Rice MYC2 (Osmyc2) Modulates Light-Dependent Seedling Phenotype, Disease Defence but Not

Rice MYC2 (OsMYC2) modulates light-dependent seedling phenotype, disease defence but not ABA signalling

Mrunmay Kumar Giri,Janesh Kumar Gautam, V. BabuRajendra Prasad, Sudip Chattopadhyay andAshis Kumar Nandi

Supplementary figure 1.tBLASTn search result using AtMYC2 amino acid sequences as query from rice genome.

OsMYC: 52 SMNLWT-DDNASMMEAFMASADLPAFPWGAASTPPPPPPPPHHHHQQQQQQVLPPPAAA- 109

+MNLWT DDNASMMEAFM+S+D+ W AST + P PA

AtMYC: 9 TMNLWTTDDNASMMEAFMSSSDISTL-WPPASTTTTTATT------ETTPTPAMEI 57

OsMYC: 110 PAAAAFNQDTLQQRLQSIIEGSRETWTYAIFWQSSIDVSTGASLLGWGDGYYKGCDDDKR 169

PA A FNQ+TLQQRLQ++IEG+ E WTYAIFWQ S D S GAS+LGWGDGYYKG +D

AtMYC: 58 PAQAGFNQETLQQRLQALIEGTHEGWTYAIFWQPSYDFS-GASVLGWGDGYYKGEEDKAN 116

OsMYC: 170 -KQRSSTP--AAAAEQEHRKRVLRELNSLIAGAGAAPDEAVEEEVTDTEWFFLVSMTQSF 226

++RSS+P + A+QE+RK+VLRELNSLI+G A D+AV+EEVTDTEWFFLVSMTQSF

AtMYC: 117 PRRRSSSPPFSTPADQEYRKKVLRELNSLISGGVAPSDDAVDEEVTDTEWFFLVSMTQSF 176

OsMYC: 227 PNGLGLPGQALFAAQPTWIATG--LSSAPCDRARQAYTFGLRTMVCLPLATGVLELGSTD 284

G GL G+A W++ LS + C+RA+Q FG+ T+ C+P A GV+E+GST+

AtMYC: 177 ACGAGLAGKAFATGNAVWVSGSDQLSGSGCERAKQGGVFGMHTIACIPSANGVVEVGSTE 236

OsMYC: 285 VIFQTGDSIPRIRALFNLSAAAAS----SWPPHPDAASADPSVLWLADAPPMDMKDSISA 340

I Q+ D I ++R LFN A +W PD DPS +W+ D P+ S

AtMYC: 237 PIRQSSDLINKVRILFNFDGGAGDLSGLNWNLDPDQGENDPS-MWIND--PIGTPGSNEP 293

OsMYC: 341 ADISVSKPPPPPPHQIQHFENGSTSTLTENPSPSVHAPTPSQPAAPPQRQQQQQQSSQAQ 400

+ + S IQ FENGS+ST+TENP+ PTPS P Q Q + +

AtMYC: 294 GNGAPSSSSQLFSKSIQ-FENGSSSTITENPNLD---PTPS----PVHSQTQNPKFNNT- 344

OsMYC: 401 QGPFRRELNFSDFASNGGAAAPPFFKPETGEILNFGNDSSSGRRNPSPAPPAATASLTTA 460

F RELNFS +S KP +GEILNFG++ NP P

AtMYC: 345 ---FSRELNFSTSSST------LVKPRSGEILNFGDEGKRSSGNPDP------382

OsMYC: 461 PGSLFSQHTPTLTAAANDAKSNNQKRSMEATSRASNTNNHPAATANEGMLSFSSAPTTRP 520

S +S T N +KRSM + +LSF

AtMYC: 383 --SSYSGQTQF------ENKRKRSM------VLNEDKVLSF------409

OsMYC: 521 STGTGAPAKSESDHSDLEAS-VREVESSRVVAPPPEAEKRPRKRGRKPANGREEPLNHVE 579

G ESDHSDLEAS V+EV EKRP+KRGRKPANGREEPLNHVE

AtMYC: 410 ----GDKTAGESDHSDLEASVVKEV------AVEKRPKKRGRKPANGREEPLNHVE 455

OsMYC: 580 AERQRREKLNQRFYALRAVVPNVSKMDKASLLGDAISYINELRGKLTALETDKETLQSQM 639

AERQRREKLNQRFYALRAVVPNVSKMDKASLLGDAI+YINEL+ K+ E++K +++Q+

AtMYC: 456 AERQRREKLNQRFYALRAVVPNVSKMDKASLLGDAIAYINELKSKVVKTESEKLQIKNQL 515

OsMYC: 640 ESLKKERDARPPAPSGGGGDGGARCHAV-----EIEAKILGLEAMIRVQCHKRNHPAARL 694

E +K E R S GGD + C ++ EIE KI+G +AMIRV+ KRNHPAARL

AtMYC: 516 EEVKLELAGR--KASASGGDMSSSCSSIKPVGMEIEVKIIGWDAMIRVESSKRNHPAARL 573

OsMYC: 695 MTALRELDLDVYHASVSVVKDLMIQQVAVKMASRVYSQDQLNAALYTRI 743

M+AL +L+L+V HAS+SVV DLMIQQ VKM R+Y+Q+QL A+L ++I

AtMYC: 574 MSALMDLELEVNHASMSVVNDLMIQQATVKMGFRIYTQEQLRASLISKI 622

Supplementary figure 2. BLAST2Seq analysis between AtMYC2 and OsMYC2-1.Score = 537 bits (1384), Expect = e-157. Identities = 342/709 (48%), Positives = 426/709 (60%), Gaps = 112/709 (15%)

Supplementary figure 3. Generation of transgenic Arabidopsis plants expressing OsMYC2-1 in atmyc2 mutant background. A, Diagram of the vector construct used. OsMYC2-1 was cloned under CaMV35S promoter in a vector that was derived from pCAMBIA1303 vector (details given in methods section). B, PCR showing the presence of OSMYC2-1 in transgenic plants. C, GUS expression in one complementation line. D, RT-PCR showing OsMYC2-1 expression in one complementation line.

Supplementary figure 4. Generation of transgenic rice plants expressing OsMYC2-1. A, Diagram of the vector construct used. OsMYC2-1 was cloned under Ubiquitn (Ubi-1) promoter in a vector that was derived from pCAMBIA1303 vector (details given in methods section). B, PCR showing the presence of hpt gene that confers hygromycin resistance. C, GUS expression in the individual transgenic lines.

Supplementary figure 5. cDNA sequence of OsMYC2-1. The bold and underlined sequence denotes the 397 bp region that was used as siRNA target.

Supplementary figure6. Generation of transgenic rice plants with OsMYC2-1 siRNA construct. A, Diagram of the vector construct used. B, GUS expression in the individual transgenic lines C, PCR showing the presence of hpt gene that confers hygromycin resistance.

Supplementary table 1. Primers used in the study

Ub RP – AAGAAGCTGAAGCATCCAGC

Ub FP - CCAGGACAAGATGATCTGCC

Actin 2 - ATGCCATCCTCCGTCTTGAC

Actin 2 – CGCTCTGCTGTTGTGGTGAA

CAB1 FP- GTTAACAACAACGCATGGC

CAB1 RP - CCTCTCACACTCACGAAGCA

RBCS-1A FP - TCGGATTCTCAACTGTCTGATG

RBCS-1A RP - ATTTGTAGCCGCATTGTCCT

OsMYC2-1 FP - CTCAACCAGCGCTTCTAC

OsMYC2-1 RP - GCTCGTTGATGTAGGAGATG