required for nonselective autophagy and those necessary for pexophagy, mitophagy, or the Cvt pathway
Test role of each gene in plant infection by
M. oryzae
Targeted gene replacements using the delta ku70 mutant as
recipient strain.
Able to generate a set of 22 isogenic mutants differing with respect to a single ATG gene
2 putative orthologues of ATG22 encode M.oryzae genome
Test efficacy of using the ku70 mutant
deleted MoATG8, MoATG4, MoATG9, and MoATG12 in Guy11 - comparative phenotype analysis can be carried out
Role of these genes in autophagy test
GFPMoATG8 gene fusion was introduced into a subset of the ATG gene deletion set and the distribution of autophagosomes was assessed
M. oryzae delta Moatg4 mutant expressing GFP-MoATG8,
autophagosomes did not accumulate in fungal spores or
appressoria
Large aggregates of GFP-MoAtg8 were observed
and were excluded from vacuoles
MoATG4 encodes a cysteine protease necessary for processing of Atg8 required for phagophore initiation, autophagosome formation, and vacuole fusion
conidia did not collapse & die during appresorium formation and no intense burst of autophagic activity in appresorium
Nonselective autophagy is necessary for rice blast
disease
Spore collected used to inoculate 21-day-old seedlings
of the blast-susceptible rice cultivar CO-39
Atq mutants impaired with nonselective autophagy - nonpathogenic/ highly reduced in virulence
Only the Moatg13 and Moatg18 mutants able to cause any disease symptoms, and lesion numbers significantly reduced compared to the wild type
Cytological analysis of prepenetration structures - conidial cell collapse prevented in all cases by inhibition of nonselective autophagy
Functional Analysis of Genes Associated with Selective Autophagy.
Presence 6 gene associated with pexophagy or
mitophagy
no evidence of functional Cvt pathway in
M. oryzae
based on absence of orthologues of ATG19, ATG20, ATG21 and ATG23 genes
carried out targeted gene deletions
used to inoculate rice seedlings and
disease symptoms were evaluated and quantified
Moatg11, Moatg24, Moatg26, Moatg27, Moatg28 and Moatg29 mutants were able to cause rice blast disease and did not affect conidial or appressorial autophagy
Conclude
that selective autophagy is dispensable for appressorium mediated plant infection.
MATERIALS AND METHOD
3. Generation of Moatg Mutants
Targeted gene replacement of the M. oryzae
MoATG genes was performed using the split marker strategy
Vectors were
constructed using a hygromycinB resistance selectable marker, hph, for transformation of Moku70. hph gene cassette was cloned into pBluescript (Stratagene) as a 1.4 kb EcoRI-XbaI fragment
To amplify the split hph templates
the primers used were M13 F with HY and M13R with YG
M. oryzae autophagy genes were identified by homology
to S. cerevisiae ATG genes obtained from the Saccharomyces genome database. The
sequence data for each M. oryzae autophagy gene was retrieved from the M. oryzae genome database at the Broad Institute and used to design specific primers
The M. oryzae ku70 mutant
was transformed with each Mgatg:hph deletion cassette (2 ug of DNA of each flank)
Transformants were selected in the presence of hygromycin B (200 ug mL^-1)
Two independent deletion mutants were obtained for each autophagy gene as assessed by Southern blot followed by completion analysis
4. GFP:MoATG8 Gene Fusion Construction
The MoATG8 gene was amplified as a
1.6 kb fragment using primers ATG8.5 and ATG8.3. creating ClaI and XhoI sites at the ends of the fragment
The amplicon was digested and
cloned into pCB1532, which carries a selectable marker bestowing resistance to sulfonyurea
The promoter region of the MoATG8 gene was amplified as a 1.4 kb fragment using primers ATG8p5 and Atg8p3 creating SpeI and NcoI sites
The fragment was sub cloned into pMJK142.2 in frame with GFP 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. Transformants were selected in the presence of chlorimuron ethyl (100 ug mL-1)
2. Generation of the ku70 Mutant of M. oryzae
MoKu70 gene was identified from the published genome sequence and primers designed to amplify the 2 regions flanking the gene
primers used were Ku70Ff and Ku70LFr to amplify a 1.0 kb region upstream from the start codon.
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 into the vector with the right flank, giving pMG12.1
ILV1 allele conferring
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 the gene disruption cassette from pMJG2
Transformants were selected in the
presence of chlorimuron ethyl (100 ugml^-1)
Two independent Moku70
deletion mutants were obtained as assessed by Southern blot
5. 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 transformants, 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-MergeSystem 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
Devices).
1. Fungal Strains, Growth Conditions, and DNA Analysis
Growth, maintenance,
and storage of M.
oryzae
isolate, media composition, nucleic acid extraction and transformation
Gel electrophoresis,
restriction enzyme digestion, gel blots, and sequencing
Discussion
Autophagy is a cell survival response that is triggered normally by starvation stress and used to recycle cytoplasm, organelles, and proteins within cells.
Autophagy is necessary for cellular differentiation, defense from infection, and many aspects of development in multi cellular organisms.
Besides, autophagy is important in
heterokaryon incompatibility
in
Podospora anserina
and for
metal ion homeostasis
in
Aspergillus fumigatus
Based on previous work, M. oryzae demonstrated a role for
MoATG8
in autophagic cell death of conidia during appressorium development
MoATG8
has also been shown to be involved in regulation of glycogen metabolism during conidiogenesis
MoATG1
gene was independently identified as a differentially expressed gene during appressorium formation and shown to be necessary for pathogenicity
Moreover,
autophagosomes
and are highly enriched in appressoria and a large central autophagic vacuole acts as the lytic compartment in maturing appressoria, consistent with previous studies of lipolysis during appressorium maturation.
During, infection-related morphogenesis in
M. oryzae
, autophagy is necessary for programmed cell death in the conidium and for differentiation and active growth in the appressorium.
Autophagy are usually considered a
pro-survival response
that is essential for cells to contend with nutrient shortage, therefore up-regulated when the nutrient supply is insufficient