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Online Resource 1. Primers used in this work
Online Resource 2. Assignment for functional traits of HOG1 homologs in Pezizomycotina
Online Resource 3. Aligment of amino acid sequences of HOG1 homologs
Online Resource 4. Protoplasting efficiencies of the WT strain and the AlHOG1-disrupted mutants
Article title: Conserved and divergent roles of the HOG1 kinase of Alternaria longipes in mycelial and
conidial development, multi-stress responses, melanin production and pathogenicity
Journal name: European Journal of Plant Pathology
Author names: Zhiqun Yina, Wei Bib, Qili Mic, Ziteng Kanga, Chenjian Liua, Jinkui Yangb*, Yiyong
Luoa*
Affiliation:
a
Laboratory of Applied Microbiology, Faculty of Life Science and Technology, Kunming University of
Science and Technology, Kunming 650500, China
b
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan
University, Kunming 650091, China
c
Technology Center, China Tobacco Yunnan Industrial Co., Ltd., Kunming, 650231, China
Corresponding author: *E-mail address: [email protected] and [email protected]
Online Resource 1. Primers used in this work
a
Primers
Oligo sequence (5'–3')a
Description
AAP1
AAGAAGATCATGAAGCC
Amplify
AAP2
ACATCAT(CG)A(CT)(CT)TTCCA(ACGT)GT
region
TSP5-1
TGGTCCTTGGCTGAACTGTG
Amplify the 5' flanking
TSP5-2
CTGCTGGTAATCTCGAAAGTGG
unknown sequence
TSP5-3
TTCCGCCATGATTGTTGG
TSP3-1
TGCGATTCGTCCAGTCACTTC
Amplify the 3' flanking
TSP3-2
GAGGAGAAGTTTGACTGGTCGTTC
unknown sequence
TSP3-3
GTTGACACGTGGAAAATCATGA
HOG-5f
GTAACGCCAGGGTTTTCCCAGTCACGACGTACCCCACGACGTCCTACTC
Amplify the 5' flanking
HOG-5r
ATCCACTTAACGTTACTGAAATCTCCAACGGAGAACTGGTTCACGTGGT
fragment
HOG-3f
CTCCTTCAATATCATCTTCTGTCTCCGACGCGGAGGAGAAGTTTGACTG
Amplify the 3' flanking
HOG-3r
GCGGATAACAATTTCACACAGGAAACAGCAGCCATGTTTCCACTTCTGG
fragment
hphF
GTCGGAGACAGAAGATGATATTGAAGGAGC
Amplify the HPH cassette
hphR
GTTGGAGATTTCAGTAACGTTAAGTGGAT
YZZJ-F
GATACTACCGAGCCCCTG
Confirm
YZZJ-R
ACTTCTCCTCCGCAATCG
candidate strains
P-hogf
TCTTCCACCACCACAACC
Make Southern blot probe
P-hogr
AGACCGAAGTCGCAAATC
the
the
conserved
disrupted
The nucleotides marked with underlines are tails homologous to the HPH cassette and the linearized
vector pRS426.
Online Resource 2. Assignment for functional traits of HOG1 homologs in Pezizomycotina
Functional traitsa
Assignment
Sensitivity to salts (SA)
No=1
Yes=2
Sensitivity to sugars (SU)
No=1
Yes=2
Whether
No=1
Yes=2
Whether affects conidial
No=1
Yes=2
affects
conidial
germination rates (WACG)
Resistance to dicarboximide
numbers (WACN)
No=1
Yes=2
and phenylpyrrole (RDP)
Sensitivity to H2O2 (SH)
spores=3
Whether affects melanin
No=1
Yes=2
production (WAPP)
No=1
Yes=2
No=1
Yes=2
Sensitivity to menadione
(WAHG)
Whether related to carbon and
No=1
Yes=2
No=1
Yes=2
(SHSS)
a
affects
hyphal
No=1
Yes=2
Resistance to cell
Yes=2
Whether
wall
reduces
pathogenicity (WRP)
Yes and mutants cannot switch
to filamentous growth=3
No=1
Yes=2
perturbing agents (RCWP)
No=1
No and mutants resistance to
menadione=3
morphology (WAHM)
nitrogen sensing (WRCN)
Sensitivity to heat shock stress
Whether
Yes and mutants produce more
melanin =3
(SM)
Whether affects hyphal growth
Yes and mutants produce more
No and mutants sensitivity to
cell wall stress=3
No=1
Yes=2
Yes
and
virulence=3
The letters in brackets were abbreviation for the corresponding functional traits description.
mutants
loss
Online Resource 3. Alignment of amino acid sequences of HOG1 homologs
Online Resource 3 Aligment of amino acid sequences of three HOG1 homologs from the genus
Alternaria, AlHOG1 (GenBank: KT362180), AaHOG1 (GenBank: ADC35362) and AbHOG1
(GenBank: Q52PH6), as well as Saccharomyces cerevisiae HOG1 (GenBank: P32485) using the
DNAman software package. Identical and similar residues are shadowed in black and gray,
respectively. Important regions and sites are marked by two adjacent vertical lines or indicated with the
# sign above them, in the following order: the ATP binding region, the Lys and Asp active sites,
activation loop containing the TGY motif with the T and Y phosphorylation site residues marked by
black arrows, the CD domain responsible for binding Pbs2, and the second Pbs2-binding site,
containing motif necessary for HOG1 autophosphorylation marked by grey box. The annotations are
based on data from the literatures (Maayan et al. 2012; Murakami et al. 2008)
Online Resource 4. Protoplasting efficiencies of the WT strain and the AlHOG1-disrupted mutants
C-00 (WT)
AlHOG1Δ
Standard protoplasting conditions
Enzymolysis stepa
AlHOG1Δ
Low salt protoplasting conditions
2 hb
7.4×106c
6.0×106
2.4×105
0
3h
8.5×106
2.5×106
3.8×105
0
4h
1.8×107
1.9×106
5.5×106
0
5h
8.0×106
1.1×106
2.9×105
0
(2.3–12.0) ×106
0
(1.0–88.0)×104
0
Suspension stepd
a
C-00 (WT)
For standard conditions, 0.5–1.0 g young tender hyphae was incubated at 33 °C in 10 ml lytic solution
(0.7 M NaCl, 5.0 mg/ml Driselase, 12.5 mg/ml Snailase, 12.5 mg/ml Cellulase, 7.5 mg/ml
Lywallzyme); For low salt conditions, the lytic solution contained 0.35 M NaCl instead of 0.7 M NaCl.
b
The time of hyphae enzymolysis in lytic solution. cThe number of protoplasts (unit: n/ml) was counted
under a light microscope after 2–5 h enzymolysis. dProtoplasts were sequentially washed once with 0.7
M NaCl and SSTC (0.7 M NaCl, 1.2 M sorbitol, 10 mM Tris pH 7.5, 50 mM CaCl2), and suspended in
STC (SSTC without NaCl).