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MoTlg2, a t-SNARE component is important for


formation of the Spitzenkrper and polar
deposition of chitin in Magnaporthe oryzae
ARTICLE in PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY JULY 2014
Impact Factor: 1.41 DOI: 10.1016/j.pmpp.2014.04.006

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Physiological and Molecular Plant Pathology 87 (2014) 9e18

Contents lists available at ScienceDirect

Physiological and Molecular Plant Pathology


journal homepage: www.elsevier.com/locate/pmpp

MoTlg2, a t-SNARE component is important for formation of the


Spitzenkrper and polar deposition of chitin in Magnaporthe oryzae
Yu-Shan Zuo a, Jun Yang a, Da-Wei Wang a, Dan He a, Yu Chu a, Xiao-Lin Chen a, Wei Zhou a,
Tom Hsiang b, You-Liang Peng a, *
a
b

MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, China Agricultural University, Beijing 100193, China
School of Environmental Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2 W1

a r t i c l e i n f o

a b s t r a c t

Article history:
Accepted 28 April 2014

T-SNAREs are a family of conserved proteins involved in intracellular transport of membrane-coated


cargo among subcellular compartments. In this study, we identied a putative t-SNARE gene, MoTLG2,
in Magnaporthe oryzae via insertion mutagenesis. Deletion of MoTLG2 resulted in slower vegetative
growth and less conidiation relative to the wild-type strain, but the DMotlg2 null mutant was as virulent
as the wild-type strain. MoTlg2 has 30% overall amino acid identity with Saccharomyces cerevisiae Tlg2,
and rescued the defect of monensin de-sensitivity in the yeast strain where TLG2 had been deleted. More
importantly, apical regions of the hyphae of the DMotlg2 null mutant were only weakly stained by FM464, which was reported as an excellent vesicle tracer, suggesting that the Spitzenkrper was not well
formed in the DMotlg2 null mutant. In addition, more uneven lateral deposition of chitin was observed in
the cell wall of vegetative hyphae of the DMotlg2 null mutant. Taken together, this study shows that the
t-SNARE Tlg2 is important for both vegetative hyphal growth and conidiation, but dispensable for plant
infection in lamentous fungi, and suggests that Tlg2 is important for formation of the Spitzenkrper and
polar distribution of chitin.
2014 Elsevier Ltd. All rights reserved.

Keywords:
SNARE
Tlg2
Cell polarity
The rice blast fungus

Introduction
SNAREs are a large protein superfamily that mediates membrane fusion in almost all trafcking steps of the secretory and
endocytic pathways [1]. Based on localization in donor or
acceptor compartments, SNAREs can be divided into two major
subgroups, v-SNAREs (vesicle-membrane SNAREs) that are associated with vesicles, and t-SNAREs (target-membrane SNAREs) that
are associated with target compartments [2]. v-SNAREs usually
consist of a tail-anchored SNARE having one SNARE motif, and tSNAREs usually have additional two or three polypeptides [3]. A
heterodimeric t-SNARE is usually comprised of a member of the
syntaxin subfamily that contributes one SNARE motif as the tAbbreviations: ATMT, Agrobacterium tumefaciens mediated-transformation; BIC,
biotrophic interfacial complex; bp, base pair(s); CFW, calcouor white; CM, complete media; HAP, high afnity purication; HBSS, Hanks balanced salt solution;
hph, hygromycin phosphotransferase gene; kb, kilobase pair(s); ORF, open reading
frame; OTA, oatmeal tomato agar; SC, synthetic complete media; SNARE, soluble Nethylmaleimide-sensitive-factor attachment protein receptors; TAIL-PCR, thermal
asymmetric inter-laced polymerase chain reaction; UDM, uracil-decient media.
* Corresponding author. Tel.: 86 10 6273 2541; fax: 86 10 6273 3607.
E-mail address: pengyl@cau.edu.cn (Y.-L. Peng).
http://dx.doi.org/10.1016/j.pmpp.2014.04.006
0885-5765/ 2014 Elsevier Ltd. All rights reserved.

SNARE heavy chain, and a member of the SNAP-25 subfamily that


contributes two SNARE motifs as two t-SNARE light chains. When
membrane fusion occurs, interactions between v-SNARE and tSNARE lead to the vesicle and target compartment joining together
and forming a trans-SNARE complex called SNAREpin, where the
four twisted parallel SNARE motifs bundle together. Formation of
this bundle is thought to provide energy to catalyze the fusion of
vesicles within the target membranes [1,4e7].
Hyphal tip growth is a major distinguishing feature of lamentous fungi. There are a number of processes that are well coordinated to create the tapered morphology of hyphal tips. Among
them are highly polarized apical synthesis and secretion, which
require the SNAREs to cause the fusion of vesicles carrying cell wall
components [8]. The Spitzenkrper, a structure commonly detected
in growing hyphal apices and marked with an aggregate of vesicles,
is supposed to act as a vesicle supply center for generating the
exocytosis gradient in lamentous fungi [9]. In Aspergillus oryzae,
SNAREs destined to be recycled to the tip by endocytosis are mostly
internalized from the subapical tip region of plasma membranes to
the endocytic recycling compartment, and thereafter are transported to the Spitzenkrper to complete the recycling of certain
components required for hyphal tip growth [10]. However, not all

10

Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

tip-growing cells contain the Spitzenkrper in their apices [11], and


the relationship between the Spitzenkrper and SNAREs is still not
clear.
Over the past decades, SNAREs have been widely studied in
different eukaryotic organisms, and more than 60 SNAREs in yeast
and mammalian cells and 21 putative SNAREs in A. oryzae have
been characterized [1,7,12,13]. In the pathogenic basidiomycetous
fungus Ustilago maydis, a t-SNARE Yup1, has been found to mediate
endocytic recycling through early endosomes, and is required for
hyphal morphogenesis and pathogenesis [14,15]. Although
increasing numbers of studies focus on SNAREs, the role of these
proteins in asexual development and pathogenesis in lamentous
fungi remains to be investigated.
The haploid ascomycetous fungus Magnaporthe oryzae is the
causal agent of rice blast that occurs throughout the world and has
been developed as a model system to investigate interactions between pathogens and host plants [16e19]. Whole genome
sequencing revealed multiple SNAREs in the M. oryzae genome.
Two SNARE genes from M. oryzae, MoSEC22 and MoVAM7, played
important roles in growth, differentiation, and virulence [20,21].
Another t-SNARE gene MoSSO1 was also recently reported to be
required for full virulence and normal development of the BIC for
accumulation of effectors [22]. However, the roles of other putative
SNARE genes in M. oryzae have not been reported. In this study, we
identied a t-SNARE gene MoTLG2 from M. oryzae. This gene was
important for vegetative hyphal growth and conidiation in
M. oryzae and could substitute for TLG2 to restore monensin
resistance in deletion mutants of Saccharomyces cerevisiae. In
addition, we found that deletion of MoTLG2 from M. oryzae affected
formation of the Spitzenkrper and distribution of chitin among
hyphal compartments.

that a single copy of T-DNA was integrated into the genome of the
mutant (Fig. 1B).
Through TAIL-PCR, the sequences anking the integrated T-DNA
were obtained from SX1548. Sequence analysis revealed that the
anking sequences of the integrated T-DNA were continuous, and
the T-DNA was integrated at 550 bp upstream of the translation
start site of the predicted gene MGG_06883.6 (named as MoTLG2 in
this study) (Fig. 1C). In the genome of the wild-type P131, it has one
copy of MoTLG2 by DNA gel blot analysis (Fig. S1). The full-length
cDNA of MoTLG2 was isolated, and it contains one 72 bp intron
compared to the genomic DNA (Fig. 1C).
MoTLG2 encodes a protein with 357 amino acid residues, which
is similar to syntaxin that is thought to facilitate t-SNARE complex
formation and mediate fusion of endosome-derived vesicles with
the late Golgi [24]. The N-terminus of MoTlg2 was predicted to
contain a syntaxin homologous domain and an SNARE domain
(known as H3), and an N-terminal regulatory domain (Habc). The
C-terminus of MoTlg2 contained an SNARE motif, and the central
position (0-layer) of the heptad repeats of the SNARE motif was a
glutamine (Gln) residue, indicating that MoTlg2 belongs to the QSNARE superfamily [25]. The homologs for MoTlg2 are widely
distributed among eukaryotic organisms, including fungi, plants,
and animals. A phylogenetic tree was constructed based on MoTlg2
and its homologous proteins (Fig. 2).
To conrm that the phenotype defects in SX1548 were due to
disruption of MoTLG2, a gene complementation vector pHBP10
containing MoTLG2 and its 2.5-kb upstream sequence was constructed and introduced into the mutant. All the resulting 25
neomycin-resistant transformants, including TMT4, showed the
wild-type colony phenotype (Table 1).
MoTLG2 is important for vegetative hyphal growth and conidiation

Results
Identication of MoTLG2 from a hyphal growth defective mutant
SX1548, a mutant defective in hyphal growth was isolated from
an ATMT mutant library of the wild-type strain P131 that was
generated in a previous study [23]. The colony size of this mutant
was 88% of the wild type (Table 1), and the colony margin of the
mutant was more compact (Fig. 1A). A crossing was made between
this mutant (MAT 1-1) and a wild-type strain S1528 (MAT 1-2).
From the crossing, 39 progeny were obtained, including 21
hygromycin-resistant progeny that grew as slowly as the mutant
parent, and 18 hygromycin-sensitive progeny that formed wildtype colonies. These results suggested that the mutant had a single T-DNA insertion and indicated that the phenotypic change in
the mutant co-segregated with the hygromycin-resistance marker.
DNA gel blot analysis of the genomic DNA from SX1548 conrmed

To further characterize MoTLG2, a gene replacement vector pYZ1


was constructed by replacing the ORF of MoTLG2 with the
hygromycin cassette (Fig. 3A). After linearization by NotI, pYZ1 was
introduced into wild-type P131. One transformant, DMT1 was
conrmed as the DMotlg2 null mutant by PCR with primer pair Z9/
Z10 and Z11/Z12 (data not shown) and by DNA gel blot (Fig. 3B).
Loss of MoTLG2 transcript was further conrmed by RT-PCR with
the primer pair in1/in2 (Fig. 3C).
Similar to the ATMT mutant SX1548, the DMotlg2 null mutant
DMT1 also grew slower. After culture on OTA at 28  C for 5 days, the
colony diameter of DMT1 was 34 mm. Under the same conditions,
the wild-type strain P131 grew signicantly larger with a 40-mmdiameter colony (Fig. 3D; Table 1). Moreover, the margin of the
mutant DMT1 colony was more compact and clearer, and the
mycelium was darker (Fig. 3D) than the wild type. While the wild
type produced 8  107 conidia per plate after 4 days, the DMotlg2

Table 1
The diameter of colonies (mm) grown on different media for 5 days.
Strains

OTAa

P131
SX1548
TMT4
DMT1
CMT1

40.0
32.5
40.0
31.0
40.0

a
b
c
d
e
f
g







0
0.5
0.4
0.9
0.3

CMb

CM monensinc

CM CFWd

CM SDSe

CM CRf

38.8  0.5
eg
e
29.0  0.5
38.9  0.5

22.0  0.9
e
e
14.3  0.5
21.7  0.9

35.3  0.2
e
e
21.7  0.4
35.0  0.5

21.3  0.6
e
e
12.7  0.6
21.0  0.7

36.7  0.9
e
e
27.7  0.5
36.5  0.8

OTA oatmeal tomato media.


CM complete media.
CM monensin complete media with 50 mM monensin.
CM CFW complete media with 200 mg/ml calcouor white.
CM SDS complete media with 0.01% sodium dodecyl sulfate.
CM CR complete media with 200 mg/ml Cong Red.
The dash e represents not tested. The means and standard deviations were calculated from three experiments with four replicates per experiment.

Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

11

Fig. 1. An ATMT mutant SX1548 disrupted in MoTLG2. (A) Colonies of the wild-type strain P131 and the mutant SX1548 cultured on OTA for 120 h. (B) DNA gel blot analysis of
genomic DNA of P131 and SX1548 probed with the hygromycin resistance gene. The estimated sizes of each band are labeled at right in kb. E, EcoRI; H, HindIII. (C) Schematic
diagram showing T-DNA integrated into the MoTLG2 (MGG_06883.6) genomic region. Black rectangles represent the exons. L and R represent the left and right border of T-DNA
(5.0 kb), respectively, and the integration site of T-DNA was shown based on the translation start site of MoTLG2. The recognition sites for EcoRI (E) and HindIII (H) are labeled, and
the calculated sizes of the fragments digested with EcoRI and HindIII in the wild-type P131 were shown above the horizontal lines.

null mutant DMT1 only produced 4  107 conidia per plate in the
same time period. Re-introduction of the wild-type MoTLG2 into
the DMotlg2 null mutant DMT1 could complement all of these
defects (Table 1). These data indicated that MoTLG2 was important
for vegetative hyphal growth and conidiation.

MoTLG2 could substitute the function of TLG2 in S. cerevisiae


A BLAST search indicated that in S. cerevisiae, no homolog of
MoTlg2 other than Tlg2 had greater than 30% amino acid sequence
identity with MoTlg2. This led to the speculation that MoTlg2 may
have similar functions as Tlg2 in S. cerevisiae. Tlg2 is important in
S. cerevisiae for tolerance to monensin, which is a Na/H ionophore blocking intracellular transport in both trans-Golgi and cisGolgi compartments [26]. To test this hypothesis, the construct
containing the full-length cDNA of MoTLG2 driven by the galactoseinducible GAL1 promoter in pYES2 was introduced into the Dtlg2
null mutant DYMT1, and two independent transformants, CYMT4
and CYMT5, were obtained. As shown in Fig. 4, the Dtlg2 null
mutant DYMT1 grew slower and smaller than the wild-type
S. cerevisiae strain BY4741 on the SC medium with 50 mM monensin dissolved in ethanol [26], although the two strains grew
similarly on SC medium amended with the same amount of
ethanol, conrming the previous ndings that Tlg2 is important for
tolerance to monensin [27]. However, the transformants CYMT4
and CYMT5 expressing MoTLG2 in DYMT1 could complement the
growth defects of DYMT1 on SC medium with 50 mM monensin
(Fig. 4B), indicating that MoTlg2 was a functional counterpart of
Tlg2 in M. oryzae.
MoTlg2 is important for transport among Golgi apparatus

Fig. 2. MoTlg2 orthologous are widely distributed among eukaryotic organisms.


Phylogenetic analysis of MoTlg2 from M. oryzae and orthologous proteins PaTlg2
(Podospora anserine, XP_001912046), NcTlg2 (Neurospora crassa, CAD36979), GzTlg2
(Gibberella zeae, XP_382066), SsTlg2 (Sclerotinia sclerotiorum, XP_001589036), AnTlg2
(Aspergillus niger, XP_001402063), UmTlg2 (Ustilago maydis, XP_762319), SpTlg2
(Schizosaccharomyces pombe, CAB90150.1), Tlg2 (Saccharomyces cerevisiae,
EEU06808.1), Sx41 (Arabidopsis thaliana, NP_198050), HsSx16 (Homo sapiens,
AAC05647), MmSx16 (Mus musculus, NP_001095893), DmSx16 (Drosophila melanogaster, NP_523420), and CeSx16 (Caenorhabditis elegans, AAD31934). Sequence
alignments were performed by using the Clustal_W program and the phylogenetic tree
was viewed by using Mega 4.0 program neighbor-joining algorithm. The numbers at
nodes represent the percent branch support out of 1000 bootstrap replicates, and the
scale bar indicates the number of amino acid differences per site. The tree was rooted
on the animal clade at the bottom.

In S. cerevisiae, Tlg2 is involved in retrograde transport from


endosome to Golgi, for maintaining Golgi at a low pH, and
monensin is an inhibitor of retrograde transport among Golgi
apparatus [26]. To address whether MoTlg2 has a similar function as Tlg2, we compared colony growth of the DMotlg2 null
mutant DMT1 with the wild-type P131. The colony diameters of
DMT1 on CM plates with and without 0.5 mM monensin were
1.4 cm and 2.9 cm, respectively (Table 1). Under the same conditions, the colony diameters of P131 were 2.2 cm and 3.9 cm,
respectively (Table 1). These data indicated that DMT1 was more
sensitive to monensin. The growth inhibition of DMT1 on 0.5 mM

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Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

Fig. 3. Generation of the MoTLG2-deleted mutants. (A) The MoTLG2 gene replacement strategy. The upstream and downstream anking sequences were amplied with primer pairs
Z5/Z6 and Z7/Z8 from the wild-type P131 and sub-cloned into the pYZ1. The hygromycin-resistant genes hph was put between the two anking sequence to replace MoTLG2. The
probe was the fragment amplied with primer pair Z5/Z6. The calculated sizes of the fragments hybridized with probe A in the wild-type P131 and the DMotlg2 null mutant DMT1
were shown above the horizontal lines. Restriction enzymes were used as follows: H, HindIII; P, PstI; S, SpeI; SA, SalI, X, XhoI. (B) DNA gel blot of XhoI-digested genomic DNA of the
wild-type P131 and the DMotlg2 null mutant DMT1. Genomic DNA was hybridized with probe A, and the estimated size of each band is labeled at right in kb. (C) RT-PCR analyses
were used to conrm the deletion and rescue events of the MoTLG2 transcripts from the wild-type P131, the DMotlg2 null mutant DMT1, and one transformant, CMT1, expressing the
wild-type MoTLG2 in DMT1. Actin was used as the constitutive control to calculate relative expression. (D) Colonies of the wild-type P131 and the DMotlg2 null mutant DMT1
cultured on OTA for 120 h.

monensin was 52%, which was signicantly higher than the wildtype P131 at 46% (Fig. 5A). Moreover, the transformant CMT1
expressing the wild-type MoTLG2 in DMT1 exhibited the wildtype phenotype for monensin tolerance. Therefore, MoTlg2
likely plays important roles in retrograde transport among Golgi
apparatus.
Deletion of MoTLG2 alters cell wall integrity
Because Tlg2 is supposed to be involved in mediating trafcking
of chitin synthase III in S. cerevisiae [28], we investigated the effects
of various cell-wall perturbing agents on the DMotlg2 mutant
DMT1. As shown in Table 1, DMT1 showed more sensitivity to 0.01%
SDS and 200 mg/ml CFW in comparison to the wild type, and the
growth inhibition of DMT1 was much higher than the wild type
under these conditions (Fig. 5A). However, there was no signicant

difference in growth between DMT1 and the wild type on CM plates


containing Congo Red (Table 1). To visualize differences on chitin
distribution between P131 and DMT1, vegetative hyphae of each
strain were stained with CFW and at least 50 hyphal tips were
measured. Intriguingly, chitin distribution in DMT1 was visually
different from that in the wild type. In all of the wild-type hyphae,
septa and hyphal tips had stronger staining with CFW, and the
hyphal compartments had weak and uniform uorescence signals.
But in over 90% of DMT1 hyphae, which showed fainter background
staining throughout the hyphal strand, there were patches of bright
uorescence observed on the lateral walls away from septa
(Fig. 5B). Since CFW is a uorescent dye that intercalates with
nascent chitin chains, these ndings suggest that altered and
generally decreased distribution of chitin on cell walls of vegetative
hyphae in the DMT1 mutant was due to aberrant activities during
cell wall synthesis.

Fig. 4. MoTLG2 rescued S. cerevisiae Dtlg2 mutant DYMT1 and conferred monensin resistance. DYMT1 was transformed with the pYES2-MoTLG2 construct, and two resulting
transformants, CYMT4 and CYMT5, were used for analyses. Serial dilutions of DYMT1, CYMT4, and CYMT5 (106, 105, 104, 103, and 102 cells/ml) were inoculated on SC plates without
(left panel) or with (right panel) monensin (50 mM) and cultured at 30  C for 2 days. As control, the wild-type S. cerevisiae strain BY4741 was transformed with the empty pYES2
vector.

Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

13

Fig. 5. MoTLG2 contributes to tolerance to monensin, CFW, and SDS and affects distribution of chitin. (A) Growth inhibition of P131, DMT1, and CMT1 by different chemicals. Each of
strains was cultured in CM containing one of the chemicals: 0.5 mM monensin, 200 mg/ml CFW, 0.01% SDS, or 20 mg/ml Congo Red for 5 days, and was compared with the growth in
CM. The means and standard deviations were calculated from three replicates. (B) Deletion of MoTLG2 altered the distribution of chitin within the cell compartments. In P131 and
CMT1, CFW uorescence was mainly distributed at the hyphal and septal apices (arrow heads). In the DMotlg2 null mutant DMT1, uorescence was detected not only at the growing
apices, but also at the lateral walls along the hyphal axes (white arrow). Bar 25 mm.

Deletion of MoTLG2 affects formation of the Spitzenkrper


In S. cerevisiae, Tlg2 as an SNARE participates in endocytosis
[29]. To determine whether MoTlg2 was also involved in the process, hyphal tips of the wild-type P131, the DMotlg2 mutant DMT1
and the rescued mutant CMT1 were compared by staining with
FM4-64, which has been reported as an excellent dye for endocytic
vesicles [30,31]. For the wild-type P131 and the rescued mutant
CMT1, 60 hyphal apexes were measured and all of them showed
strong staining of FM4-64 (Fig. 6). In contrast, none of the 60 hyphal
apexes of DMT1 were well stained by FM4-64 (Fig. 6), indicating
that the apical vesicle cluster or the Spitzenkrper was not formed
in the hyphal apex of the DMotlg2 mutant.
Deletion of MoTLG2 delays appressorium formation
To investigate the role of MoTLG2 in infection-related processes,
we inoculated the conidia of the wild-type P131 and the DMotlg2
mutant DMT1 onto hydrophobic cover slips and onion epidermal
cells. There was no signicant difference in conidial germination
rate on cover slips (Fig. 7A). At 4 h after inoculation (hpi) on cover
slips, 51% of the germinated conidia in the wild-type could form
appressoria. However, under the same conditions, only 38% of the
DMT1 germinated conidia formed appressoria (Fig. 7A and B).
DMT1 initially lagged behind the wild type, but by 24 hpi, DMT1

formed as many appressoria as the wild type. These results suggest


that MoTLG2 is important for appressorial formation. Interestingly,
about 71% of DMT1 conidia formed curved germ tubes, whereas no
curved germ tubes formed by the wild type (Fig. 7A), suggesting
that MoTLG2 might be related to abnormal surface recognition. We
also observed that DMT1 developed as many bulbous infection
hyphae as the wild type inside onion epidermal cells (data not
shown), indicating that MoTLG2 plays some minor roles in development of infection hyphae.
MoTLG2 is dispensable for plant infection
To determine if MoTLG2 might be essential or important for
plant infection, the conidia of the wild-type P131, the DMotlg2
mutant DMT1, and the rescued mutant CMT1 were sprayed onto
seedlings of rice cultivar LTH. As shown in Fig. 8, numerous typical
disease lesions were formed on inoculated leaves, but no signicant
differences were observed between the three strains. These results
indicated that MoTLG2 was not important for plant infection.
Discussion
Over the past decades, the rice blast fungus M. oryzae has been
developed as a model fungus for studying the interactions between
pathogen and host and infection-related morphogenesis [16e19].

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Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

Fig. 6. MoTLG2 is important for formation of the Spitzenkrper at hyphal tips. The wild-type P131, the DMotlg2 null mutant DMT1, and the transformant CMT1 expressing the wildtype MoTLG2 were stained with the endocytic tracer FM4-64. The wild-type strain (P131) and the rescued mutant (CMT1) show the presence of intact the Spitzenkrpers (arrow
heads) at the hyphal tips, but the DMotlg2 null mutant seems to have lost the Spitzenkrper (white arrow). The photographs were taken with uorescent microscope by 400- (left
panel) and 1000-fold (right panel) magnication. Bar 25 mm (left panel) and 5 mm (right panel), respectively.

By using both forward and reverse genetics approaches, hundreds


of genes have been cloned from M. oryzae, and these genes can be
mainly classied into three major categories based on their roles on
development and pathogenicity: vegetative hyphal growth, conidiation, and plant infection [32]. In this study, we identied a gene,
MoTLG2, encoding a putative t-SNARE from an ATMT mutant.
MoTlg2 contains two highly conserved domains: one putative
syntaxin homology domain at its N-terminal and one SNARE motif
at its C-terminal [7,33] (Fig. S2). Based on sequence comparisons,
MoTlg2 was classied as Qa-SNARE, which contains a conserved
glutamine [4,7,25]. MoTlg2 shares an overall of 30% identity with
S. cerevisiae Tlg2, and the identity between MoTlg2 and Tlg2 within
the syntaxin domain (pfam00804) and the SNARE domain
(pfam05739) was as high as 55% and 56%, respectively. The fulllength cDNA of MoTLG2 can rescue the defects of the Dtlg2 null
mutant for monensin tolerance, indicating that MoTlg2 may
perform a similar function as Tlg2 in M. oryzae. In S. cerevisiae, Tlg2
is involved in retrograde transport from endosome to Golgi for
maintaining Golgi at a relative low pH, and is important for resistance to monensin, an ionophore that interferes with retrograde
transport among Golgi apparatus [26,34]. Consistently, the DMotlg2

null mutant showed more sensitivity to monensin and a dramatic


reduction in colony growth in comparison to the wild-type
M. oryzae. Deletion of MoTLG2 also leads to altered distributions
of chitin and fewer the Spitzenkrpers in the apices. Unfortunately,
no uorescent signals could be observed when fusing MoTLG2 with
GFP at its N- or C-terminus, although the corresponding transformants could be restored to the wild-type level of hyphal growth
and conidiation. Similar to the previous ndings on Com1, we
speculated that the expression levels of the GFPeMoTLG2 and
MoTLG2eGFP constructs were too low to be detected, or that fusion
of MoTLG2 might have an adverse effect on the folding and uorescence of the GFP fusion protein [35].
Recently, two SNARE genes from M. oryzae, MoSEC22 and
MoVAM7, were reported to play important roles in hyphal growth,
differentiation of infection-related morphogenesis, and pathogenesis [20,21]. In S. cerevisiae, Sec22 functioned in both anterograde
and retrograde trafc between the ER and the Golgi apparatus, and
Vam7 played a role in vacuole formation and assembly [36e38].
Moreover, a t-SNARE MoSso1 was reported to be required for full
virulence and normal development of the BIC [22]. In S. cerevisiae,
Sso1 was involved in fusion of secretory vesicles at the plasma

Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

15

M. oryzae and other pathogenic lamentous fungi may yield


important insights.
Cell polarization is important for many cellular processes in both
unicellular and multi-cellular organisms, including localized
membrane growth, directional cell migration, and differentiation
[33]. Small-bud formation of the budding yeast S. cerevisiae requires Tlg2, and there is a mechanism that promotes polarized
growth by local recycling of endocytic vesicles [40]. Because of
advantages of its staining pattern, greater photo-stability and low
cyto-toxicity, the endocytic tracer FM4-64 was found best for imaging dynamic changes in size, morphology and position of apical
vesicle clusters within growing hyphal tips. FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the
Spitzenkrper, the vacuolar membrane, and mitochondria [31]. We
observed that the hyphal tips of the DMotlg2 null mutant were less
stained by FM4-64, which implies that MoTLG2 was required for
maintaining the normal formation of the Spitzenkrper. Because
MoTLG2 is dispensable for virulence, we speculate that formation of
the Spitzenkrpers was also dispensable for plant infection.
Chitin is a crucial component of fungal cell wall, and synthesis of
chitin depends on the activity of chitin synthases [41]. In
S. cerevisiae, Tlg1 mediates trafcking of the enzyme Chs3 to
polarized growth sites. Chs3 accounts about 90% of chitin synthesis
activities, and Tlg2p can also perform a similar function with lower
efciency [28] with considerable overlap between the distributions
of Tlg1 and Tlg2 [24,28]. Deletion of MoTLG2 leads to abnormal
accumulation and uneven lateral distribution of chitins on cell
walls of vegetative hyphae. However, it seems no obvious differences in chitin distribution between the wild type and the DMotlg2
null mutant during appressorium formation (Fig. S3). Because there
are seven genes encoding chitin synthases in M. oryzae [42,43],
more investigation of the expression pattern and sub-cellular
localization of these genes, especially the homologous gene of
CHS3, in the DMotlg2 null mutant, is needed. Based on the observation that abnormal distribution of chitin and formation of the
Spitzenkrpers in the DMotlg2 null mutant, we speculate that formation of the Spitzenkrpers might contribute to normal distribution of chitin in the apex.
Materials and methods
Fungal strains and growth conditions

Fig. 7. Deletion of MoTLG2 results in delay on appressorium formation. (A) Microscopic


images of germinated spores and appressoria of the wild-type P131 and the DMotlg2
mutant DMT1 at 4, 6, 12, and 24 h post-inoculation on hydrophobic cover slips. The
percentage of appressoria formed by DMT1 was lower than that of the wild type at the
same time after inoculation, and DMT1 formed more curved germ tubes compared to
the wild type. The black arrow shows the formed germ tubes. Bar 25 mm. (B) Percent
appressorial formation of the wild-type P131 and the DMotlg2 mutant DMT1 on hydrophobic cover slips at the time points shown in (A). The means and SD were
calculated from over 100 conidia per sample in three independent replicates.

membrane and in vesicle fusion during sporulation [39]. In our


study, MoTLG2 was shown to be dispensable for virulence, although
it plays important roles in vegetative hyphal growth and sporulation. Moreover, two other SNARE genes, MoTLG1 and MoSSO2, were
preliminarily demonstrated to play similar roles as MoTLG2 in
asexual development and plant infection in M. oryzae (data not
shown). Together, we hypothesize that different SNARE genes play
various roles at different developmental stages of the infectionrelated processes. Dissecting their roles in plant infection by

M. oryzae isolate P131 was originally isolated from a rice eld in


Japan [44e46]. This isolate and all of its corresponding transformants generated in this study (Table S1) were cultured and
maintained on OTA at 25  C. Mycelia collected from 2-day-old
colonies in complete medium (0.6% yeast extract, 0.3% enzymatic
casein hydrolyzate, 0.3% acidic casein hydrolyzate, 1% glucose)
shaken at 150 rpm were used for isolation of fungal DNA and
protoplasts. Protoplasts were then transformed with a CaCl2/PEG
method [35]. Media were supplemented with 250 mg/ml hygromycin B (Roche, USA) or 400 mg/ml neomycin (Ameresco, USA) for
selecting hygromycin-resistant or neomycin-resistant transformants, respectively.
Molecular manipulation with nucleic acids
Genomic DNA was extracted from vegetative hyphae with the
CTAB protocol [47]. Standard molecular biology procedures were
followed for plasmid isolation, DNA gel blot analyses, and enzymatic manipulation with DNA [48]. Probes were labeled with the
Random Primer Labeling Kit (TaKaRa, Japan). Total RNA was
extracted with the Trizol reagent (Invitrogen, USA). Reverse

16

Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

were sequentially ligated into the SpeIePstI and HindIIIeSalI sites of


pKOV21. The vector pKOV21 was generated by sub-cloning a 1.4-kb
HpaI fragment with the bacterial hygromycin resistance gene from
the plasmid pCB1004 into EcoRV-digested pKN with neomycin
resistance to provide hygromycin resistance [55e57]. After transforming the linearized vector into the protoplasts of the wild-type
strain P131, hygromycin-resistant transformants were isolated and
tested for resistance to neomycin, and screened by PCR with the
primer pairs Z9/Z10 and Z11/Z12. To conrm deletion of MoTLG2,
15 mg XhoI-digested genomic DNA of the putative null mutant
DMT1 was gel blotted onto Nylon membrane (Amersham, USA),
and hybridized with the probe amplied by primer pair Z7/Z8 according to the manufacturers instruction (TaKaRa, Japan).
Complementation assay
For complementation assays, MoTLG2 along with its 2.5-kb upstream and 0.5-kb downstream sequences was amplied with
primers Z13/Z14 and cloned into the ApaI and HindIII sites of pKN to
generate pHBP10. Linearized pHBP10 was then used to transform
the DMotlg2 null mutant, and the transformants selected on CM
with 400 mg/ml neomycin were used for further phenotypic
analyses.
Microscopy
Fig. 8. MoTLG2 is dispensable for plant infection. Four-week-old seedlings of rice were
sprayed with 30 ml of 105 spores/ml of the wild-type P131, the DMotlg2 null mutant
DMT1, or transformant CMT1 expressing the wild-type MoTLG2 in DMT1. Infected
leaves were photographed at 7 days after inoculation.

transcription assays were performed by using the PrimeScript RTPCR Kit (TaKaRa, Japan). The actin gene (MGG_03982.5) was
amplied with primers Z1/Z2 [49], and used as an internal control
for semi-quantitative RT-PCR. All plasmid constructs were
sequenced with an ABI 3730 sequencer (SunBio, China). All primers
(Table S2) were synthesized and puried with the HAP approach
(Sangon, China). BLAST programs were used to search for MoTlg2
homologs at the NCBI and Broad databases, and the highest
matching sequences downloaded. Protein sequence alignments of
MoTlg2 and its closest matching sequences were done using ClustalX 2.0.11 [50]. Phylogenetic analyses were conducted using Mega
4.0 with the neighbor-joining algorithm [51]. Protein domains were
predicted by Pfam [52], and protein domain structures were illustrated by DOG 1.0 [53].
Identication of MoTLG2
The mutant SX1548 was obtained from a mutant library of
M. oryzae strain P131 that was generated by ATMT [23]. Flanking
sequences of the insertion site were isolated using the TAIL-PCR
method [54]. The PCR products were cloned into pMD 18-T Vector (TaKaRa, Japan) and sequenced. The sequences were used to
search against the M. oryzae database at the Broad Institute and
NCBI. A full-length cDNA fragment for MoTLG2 was amplied from
the wild-type strain P131 using primer pairs Z3/Z4 and cloned into
pMD19-T Vector (TaKaRa, Japan) to generate pMD-MoTLG2, and
then veried by sequencing.
Targeted gene deletion of MoTLG2
The MoTLG2 gene replacement vector pYZ1 was constructed by
using 1.32-kb upstream and 1.28-kb downstream sequences
around the predicted coding sequence. These two regions were
amplied with the primer pairs Z5/Z6 and Z7/Z8, respectively, and

Conidia were inoculated onto hydrophobic cover slips (VWR,


USA; 22  22 mm) in a humid chamber at 28  C, and were observed
after 0.5, 4, 12, and 24 h, under a Nikon Ni90 uorescence microscope (Nikon, Japan). For cell wall and septa staining, the strains
were inoculated onto OTA, and cover slips placed near the inoculum. After hyphal growth onto the cover slips, some slips were
removed and stained in 10 mg/ml CFW (Sigma, USA) for 1 min. The
cover slips were washed three times with sterilized distilled water
and observed under a Nikon Ni90 uorescence microscope using a
450e490 nm laser line.
For the FM4-64 (Invitrogen, USA) membrane-staining assay, a
staining solution of 7.5 mM dye was prepared in ice-cold HBSS
(0.14 g/L CaCl2, 0.40 g/L KCl, 0.06 g/L KH2PO4, 0.1 g/L MgCl2$6H2O,
0.10 g/L MgSO4$7H2O, 8.00 g/L NaCl, 0.35 g/L NaHCO3, 0.048 g/L
Na2HPO4, 1.00 g/L D-glucose) without magnesium or calcium and
kept on ice. Some cover slips with attached hyphae were removed
and quickly immersed in 10 ml of staining solution on ice separately for 1 min, after which, the stained material was xed in icecold 4% formaldehyde in HBSS for 10 min on ice. The stained cover
slips were rinsed twice with HBSS, and then observed under uorescence microscopy using a 565-nm laser line.
Plant inoculation
Conidia were harvested from 7-day-old OTA plates and adjusted
to 5  104 per ml in 0.025% Tween-20 solution. Four-week-old
seedlings of the rice cultivar LTH were used for infection assays.
Plant inoculation was performed by spraying the conidial suspension onto the intact rice leaves [45]. Photographs were taken seven
days after inoculation and later examined.
Yeast complementation assay
The full-length cDNA of MoTLG2 in pMD19-T was digested with
HindIII and EcoRI, and ligated into the yeast expression vector
pYES2 (Invitrogen, Carlsbad, CA, USA) as pYT5. The resulting
vector was transformed into an S. cerevisiae Dtlg2 mutant strain
(Acc. No: Y01709, BY4741: Mat a; his3D1; leu2D0; met15D0;
ura3D0; YOL018c::kanMX4) with the lithium-acetate method [9].

Y.-S. Zuo et al. / Physiological and Molecular Plant Pathology 87 (2014) 9e18

Yeast cells were incubated in liquid YPD medium (2% glucose, 2%


peptone, and 1% yeast extract) supplemented with the amino
acids required by the respective strains. Transformants were
selected at 30  C on UDM containing 0.08% Urea DO Supplement
(TaKaRa, Japan), 0.67% Difco Yeast Nitrogen Base w/o Amino
Acids (BD, USA), 2% dextrose and 1.4% Bacto agar (BD, USA). Two
of the resultant colonies, CYMT4 and CYMT5, were randomly
selected and grown on UDM or uracil plus minimal media with or
without the addition of uracil at 30  C and 37  C, respectively. Rich
media (YPD) and SC were prepared as described [58], but contained twice the recommended amount of leucine in the SC media. Monensin (Sigma, USA) was rst dissolved in ethanol, and
then added to either SC or CM plates to a nal concentration of
50 mM.
Acknowledgments
We thank Lu-Jia Peng for assisting in analysis of the ATMT
mutant SX1528, and EUROSCARF (EUROpean Saccharomyces Cerevisiae ARchive for Functional Analysis) at Frankfurt for providing
the wild-type yeast strain BY4741 and its null mutant strain with
deletion of TLG2. This work was supported by the 973 program
(Grant No. 2012CB114002) from the Ministry of Sciences and
Technology, China.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.pmpp.2014.04.006.
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