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%FQBSUNFOUPG#JPDIFNJTUSZBOE.PMFDVMBS#JPMPHZ

3BUJPOBMF 4UBSDI"DDVNVMBUJPOJO5SBOTHFOJD1MBOUT
5IJTTVCQSPKFDUGPDVTFTPOJODSFBTJOHUIFZJFMEPGFBTJMZEFHSBEFEQPMZNFST 
TVDIBTTUBSDI JOMFBWFT5IFHPBMJTUPNBLFMFBWFTBCFUUFSGFFETUPDLGPSB
DFMMVMPTJD FUIBOPM GFSNFOUPS5IF NBKPS BEWBOUBHFT PG UIF BQQSPBDI JO UIJT
TVCQSPKFDUJT 
QPUFOUJBMGPSIJHIFSZJFMEBOE 
FBTFBOEDPNQMFUFOFTTPG
GFSNFOUBUJPO 5IF NBKPS CPUUMFOFDL JT SFEVDFE ZJFME JO NPTU TUBSDI
BDDVNVMBUJOHNVUBOUT

0WFSDPNJOHUIF:JFME1FOBMUZ
5PBUUFNQUUPPWFSDPNFUIFZJFMEQFOBMUZXFCMPDLFETUBSDIEFHSBEBUJPOBUB
TQFDJmDEFWFMPQNFOUBMUJNFQPJOUMBUFJOUIFMJGFDZDMFPGUIFQMBOU&BSMZJO
QMBOU HSPXUI DBSCPO  GSPN TUBSDI  SFJOWFTUFE JO MFBWFT DBVTFT FYQPOFOUJBM
HSPXUI 0ODF TVöDJFOU QMBOU NBTT JT QSFTFOU UP DPOUBJO UIF DBSCPO 
NFUBCPMJTNDBOCFTXJUDIFEUPDBVTFBDDVNVMBUJPOPGTUBSDI 'JHVSF4FOFTDFODF*OEVDFE3/"J4UBSDI"DDVNVMBUJOHQMBOUT
1IPUPHSBQIT XFSF UBLFO BOE MFBWFT XFSF TUBJOFE XJUI *,* GPS TUBSDI JO UIF
5PEPUIJTXFVTFE3/"JOUFSGFSFODF 3/"J
BHBJOTUUIFHMVDBOXBUFSEJLJOBTF NPSOJOHXIFOTUBSDIMFWFMTBSFUIFMPXFTU5IF(8%LOPDLPVUQMBOUIBTIJHI
FO[ZNF (8%
 BOE UIF QIPTQIPHMVDBO QIPTQIBUBTF #PUI FO[ZNFT BSF TUBSDIMFWFMTCVUBTJHOJmDBOUHSPXUIQFOBMUZ5IFQMBOUXJUIUIFTFOFTDFODF
OFDFTTBSZ GPS TUBSDI EFHSBEBUJPO JO MFBWFT 5IFTF 3/"J DPOTUSVDUT XJMM CF JOEVDFE 3/"J HFOF BHBJOTU (8% EPFT OPU FYIJCJU B EFDSFBTF JO HSPXUI BOE
DPVQMFEUPBTFOFTDFODFJOEVDFEQSPNPUFS 4"(
PSBOBMDPIPMJOEVDJCMF BGUFSNPOUITDPOUBJOTIJHIMFBGTUBSDIMFWFMTDPNQBSBCMFUPUIF(8%,0MJOF
QSPNPUFS 'JH
$POTUSVDUTGPSUIFWBSJPVTQSPNPUFSTBOEJOWFSUFESFQFBUT
XFSFNBEFCZ#JP#BTJDJO.BSLIBN0OUBSJP$BOBEB XXXCJPCBTJDDPN

Starch (µmol Glu g-1FW)

GWD KO
a. Sag12p GWD 400bp Intron 160
120
Ai
RN
b. 35S alcR alcA GWD 400bp Intron 80 WD
-G
Alc Spray

Alc
ALCR
GWD 400bp 40
ALCR E
GWD 400bp WT
Ethanol E Dicer
0
added
E
GWD 400
400
bp
bp
0 2 4 6 8 10 12 14
Day
GWD

Target GWD DNA or mRNA RISC


Starch (µmol Glu g-1FW)

120
'JHVSF4FOFTDFODF*OEVDJCMFBOE"MDPIPM*OEVDJCMF3/"J$POTUSVDUT
B 5IF QSPNPUFS PG UIF 4"( HFOF  B DZTUFJOF QSPUFBTF  EJTDPWFSFE CZ 80 Ai
:PP4VO/PIBOE3JDL"NBTJOP 
JTBDUJWFPOMZBUUIFUJNFPGTFOFTDFODF Phosphatase KO RN
se
JO"SBCJEPQTJTBOEXJMMCFVTFEUPESJWF3/"JUBSHFUFEUPUIF(8%FO[ZNFPS ata
ph
Alc Spray

UIFQIPTQIPHMVDBOQIPTQIBUBTF 40 -P
hos
C "O BMDPIPMJOEVDJCMF QSPNPUFS XJMM BMTP CF VTFE UP ESJWF 3/"J 5IF Alc
BEWBOUBHF PG UIF BMDPIPMJOEVDJCMF QSPNPUFS JT UIBU UIF EFWFMPQNFOUBM WT
UJNJOHPGTUBSDIBDDVNVMBUJPODBOCFQSFDJTFMZDPOUSPMMFEBOEUIJTQSPNPUFS
0
XJMMXPSLJOPUIFSTQFDJFTCFTJEFT"SBCJEPQTJT
0 2 4 6 8 10 12 14
'JSTU"SBCJEPQTJTUIFO$PSO Day

'JHVSF"MDPIPM*OEVDFE3/"J4UBSDI"DDVNVMBUJPO
8JMEUZQF 1SJNBSZUSBOTGPSNBOUQMBOUTDPOUBJOJOHUIFBMDPIPMJOEVDJCMF3/"J
BHBJOTUUIFHMVDBOXBUFSEJLJOBTF (8%
PSUIFQIPTQIPHMVDBOQIPTQIBUBTF 
CPUIOFDFTTBSZGPSMFBGTUBSDIEFHSBEBUJPO BOEUIFDPSSFTQPOEJOHLOPDLPVU
MJOFTXFSFHSPXOGPSEBZT"MDPIPMJOEVDJCMFQMBOUTXFSFUIFOTQSBZFEXJUI
FUIBOPM-FBWFTXFSFUBLFOGPSTUBSDIEFUFSNJOBUJPOTJOUIFNPSOJOH5IF
BMDPIPMJOEVDJCMFQMBOUTRVJDLMZBDDVNVMBUFBTNVDITUBSDIBTUIFLOPDLPVU
$PODVSSFOU XJUI UIF "SBCJEPQTJT XPSL  3/"J DPOTUSVDUT UBSHFUFE UP UIF DPSO MJOF *O UIF DBTF PG UIF "MD JOEVDJCMF 3/"J BHBJOTU UIF QIPTQIPHMVDBO
IPNPMPHVFPG(8%BOE4FYIBWFCFFONBEF5IFTFXJMMCFQMBDFECFIJOEUIF QIPTQIBUBTFUIFTUBSDIMFWFMTFYDFEFUIPTFPGUIFLOPDLPVUMJOF
BMDPIPMJOEVDJCMFQSPNPUFS BOECFIJOEUIFDPOTUJUVUJWFQSPNPUFSPGVCJRVJUJO
5IFUSBOTGPSNBUJPOTBSFCFJOHDBSSJFEPVUCZ%S)FJEJ,BFQQMFSBUUIF6OJWFSTJUZ
PG8JTDPOTJO.BEJTPO

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