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