advances.sciencemag.org/cgi/content/full/6/48/eabb7719/DC1

Supplementary Materials for

A fungal effector targets a heat shock–dynamin protein complex to modulate

mitochondrial dynamics and reduce plant immunity

Guojuan Xu, Xionghui Zhong, Yanlong Shi, Zhuo Liu, Nan Jiang, Jing Liu, Bo Ding, Zhiqiang Li, Houxiang Kang, Yuese Ning, Wende Liu, Zejian Guo, Guo-Liang Wang*, Xuli Wang*

*Corresponding author. Email: wang.620@osu.edu (G.-L.W.); wangxuli@caas.cn (X.W.)

Published 25 November 2020, Sci. Adv. 6, eabb7719 (2020)

DOI: 10.1126/sciadv.abb7719

This PDF file includes:

Figs. S1 to S8 Table S1

NPB

#10

#16

#22

0

50

100

***

***

***

OX-MoCDIP4ΔSP

P<0.0001

P<0.0001

P<0.0001

Lesi

on a

rea(

mm

2 )

NPB

#10

#16

#22

NPB

#10

#16

#22

0.0

0.7

1.4

OX-MoCDIP4ΔSP OX-MoCDIP4ΔSP

****** **

*********

WRKY45PAL1

P=0.0001

P=0.0001

P=0.0002

P=0.0008

P=0.0011

P=0.0003

Rel

ativ

e tr

ansc

ript

ion

(/NP

B)

CM

D-W

Y

PR

AA

YTK12 Mg87 Avr1b MoCDIP4

1 18 295 226 260 GH61 CBD SP

MoCDIP4

OX-MoCDIP4ΔSP

OsUbq

NPB #10 #16 #22

A

B

C

NPB #10 #16 #22 OX-MoCDIP4ΔSP

E F

I

MoCDIP4

Beta-tubulin

D

NPB #10 #16 #22 OX-MoCDIP4ΔSP

NPB

#10

#16

#22

0

80

160

240

320

OX-MoCDIP4ΔSPP=0.0005

***

* **

P=0.0128

P=0.0034

Rel

ativ

e fu

ngal

bio

mas

s(MoPot2/OsUbq

)

G H

Fig. S1. MoCDIP4 is a virulent effector of M. oryzae. (A) The structure of MoCDIP4. (B) Secretory

ability of the MoCDIP4 signal peptide. Avr1b and Mg87 were used as a positive and a negative

control, respectively. (C) MoCDIP4 transcript levels in the MoCDIP4 mutant. Guy11 was the wild-

type strain, Beta-tubulin was used as the loading control. (D) MoCDIP4 transcript levels in MoCDIP4

transgenic rice. Rice ubiquitin gene OsUbq was used as the loading control. (E) Spray inoculation of

MoCDIP4 overexpression plants with M. oryzae. (F) Lesion area of spray-inoculated leaves in (E).

Bars represent means ± SD, n = 5. Asterisks indicate significant differences between the transgenic line

and NPB according to a Student's t-test. (G) Punch inoculation of MoCDIP4 overexpression plants

with M. oryzae. (H) Relative fungal biomass of punch-inoculated leaves in (G). (I) Transcript levels of

PR genes in MoCDIP4 transgenic rice at 2 days after inoculation. Bars (H, I) represent means ± SD, n

= 3. Asterisks (H, I) indicate significant differences between the transgenic line and NPB according to

a Student's t-test.

Fig. S2. MoCDIP4 interacts with OsDjA9 in LCI assay. (A) Fluorescent image of the MoCDIP4-

OsDjA9 interaction in LCI assay. AvrPiz-t and OsDjA11 were used as the specific control. (B)

Quantification of luciferase activity in the leaves shown in (A). Bars represent means ± SD, n = 3.

Bright field

1.nLUC+cLUC 2.nLUC+OsDjA9-cLUC 3.MoCDIP4ΔSP-nLUC+cLUC 4.MoCDIP4ΔSP-nLUC+OsDjA9-cLUC 5.AvrPiz-t-nLUC+OsDjA9-cLUC 6.MoCDIP4ΔSP-nLUC+OsDjA11-cLUC

Dark field

1 2 3 4 5 60

20

401,000

2,000

3,000

4,000

Lu

cif

era

se

ac

tiv

ity1 2

4 3

5 6

A B

NPB

CR-OsDjA9-16 1bp insertion

CR-OsDjA9-11 54bp deletion

(partial deletion in DnaJ domain)

B

A

NPB 5’-CTCATAAGCTCGCTGAACCTCTTGAAACTTCCTCTCTGCATCAGAATCACCTTTATTTGTATCCGGATGGAC-3’ CR-OsDjA9-11 5’-C------------------------------------------------------TTTGTATCCGGATGGAC-3’ CR-OsDjA9-16 5’-CTCATAAGCTCGCTGAACCTCTTGAAACTTCCTCTCTGCATCAGAATCACCTTTAATTTGTATCCGGATGGAC-3’

D P H

G

NPB

#18

#22

#25

NPB

#18

#22

#25

NPB

#18

#22

#25

0

2

4

6

8PAL1 PBZ1 PR10

OX-OsDjA9 OX-OsDjA9 OX-OsDjA9

*** ***

**

***

**

***

**

***P=0.0006

P=0.0002

P=0.002

P=0.0353

P=0.0011

P=0.0012

P=0.0001

P=0.0005

P=0.0011

Rel

ativ

e tr

ansc

ript

ion

(/NP

B)H

NPB

#32

#38

#41

NPB

#32

#38

#41

NPB

#32

#38

#41

0.0

0.6

1.2

1.8

PAL1

PBZ1 PR10

RNAi-OsDjA9 RNAi-OsDjA9 RNAi-OsDjA9

******

*

*** *** ******

** **

P=0.0001

P=0.0003 P=0.0142

P=0.0004

P=0.0002

P=0.0007

P=0.0002

P=0.0011

P=0.0014

Rel

ativ

e tr

ansc

ript

ion

(/NP

B)I

0 5 10 15 20 25 300

4,000

8,000

12,000RNAi-OsDjA9-chitinOX-OsDjA9-chitin

RNAi-OsDjA9-H2OOX-OsDjA9-H2ONPB-chitin

NPB-H2O

Times(Mins.)

Chemiluminescence

C

NPB

#18

#22

#25

NPB

#32

#38

#41

0.000.010.020.030.040.05

1.0

2.1

***

***

***

OX-OsDjA9

*******

RNAi-OsDjA9

P<0.0001

P<0.0001

P=0.0003

P=0.0012

P=0.0035

P=0.0009

Rel

ativ

e tr

ansc

ript

ion

(OsDjA9/OsUbq

)

NPB #18 #22 #25 OX-OsDjA9

NPB #32 #38 #41 RNAi-OsDjA9

NPB

#18

#22

#25

NPB

#32

#38

#41

0

80

160

240

320

******

OX-OsDjA9 RNAi-OsDjA9

*****

P=0.0038

P=0.0116

P=0.0053

P=0.0047

P=0.0033

P=0.0023

Rel

ativ

e fu

ngal

bio

mas

s(MoPot2/OsUbq

)

D

E

F

Fig. S3. OsDjA9 positively regulates rice immunity against M. oryzae. (A) Schematic representation of

the OsDjA9 structure and gene editing sites. Bold letters indicate the target sequences of single guide RNA,

green letters indicate PAM, - indicates nucleotide deletion, and red letter indicates nucleotide insertion. (B)

Sanger sequencing chromatograph of the target site in OsDjA9. (C) OsDjA9 transcript levels in OsDjA9

transgenic plants. Punch inoculation of OsDjA9 (D) overexpression and (E) RNAi plants with M. oryzae.

(F) Relative fungal biomass of punch-inoculated leaves in (D, E). (G) ROS accumulation in OsDjA9

transgenic plants after chitin treatment. Transcript levels of PR genes in OsDjA9 (H) overexpression and (I)

RNAi plants at 2 days after inoculation. Bars (C, F-I) represent means ± SD, n = 3. Asterisks (C, F, H, I)

indicate the significant differences between the transgenic line and NPB according to Student's t-test.

Fig. S4. MoCDIP4 and OsDjA9 are co-localized in the ER in N. benthamiana. (A) Subcellular

localization of MoCDIP4 in N. benthamiana. HDEL was used as the ER marker. (B) Subcellular

localization of OsDjA9 in N. benthamiana. COX4 was used as the mitochondrial marker; HDEL was

used as the ER marker. (C) Co-localization of MoCDIP4 and OsDjA9 in N. benthamiana. HDEL was

used as the ER marker. (D) BiFC analysis of the MoCDIP4-OsDjA9 interaction in N. benthamiana.

Yellow squares (A-D) show the enlarged ER localization. Scale bars (A-D) represent 10 µm.

B

A C MoCDIP4ΔSP-GFP HDEL-RFP Merge Merge MoCDIP4ΔSP-CFP OsDjA9-GFP HDEL-RFP

YN+ OsDjA9-YC

MoCDIP4ΔSP-YN+OsDjA9-YC

Bright Merge YFP

MoCDIP4ΔSP-YN+YC

D Merge OsDjA9-GFP COX4-RFP

Merge OsDjA9-GFP HDEL-RFP

Fig. S5. OsDjA9 interacts with the dynamin-related protein OsDRP1E. (A) Interaction between

OsDjA9 and OsDRP1E in Co-IP assay. 4*HA was a fusion of four HA tags. (B) Interaction between

OsDjA9 and OsDRP1E in LCI assay. Bars represent means ± SD, n = 3. (C) Subcellular localization of

OsDRP1E in N. benthamiana. COX4 was used as mitochondrial marker; HDEL was used as the ER

marker. Yellow squares show the enlarged ER localization. (D) Detection of OsDRP1E in different cell

fractions extracted from (C). T, total extract; MT, mitochondria; S, soluble fraction; M, membrane

fraction. (E) Co-localization of OsDjA9 and OsDRP1E in N. benthamiana. (F) Detection of OsDjA9

and OsDRP1E in different cell fractions extracted from tobacco leaves co-infiltrated with OsDjA9 and

OsDRP1E. Scale bars (C, E) represent 10 µm.

Merge OsDRP1E-GFP OsDjA9-RFP

E

Merge OsDRP1E-GFP COX4-RFP A

F

α-GFP

α-BiP

α-UGPase

OsDRP1E-GFP T MT S M

α-VDAC

51.6 kDa

80 kDa

29 kDa

110 kDa

α-UGPase

α-cLUC

α-BiP

α-VDAC

α-GFP

OsDjA9-cLUC+OsDRP1E-GFP T MT S M

51.6 kDa

80 kDa

29 kDa

71 kDa

110 kDa

D

B

OsDRP1E-GFP

GFP OsDjA9-4*HA

+ - Input

+ + - +

α-HA

α-GFP

26.9 kDa

IP: α- GFP + - + + - +

62 kDa

110 kDa

1 2 3 40

200

400

600

800

Luci

fera

se a

ctiv

ity

1.nLUC+cLUC 2.OsDRP1E-nLUC+cLUC 3.nLUC+OsDjA9-cLUC 4.OsDRP1E-nLUC+OsDjA9-cLUC

Dark field Bright field

1 2

4 3

C

OsDRP1E-GFP HDEL-RFP Merge

NPB 5’-GCGAGCATGGAGGGTCTGATCGGCCTGATGAACCGCATCCAGCGAGCCTGCACCGCCCTCGGAGACCACGGAG-3’ CR-OsDRP1E-1 5’-GCGAGCATGGAG----AGATCGGCCTGATGAACCGCATCCAGCGAGCCTGCACCGCCCTCGGAGACCACGGAG-3’ CR-OsDRP1E-9 5’-GCGAGCATGGA-----------------TGAACCGCATCCAGCGAGCCTGCACCGCCCTCGGAGACCACGGAG-3’ CR-OsDRP1E-22 5’-GCGAGCATGGA-------------------------------------------------------------G-3’

CR-OsDRP1E-22 61bp deletion

CR-OsDRP1E-9 17bp deletion

CR-OsDRP1E-1 4bp deletion and 1bp substitution

NPB

A

D

NPB #2 #3 #5

0.00

0.05

0.10

2468

***

***

***

OX-OsDRP1E

P<0.0001

P=0.0003

P=0.0002

Rel

ativ

e tr

ansc

ript

ion

(OsDRP1E/OsUbq

)B

NPB #1 #9 #22

NPB #1 #9 #22

NPB #1 #9 #22

0

1

2

3

4PAL1 WRKY45

PR10

CR-OsDRP1E CR-OsDRP1E CR-OsDRP1E

***

***** **

***

*****

**

**

P<0.0001

P=0.0002

P=0.0014

P<0.0001

P=0.0084

P=0.

0039

P=0.0017

P=0.0029

P=0.0003

Rel

ativ

e tr

ansc

ript

ion

(/NP

B)

NPB #2 #3 #5

NPB #2 #3 #5

NPB #2 #3 #5

0.0

0.5

1.0

1.5PAL1

PR10 WRKY45

OX-OsDRP1E OX-OsDRP1E OX-OsDRP1E

***

***

**

*** ****** *** ***

***

P=0.0008

P=0.0003

P=0.0011

P=0.0002

P=0.0001

P<0.0001

P=0.0002

P<0.0001

P=0.0002

Rel

ativ

e tr

ansc

ript

ion

(/NP

B)

C

F

NPB #1 #9 #22 CR-OsDRP1E

E

G H

NPB #2 #3 #5 OX-OsDRP1E

NPB #2 #3 #5

NPB #1 #9 #22

0

50

100

150

OX-OsDRP1E CR-OsDRP1E

**

**

*****

***

P=0.0011

P=0.0007

P=0.0003

P=0.0042

P<0.0055

P=0.0007

***

Rel

ativ

e fu

ngal

bio

mas

s(MoPot2/OsUbq

)

Fig. S6. OsDRP1E negatively regulates rice immunity. (A) OsDRP1E transcript levels in OsDRP1E

overexpression plants. (B) Schematic representation of the OsDRP1E structure and gene editing site.

Bold letters indicate the target sequences of single guide RNA, green letters indicate PAM, - indicates

nucleotide deletion, blue letter indicates nucleotide substitution. (C) Sanger sequencing chromatograph

of the target site in OsDRP1E. Punch inoculation of OsDRP1E (D) overexpression and (E) CRISPR/

Cas9-edited plants with M. oryzae. (F) Relative fungal biomass of punch-inoculated leaves in (D, E).

Transcript levels of PR genes in OsDRP1E (G) overexpression and (H) CRISPR/Cas9-edited plants at

2 days after inoculation. Bars (A, F-H) represent means ± SD, n = 3. Asterisks (A, F-H) indicate the

significant differences between the transgenic line and NPB according to Student's t-test.

Fig. S7. MoCDIP4 stabilizes OsDRP1E by decreasing the association of OsDjA9 with OsDRP1E

in a competitive manner. OsDRP1E transcript levels in (A) OsDjA9 transgenic plants and (B)

MoCDIP4 transgenic plants. Bars represent means ± SD, n = 3. ns indicates no significant difference

between the transgenic line and NPB according to Student's t-test. (C) OsDRP1E protein levels when

expressed in NPB and transgenic rice protoplasts. Inhibitors were added at 12 h after transfection,

respectively. Protoplasts were sampled at 12 h after the treatment. (D) Interaction between OsDRP1E

and ATG8d in yeast. (E) Transcript levels of autophagy components in transgenic rice protoplasts

when transformed with OsDRP1E-GFP plasmids. Bars represent means ± SD, n = 3. Asterisks indicate

significant differences between the transgenic line and NPB according to a Student's t-test. (F) The

dosage dependent effect of MoCDIP4 on the OsDjA9-OsDRP1E interaction in GST pull-down assay.

Relative band intensity of each lane below the panel is determined by ImageJ.

NPB

#18

#22

#25

#32

#38

#41

0.00

0.06

0.12

OX-OsDjA9 RNAi-OsDjA9

ns

nsns

ns

ns

ns

P=0.2464

P=0.1622

P=0.4996

P=0.6582

P=0.5303

P=0.9213

Rel

ativ

e tr

ansc

ript

ion

(OsDRP1E/OsUbq

)

A

NPB

#10

#16

#22

0.00

0.07

0.14

OX-MoCDIP4ΔSP

P=0.0829

ns

nsns

P=0.2135

P=0.7526

Rel

ativ

e tr

ansc

ript

ion

(OsDRP1E/OsUbq

)

B

NPB

OX-MoCDIP4ΔSP

OX-OsDjA9

CR-OsDjA9 NP

B

OX-MoCDIP4ΔSP

OX-OsDjA9

CR-OsDjA9 NP

B

OX-MoCDIP4ΔSP

OX-OsDjA9

CR-OsDjA9

0.0

0.5

1.0

1.5

2.0

2.5

ATG8dATG8c ATG9

P=0.0013

**

*

P=0.0101

P=0.1066

P=0.0176P=0.0044

P=0.0029

P=0.3054

P=0.0497

P=0.0128

**

**

**

*

nsns

P=0.0101

Rel

ativ

e tr

ansc

ript

ion

(/NP

B)

OsDRP1E pGADT7

AD

ATG8d

SD

-LW

SD

-LW

HA

BD

OsDRP1E

pGADT7

pGBKT7

OsDRP1E-GFP

OX-OsDjA9

1.00 0.97 0.41 0.90 1.00 0.80 1.69 2.37 3.29 α-Actin

α-GFP

C

D E F OsDjA9-GST+OsDRP1E-His

+MoCDIP4ΔSP-MBP 0× 0.5×1.0×2.0×3.0×4.0×

α-MBP

α-GST

α-His

α-MBP

α-GST

α-His

Input IP (α-G

ST)

91 kDa

75 kDa

78 kDa

91 kDa

75 kDa

78 kDa 1.00 0.92 0.85 0.80 0.60 0.55

Fig. S8. OsDjA9 does not affect the protein levels of MoCDIP4.

MoCDIP4 was expressed in NPB and OsDjA9 overexpression rice

protoplasts. Protoplasts were sampled at 16 h, 20 h, and 24 h after the

transfection. The actin protein was used as the internal control.

16h 20h 24h

MoCDIP4ΔSP-4*HA

α-HA

α-Actin

Table S1. Primers are used in this study.

Primers name Sequence (5’-3’) Purpose PAL1-QF AGCACATCTTGGAGGGAAGCT qRT-PCR PAL1-QR GCGCGGATAACCTCAATTTG OsUbq-QF CGCAAGAAGAAGTGTGGTCA qRT-PCR OsUbq-QR GGGAGATAACAACGGAAGCA WRKY45-QF ACGACATTATGGGTTTGAGCTT qRT-PCR WRKY45-QR GAGACGACACATCAACAAGGAA PBZ1-QF CCCTGCCGAATACGCCTAA qRT-PCR PBZ1-QR CTCAAACGCCACGAGAATTTG PR10-QF ATGAAGGAGAGGCTGGAGTTC qRT-PCR PR10-QR CCTTAGCCTTGGTGATCTCGT OsUbq-GQF TTCTGGTCCTTCCACTTTCAG Fungal biomass OsUbq-GQR ACGATTGATTTAACCAGTCCATGA MoPOT2-GQF ACGACCCGTCTTTACTTATTTGG Fungal biomass MoPOT2-GQR AAGTAGCGTTGGTTTTGTTGGAT OsDjA9-QF ATCCGACTCAACATAGTGCCTG qRT-PCR OsDjA9-QR TTGCCTGAGTCACGTTCAATAC OsDRP1E-QF CTATCCGTGCAAAATGTGAAGA qRT-PCR OsDRP1E-QR TTCCTCACCAAGTCCCTTAAAA MoCDIP4-RT-F TACATCTTCAGCATCGTCTTCG RT-PCR MoCDIP4-RT-R CTCGATGAACTCGTTGTACCAG pRHV-MoCDIP4ΔSP-F CGGGATCCATGCACTACATCTTCAGCATCG pRHV,

pRTVcHA pRHV-MoCDIP4-R CGAGCTCCAAGCACTGGCTGTAGTACTG pRHV-OsDjA9-F CGAGCTCATGCGGCTCCCCGGCGACGCT pRHV,

pRTVcHA, pRTVcMyc

pRHV-OsDjA9-R GGGGTACCTCCCGATGCTCCTGCTGCCTTT

pANDA-OsDjA9-F CGGGATCCGCGGCGGTGGAATGAATG pANDA pANDA-OsDjA9-R CCGCTCGAGCCTTCACAGTCTTCCCAC U6a-OsDjA9-F GCCGGTCCATCCGGATACAAATAA pYLCRISPR/Cas

9Pubi-H U6a-OsDjA9-R AAACTTATTTGTATCCGGATGGAC U6a-OsDRP1E-F GCCGCGAGCATGGAGGGTCTGAT pYLCRISPR/Cas

9Pubi-H U6a-OsDRP1E-R AAACATCAGACCCTCCATGCTCG pRHV-OsDRP1E-F CGGGATCCATGGCGAGCATGGAGGGTCT pRHV,

pSPYNE(R)173 pRHV-OsDRP1E-R GGGGTACCCCTGGTCCATGCGACAGAGT pMAL-c2-MoCDIP4-F AGAATTCATGAAGTCGACAACCTTCCT pMAL-c2 pMAL-c2-MoCDIP4-R GCTCTAGACTACAAGCACTGGCTGTAGTA pMAL-c2-MoCDIP4ΔSP-F AGAATTCATGCACTACATCTTCAGCATCG pMAL-c2 pGEX-6p-1-OsDjA9-F TCCCCCGGGTATGCGGCTCCCCGGCGACGCT pGEX-6p-1 pGEX-6p-1-OsDjA9-R CCCTCGAGCTATCCCGATGCTCCTGCTGC pGADT7-OsDjA9-R CCCTCGAGCCTATCCCGATGCTCCTGCTGC pGADT7 pGADT7-OsDjA11-F GGAGGCCAGTGAATTCATGGCGCGCGCCGCCGCCTC pGADT7 pGADT7-OsDjA11-R TCGAGCTCGATGGATCCCTCATCCGGAGGCAGCTGCAAC pCAMBIA1300-cLUC-OsDjA11-F

TACGCGTCCCGGGGCGGTACCATGGCGCGCGCCGCCGCCTC pCAMBIA1300-cLUC

pCAMBIA1300-cLUC-OsDjA11-R

ACGAAAGCTCTGCAGGTCGACTCATCCGGAGGCAGCTGCAAC

pCAMBIA1300-cLUC-OsDjA9-F

GGGGTACCATGCGGCTCCCCGGCG pCAMBIA1300-cLUC

pCAMBIA1300-cLUC-OsDjA9-R

GCGTCGACCTATCCCGATGCTCCTGC

pCAMBIA1300-nLUC-MoCDIP4ΔSP-F

CGGGATCCAATGCACTACATCTTCAGCATCG pCAMBIA1300-nLUC, pCAMBIA1300-pCAMBIA1300-nLUC- GCGTCGACCAAGCACTGGCTGTAGTAC

MoCDIP4ΔSP-R cLUC pGBKT7-MoCDIP4ΔSP-F CATGGAGGCCGAATTCATGCACTACATCTTCAGCATCG pGBKT7 pGBKT7-MoCDIP4ΔSP-R TGCAGGTCGACGGATCCCCTACAAGCACTGGCTGTAGTAC MoCDIP4-pro-F CATAGTCTATATAAGGCACGCTCATTACCATG MoCDIP4

knockout strain

MoCDIP4-pro-R TTGACCTCCACTAGCTCCAGCCAAGCCACTTCATGATGGGCGGGGGGA

MoCDIP4-3UTR-F GAATAGAGTAGATGCCGACCGCGGGTTGGCTGGGAAGATTGAGGATGTGTG

MoCDIP4-3UTR-R AGAGCTTTGGGGCAGTAAGATA pKNGT-MoCDIP4-pro-F GGGAACAAAAGCTGGGTACCTCAAACAGATCAAGAGGGATC

A MoCDIP4 complementation strain pKNGT-MoCDIP4-CDS-R CTGCAGGCATGCAAGCTTCAAGCACTGGCTGTAGTACTGG

pSPYCE(M)-OsDjA9-F GCTCTAGAATGCGGCTCCCCGGCGACGCT pSPYCE(M) pCAMBIA1300-nLUC-OsDRP1E-F

ACGGGGGACGAGCTCGGTACCATGGCGAGCATGGAGGGTCTG

pCAMBIA1300-nLUC

pCAMBIA1300-nLUC-OsDRP1E-R

CGCGTACGAGATCTGGTCGACCCTGGTCCATGCGACAGAGTC

pGADT7-OsDRP1E-F GGAATTCCATATGATGGCGAGCATGGAGGGTCT pGADT7, pGBKT7 pGADT7- OsDRP1E-R CGGGATCCCCTACCTGGTCCATGCGACAGA

pSUC2-SP-F AATTCATGAAGTCGACAACCTTCCTCAGCCTGCTGGCGGCTCCGCTGGCCGCGCAGGCGC

pSUC2

pSUC2-SP-R TCGAGCGCCTGCGCGGCCAGCGGAGCCGCCAGCAGGCTGAGGAAGGTTGTCGACTTCATG