Supporting Information
Identification of Autophagy-related Genes ATG4 and ATG8 from
Wheat (Triticum aestivum L.) and Profiling of Their Expression
Patterns Responding to Biotic and Abiotic Stresses
Dan Pei, Wei Zhang, Hong Sun, Xiaojing Wei, Jieyu Yue, Huazhong Wang
School of Life Sciences, Tianjin Normal University / Tianjin Key Laboratory of Animal and Plant
Resistance, Tianjin 300387, China
First author:
Ms. Dan Pei
School of Life Sciences, Tianjin Normal University / Tianjin Key Laboratory of Animal and Plant
Resistance, Tianjin, China
393 Binshuixi Road, Tianjin 300387, China
E-mail: 415017319@qq.com
Corresponding author:
Prof. Huazhong Wang
School of Life Sciences, Tianjin Normal University / Tianjin Key Laboratory of Animal and Plant
Resistance, Tianjin, China
393 Binshuixi Road, Tianjin 300387, China
Telephone and fax: 86-22-23766539
E-mail: skywhz@mail.tjnu.edu.cn
Journal: Plant Cell Reports
Table S1. List of primers used in this paper
Primer name
Sequence（5' to 3'）
A8a/eF
A8a/eR
A8b/dF
A8b/dR
A8cF
A8cR
A8fF
A8fR
A8gF
A8gR
A8hF
A8hR
A4a/bF
A4a/bR
GTCGCAAACCCTCGCCCAAATCC
CTCGCCCAAATCCGCCGAATCCC
ACCAAGGTAGATCGATGGCGAAGA
TTGATTGGCACGCCTTATTTACA
GCGTCGCAAACCCTCGCCCAAAT
ATAGGATAAGTCAGGATACGAATAGG
CAAGGTAGATCGATGGCGAAGAG
TTAAAGATCCTACGACGAGGCAA
GAGCGACTTCCAATCCAGTC
AATCATCCCAGAGCATGAGG
TCTATCCGCGATTCAATCAAATAC
CAGTCAGGCAGATGTCACCCAAA
GGGTGCCTGATGCCCTGATTGAC
TTCCCATCGAACCAACTTGTGCC
NotI-A8a/bF
ATAAGAATGCGGCCGCATGGCGAAGAGCTC
GTTCAAGC
NotI-A8a/bR
ATAAGAATGCGGCCGCCTAGAGCAATCCGAA
GGTGT
NotI-A8gF
ATAAGAATGCGGCCGCATGGCCAAGACTTGC
TTCAA
NotI-A8gR
ATAAGAATGCGGCCGCTTAGGCGGAGCCGA
AAGTGT
NotI-A8hF
ATAAGAATGCGGCCGCATGAAGTCCTTCAAG
AAGGA
NotI-A8hR
ATAAGAATGCGGCCGCTCACCCAAATGTCTT
CTCGC
NotI-A4aF
ATAAGAATGCGGCCGCATGACGAGCTTGCCT
GAGAG
NotI-A4aR
ATAAGAATGCGGCCGCTCAGAGAATCTGCCA
CTCAT
NotI-A4bF
ATAAGAATGCGGCCGCATGACGAGCTTGCCT
GAGAG
NotI-A4bR
ATAAGAATGCGGCCGCTCAGAGAATCTGCCA
CTCGT
SmaI-A8a/bF
TCCCCCGGGAATGGCGAAGAGCTCGTTCAA
BamHI-A8a/bR
CGGGATCCCTAGAGCAATCCGAAGGTGT
XbaI-A8gF
GCTCTAGAAGCCAAGACTTGCTTCAAGACC
SacI-A8gR
CGAGCTCTTAGGCGGAGCCGAAAGTGT
XbaI -A8hF
GCTCTAGAAATGAAGTCCTTCAAGAAGGA
SacI -A8hR
CGAGCTCTCACCCAAATGTCTTCTCGC
qA8aF
qA8aR
qA8gF
qA8gR
qA8hF
qA8hR
qA4aF
qA4aR
qA4bF
qA4bR
qTubulin-F
TTCCTGTGATCGTTGAGAAGGCTG
AAGGTCGGCAGGGACAAGGT
TTGTGAATAGCACCTTGCCACC
GTGTTCTCGCCACTGTAAGTCATG
GGAGGAGAGGGCGAATGAGTC
GCATGTCACACGGAACCAGGTAC
TATCACTGCAGCACTGTCCG
GGTGTCGATGTTGTCGGAGG
TCAGTTCAGCCGTCGAAACA
GAGCCATCAAGGTCCTCCG
GTGGAACTGGCTCTGGC
qTubulin-R
CGCTCAATGTCAAGGGA
Product
size (bp)
Application
438
Amplification of TaATG8a, 8e
full-length cDNA by RT-PCR
436
Amplification of TaATG8b, 8d
full-length cDNA by RT-PCR
559
Amplification of TaATG8c
full-length cDNA by RT-PCR
474
Amplification of TaATG8f
full-length cDNA by RT-PCR
654
Amplification of TaATG8g
full-length cDNA by RT-PCR
446
Amplification of TaATG8h
full-length cDNA by RT-PCR
1724(4a)
1727(4b)
Amplification of TaATG4a,4b
full-length cDNA by RT-PCR
390
Construction of TaATG8a and
8b yeast or prokaryotic
expression vectors
392
Construction of TaATG8g yeast
or prokaryotic expression
vectors
383
Construction of TaATG8h yeast
or prokaryotic expression
vectors
1470
Construction of TaATG4a yeast
or prokaryotic expression
vectors
1473
Construction of TaATG4b yeast
or prokaryotic expression
vectors
360
Construction of
GFP-TaATG8a or -TaATG8b
expression vectors
358
Construction of GFP
-TaATG8g expression vector
351
Construction of GFP
-TaATG8h expression vector
80
106
134
245
Quantitative RT-PCR of
TaATG8a transcripts
Quantitative RT-PCR of
TaATG8g transcripts and
amplification in Chinese
spring derived
aneuploid
Quantitative
RT-PCR
of
nulli-tetrasomic(NT)
TaATG8h
transcriptslines for
chromosomal localization of
TaATG8g RT-PCR of
Quantitative
TaATG4a transcripts
101
Quantitative RT-PCR of
TaATG4b transcripts
234
Quantitative RT-PCR of the
wheat β-tubulin gene
transcripts
Figure S1. Multiple alignment of wheat ATG8s and homologues from other species. The
alignment was produced in ClustalX 2.1 software. Sequences in the alignment include the
nine wheat ATG8s here identified, AER27507 (TdATG8) from Triticum dicoccoides,
XP_003581707 (BdATG8a) and XP_003571383 (BdATG8b) from Brachypodium
distachyon, Os07g0512200 (OsATG8a), Os04g0624000 (OsATG8b), Os08g0191600
(OsATG8c) and Os02g0529150 (OsATG8d) from Oryza sativa, ACJ73920 (ZmATG8a),
ACJ73921 (ZmATG8b), ACJ73922 (ZmATG8c), ACJ73923 (ZmATG8d), and ACJ73925
(ZmATG8e) from Zea mays, At4g21980 (AtATG8a), At4g04620 (AtATG8b), At1g62040
(AtATG8c), At2g05630 (AtATG8d), At2G45170 (AtATG8e), At4g16520 (AtATG8f),
At3g60640 (AtATG8g), At3g06420 (AtATG8h) and At3g15580 (AtATG8i) from Arabidopsis
thaliana, ACU13796 (GmATG8a), ACU14633 (GmATG8b), ACU17086 (GmATG8c),
BAH22449 (GmATG8d), ACU15101 (GmATG8e), ACU19559 (GmATG8f), ACU13862
(GmATG8g), ACU16419 (GmATG8h), BAH22448 (GmATG8i) and ACU19611 (GmATG8k)
from Glycine max, NP_009216 (HsATG8/GATE16/GABARAPL2) from Human and
NP_009475 (ScATG8) from Yeast. Names of wheat ATG8s are boxed. One hundred
percent similarity, black; 80 to 100% similarity, dark gray; 60 to 80% similarity, light gray;
<60% similarity, white. Hashes and asterisks respectively indicate conserved essential
amino acids at the N-terminal microtubule binding site and at the ATG7 binding site. The
C-terminal conservative glycine for PE-conjugation is indicated by an arrowhead.
#
#
## ##
*
Clade I
Clade II
*
Clade I
Clade II
*
Figure S2. Multiple alignment of wheat ATG4s and homologues from other species. The
alignment was produced in ClustalX 2.1 software. Sequences in the alignment include the
two wheat ATG4s here identified, Os03g0391000 (OsATG4a) and Os04g0682000
(OsATG4b) from Oryza sativa, ACJ73912 (ZmATG4a) and ACJ73914 (ZmATG4b) from
Zea mays, At2g44140 (AtATG4a) and At3g59950 (AtATG4b) from Arabidopsis thaliana,
NP_443168 (HsATG4a) from Human and NP_014176 (ScATG4) from Yeast. One
hundred percent similarity, black; 80 to 100% similarity, dark gray; 60 to 80% similarity,
light gray; <60% similarity, white. The conserved Peptidase_C54 domain is under thick
line；Arrowheads mask the canonical catalytic triad of cysteine protease: Cys, Asp and
His.
TaATG4a
TaATG4b
OsATG4a
OsATG4b
ZmATG4b
ZmATG4a
AtATG4a
AtATG4b
ScATG4
HsATG4a
MTSLPERGAAPPSNPTPSSSRE------GDAAVASSS-SSASEEQKKDGSPKQCKASILSSVLT----IFEPDQD-QSG-----RSGGHASGSYAWSRVLRRF-VGGGSMWRFLG-------CGKALTAGDVWFLGKCYKLSSEE
MTSLPERGATPPSNPTSSSSCE------GDAAVASSSASSASEEQKKDGSPKQCKASILSSVLT----IFEPDQD-QSG-----RSGGHASGSYAWSRVLRRF-VGGGSMWRFLG-------CGKALTAADVWFLGKCYKLSSEE
MTSLPGRGVSPSS---SDPLCE------GNAAPSSSS-----SSGQD---LKQLKNSILSCVFSSPFSIFEAHQD-SSAN----RSLKPHSGSYAWSRFLRRI-ACTGSMWRFLG-------ASKALTSSDVWFLGKCYKLSSEE
MTSLPDRGVSSSS---SDPLCE------GNIAPCSSS-----SEQKEDCSLKQSKTSILSCVFNSPFNIFEAHQD-SSAN----KSPKSSSGSYDWSRVLRRI-VCSGSMWRFLG-------TSKVLTSSDVWFLGKCYKLSSEE
MTTSPERGEAPPAPPRDSPCEDAAP---AAAVVASSS---SGSEREEDGGSRQPKASVLSGVFAPPLAIFEGQQQVSSTP-CDASSTKPPSGSYAWSRILRRF-VGSGSMWRLLG-------CARVLTSGDVWFLGKCYRVSPEE
MTSLPERGESP-VPPLDSLRQDSAVTVLAAAAVASSS---AGSDRKEDSGSRQPKASILSGVFSPPFAIFEGQQQGSSSPACDARSTKSSSGSYGLSRILRRF-VGSGSMWRLLG-------CGRVLTSSDVWFLGKCYKVSPEE
MKALCDRFVPQQCS--SSSKSD-------THDKSP-------LVSDSGPSDNKSKFTLWSNVFTSSSSVSQPYRESSTSG-----HKQVCTTRNGWTAFVKRVSMASGAIRRFQERVLGPNRTGLPSTTSDVWLLGVCYKISADE
MKAICDRFVPSKCS--SSSTSE-------KRDISSPT----SLVSDSASSDNKSNLTLCSDVVASSSPVSQLCREASTSG-----HNPVCTTHSSWTVILKTASMASGAIRRFQDRVLGPSRTGISSSTSEIWLLGVCYKISEGE
MQRWLQLWKMDLVQKVSHGVFE--------------------GSSEEPAALMNHDYIVLGEVYPE-------------------RDEESGAEQCEQDCRYRGEAVSDGFLSSLFG------REISSYTKEFLLDVQSRVNFTYRT
-------------------------------------------------------------------------------------------MESVLSKYEDQITIFTDYLEEYPD------------TDELVWILGKQHLLKTEK
120
121
115
118
130
133
124
127
100
42
TaATG4a
TaATG4b
OsATG4a
OsATG4b
ZmATG4b
ZmATG4a
AtATG4a
AtATG4b
ScATG4
HsATG4a
S-SSDSDSEGGHAAFLEDFSSRVWITYRKGFDVISDSKLTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRKPAQNPSDPEHTRILHLFGDSEVCAFSIHNLLQAGKSYG-LAAGSWVGP---------YAMCRAWQTLIRTNREQ
S-SSDSDSEGGHAAFLEDFSSRVWITYRKGFDAISDSKLTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRKPAQSPSDPEHTRILHLFGDSEVCAFSIHNLLQAGKSYG-LAAGSWVGP---------YAMCRAWQTLIRTNREQ
L-SNSSDCESGNAAFLEDFSSRIWITYRKGFDAISDSKYTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRKPSQKPYSPEYIGILHMFGDSEACAFSIHNLLQAGKSYG-LAAGSWVGP---------YAMCRAWQTLVCTNREH
S-SSDSDSESGHATFLEDFSSRIWITYRRGFDAISDSKYTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRRPLEKPYNPEYIGILHMFGDSEACAFSIHNLLQAGNSYG-LAAGSWVGP---------YAMCRAWQTLVRTNREQ
EESGGSDSDSGHAAFLEDFSSRIWITYRKGFDAIPGSKLTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRKPSEKPYDPDYIRVLHLFGDSEACAFSIHNLLQAGRNYG-LAAGSWVGP---------YAMCRAWQTLIRTNREQ
EESGDSESDSGHAAFLEDFSSRIWITYRKGFDAISDSKLTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRKPPEKPYNPDYIGVLHLFGDSEACAFSIHNLLQAGRNYG-LAAGSWLGP---------YAMCRAWQTLIRTNREQ
N-SGETDTGTVLAALQLDFSSKILMTYRKGFEPFRDTTYTSDVNWGCMIRSSQMLFAQALLFHRLGRAWTKK-SELPEQEYLETLEPFGDSEPSAFSIHNLIIAGASYG-LAAGSWVGP---------YAICRAWESLACKKRKQ
S-SEEADAGRVLAAFRQDFSSLILMTYRRGFEPIGDTTYTSDVNWGCMLRSGQMLFAQALLFQRLGRSWRKKDSEPADEKYLEILELFGDTEASAFSIHNLILAGESYG-LAAGSWVGP---------YAVCRSWESLARKNKEE
RFVPIARAPDGPSPLSLNLLVRTNPISTIEDYIANPDCFNTDIGWGCMIRTGQSLLGNALQILHLGRDFRVNGNESLERE-SKFVNWFNDTPEAPFSLHNFVSAGTELSDKRPGEWFGP---------AATARSIQSLIYG---S------------KLLSDISARLWFTYRRKFSPIGGTGPSSDAGWGCMLRCGQMMLAQALICRHLGRDWSWEKQKEQPKEYQRILQCFLDRKDCCYSIHQMAQMGVGEG-KSIGEWFGPNTVAQVLKKLALFDEWNSLAVYVSMD
254
255
249
252
265
268
257
261
231
174
TaATG4a
TaATG4b
OsATG4a
OsATG4b
ZmATG4b
ZmATG4a
AtATG4a
AtATG4b
ScATG4
HsATG4a
PEVINRNECFPMALYVVSGDEDGERGGAPVVCIDVAAQLCYDFNKDQSTWSPILLLVPLVLGLDKINPRYIPLLKETFTFPQSLGILGGKPGASTYIAGVQDDRALYLDPHEVQLAVNIASDNLEANTSSYHCSTVRDMPLDLID
PEVINRNESFPMALYVVSGDEDGERGGAPVVCIDVAAQLCYDFNKDQSAWSPILLLVPLVLGLDKINPRYIPLLKETFTFPQSLGILGGKPGASTYIAGVQDDRALYLDPHEVQMAVNIASDNLEADTSSYHCSTVRDMPLDLID
HEAVDGNGNFPMALYVVSGDEDGERGGAPVVCIDVAAQLCCDFNKNQSTWSPILLLVPLVLGLDKLNPRYIPLLKETLTFPQSLGILGGKPGTSTYIAGVQDDRALYLDPHEVQLAVDIAADNLEAGTSSYHCSTVRDLALDLID
HEVVDGNESFPMALYVVSGDEDGERGGAPVVCIDVAAQLCCDFNKGQSTWSPILLLVPLVLGLDKINPRYIPLLKETFTFPQSLGILGGKPGTSTYIAGVQDDRALYLDPHEVQMAVDIAADNIEADTSSYHCSTVRDLALDLID
ADAVDGKENFPMALYVVSGDEDGERGGAPVFCIDVAAQLCSNFNKGQCTWSPILLLIPLVLGLDKINPRYIPLLKETFKFPQSLGILGGKPGTSTYIAGVQEDRALYLDPHDVQMAVDIAPDNLEADTSSYHCSVVRDLALEQID
ADAVDGKENFPMALYVVSGDEDGERGGAPVVCIDVAAQLCSDFNKGPSTWSPILLLVPLVLGLDKINPRYIPLLKETFMFPQSLGILGGKPGTSTYIAGVQDDRALYLDPHEVQMTVDIALDNLEADTSSYHCSVVRALALEQID
TD--SKNQTLPMAVHIVSGSEDGERGGAPILCIEDATKSCLEFSKGQSEWTPIILLVPLVLGLDSVNPRYIPSLVATFTFPQSVGILGGKPGASTYIVGVQEDKGFYLDPHEVQQVVTVNKETPDVDTSSYHCNVLRYVPLESLD
TD--DKHKSFSMAVHIVSGSEDGERGGAPILCIEDVTKTCLEFSEGETEWPPILLLVPLVLGLDRVNPRYIPSLIATFTFPQSLGILGGKPGASTYIVGVQEDKGFYLDPHDVQQVVTVKKENQDVDTSSYHCNTLRYVPLESLD
----FPECGIDDCIVSVSSGDIYENEVEKVFAENPN--------------SRILFLLGVKLGINAVNESYRESICGILSSTQSVGIAGGRPSSSLYFFGYQGNEFLHFDPHIPQPAVEDS------FVESCHTSKFGKLQLSEMD
NTVVIEDIKKMCRVLPLSADTAGDRPPDSLTASNQSKGTSAYCS----AWKPLLLIVPLRLGINQINPVYVDAFKECFKMPQSLGALGGKPNNAYYFIGFLGDELIFLDPHTTQTFVDTEENGTVNDQTFHCLQSPQRMNILNLD
TaATG4a
TaATG4b
OsATG4a
OsATG4b
ZmATG4b
ZmATG4a
AtATG4a
AtATG4b
ScATG4
HsATG4a
PSLAIGFYCRDKDDFDDFCSRASE------LAEQANGAPLFTVVQSVQPSKQMYNQDDGSGCSGY---------------GASDNIDTEDLDGSGETGEDEWQIL------------------------------------PSLAIGFYCRDKDDFDDFCSRASE------LAEQANGAPLFTVVQSVQPSKQMYNQDDGSGCSGY---------------GVSDNIDAEDLDGSGETGEDEWQIL------------------------------------PSLAIGFYCRDKDDFDDFCSRASE------LVDKANGAPLFTVVQSVQPSKQMYNEESSSGD-------------------GMDSINVEGLDGSGETGEEEWQIL------------------------------------PSLAIGFYCRDKDDFDDFCSRATE------LVDKANGAPLFTVVQSVQPSKQMYNQDDVLGIS------------------GDGNINVEDLDASGETGEEEWQIL------------------------------------PSLAIGFYCRDKDDFDDFCSRASE------LAEKANGAPLFTVMQSVQPSKQMYKQDDGLCCCSG---------------SSMANEDY-DLDASGEAG-EEWQIL------------------------------------PSLAIGFYCRDKDDFDDFCSRASE------LAEKANGAPLFTVVQSIEPSKQMYKQDDGLG-CSG---------------SSMANDD--DLDGSGEA--EEWQIL------------------------------------PSLALGFYCRDKDDFDDFCLRALK------LAEESNGAPLFTVTQTHT------AINQSNY--------------------GFAD------DDSEDEREDDWQML------------------------------------PSLALGFYCQHKDDFDDFCIRATK------LAGDSNGAPLFTVTQSHRRNDCGIAETSSST--------------------ETST------EISGEEHEDDWQLL------------------------------------PSMLIGILIKGEKDWQQWKLEVAESAIINVLAKRMDDFDVSCSMDDVESVSSNSMKKDASNNENLGVLEGDYVDIGAIFPHTTNTEDVDEYDCFQDIHCKKQKIVVMGNTHTVNANLTDYEVEGVLVEKETVGIHSPIDEKC
PSVALGFFCKEEKDFDNWCSLVQK------EILKEN-LRMFELVQKHPSHWPPFVPPAKPEVTTT---------------GAEFIDSTEQLEEFDLEEDFEILSV-------------------------------------
Peptidase_C54 domain
399
400
394
397
410
413
400
404
352
315
483
484
474
478
492
492
467
477
494
398