The Asparagine-rich Protein NRP Facilitates the Degradation of the PP6-type

Phosphatase FyPP3 to Promote ABA Response in Arabidopsis

Tong Zhu1, Yanying Wu1, Xiaotong Yang1, Wenli Chen1, Qingqiu Gong2*, Xinqi Liu1*

1State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry

and Molecular Biology, College of Life Sciences, Nankai University, Tianjin, 300071,

China2Tianjin Key Laboratory of Protein Science, Department of Plant Biology and

Ecology, College of Life Sciences, Nankai University, Tianjin, 300071, China

Running title: NRP and FYPP3 interaction in ABA response

*Corresponding authors: Xinqi Liu, Department of Biochemistry and Molecular

Biology, College of Life Sciences, Nankai University, Tianjin, 300071, China. Phone:

86-22-23505130, email: liu2008@nankai.edu.cn; Qingqiu Gong, Department of Plant

Biology and Ecology, College of Life Sciences, Nankai University, Tianjin, 300071,

China. Phone: 86-22-23503914, email: gongq@nankai.edu.cn

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Figure S1 Transcript levels and phenotypes of transgenic lines used in this study.

(A), (C), (E), (G) The transcript levels of FyPP3 or NRP in FyPP3/FyPP1 RNAi,

FyPP3-RFP, FyPP3DN-RFP and NRP-GFP lines. Three biological replicates, each

composed of four technical repeats were carried out for each gene in each line.

EF1a was used as an internal control. Bars = SD.

(B), (D), (F), (H) Phenotypes in each line in (A), (C), (E) and (G). The representative

seedlings are shown. Arrow heads indicate abnormal root growth.

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Figure S2 Co-localization of NRP-GFP with the plasma-membrane marker PIP2;1-

RFP and the late endosome (LE) marker FAB1a-RFP. Bar = 25 μm.

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Figure S3 The relative transcript levels of NRP and FyPP3 are correlated with each

other in different tissues and organs. Roots and shoots were collected from 7-day-

old seedlings, and other tissues were collected from 6-week-old WT plants. EF1a,

TIP4;1-like and AP2M were used as internal controls in (A)-(C). The left vertical axis

was shown for NRP, and the right axis was shown for FyPP3. r = Pearson’s

correlation coefficient calculated from the transcript levels of NRP and FyPP3 in (A)-

(C). The correlation among three internal controls was shown in (D). At least 3

biological replicates, each composed of 4 technical repeats, were used for each

gene. Bars = SD.

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Figure S4 The promote activities of NRP and FyPP3 in different tissues indicated by

GFP.

(A)-(D) The promoter activities in 26-hour-old geminating seeds without (-) or with (+)

1.5 μM ABA. (A)-(C): Bar = 75 μm. (D): Bar = 25 μm.

(E)-(G) The promoter activities in 7-day-old seedlings. (E): Bar = 25 μm. (F), (G): Bar

= 75 μm.

(H)-(N) The promoter activities in 6-week-old plants. (H), (I), (K), (M): Bar = 75 μm.

(J), (L), (N): Bar = 25 μm.

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Figure S5 The relative transcript levels of NRP and FyPP3 are strongly correlated

with each other in seeds and seedlings upon ABA treatment.

(A)-(C) The transcript levels of NRP and FyPP3 in WT seeds with or without ABA

treatment. Dry seeds were immersed in water or 5 μM ABA for 24 hours before

collected. EF1a, TIP4;1-like and AP2M were used as internal controls in (A)-(C). r =

Pearson’s correlation coefficient calculated from the transcript levels of NRP and

FyPP3. At least 3 biological replicates, each composed of 4 technical repeats, were

used for each gene. Bars = SD.

(D) The correlation among the three internal controls in (A)-(C).

(E)-(G) The transcript levels of NRP and FyPP3 in WT seedlings with or without ABA

treatment. Seven-day-old seedlings were immersed in water or 5 μM ABA for 24

hours before collected. EF1a, TIP4;1-like and AP2M were used as internal controls

in (E)-(G). The left vertical axis was shown for NRP, and the right axis was shown for

FyPP3. At least 3 biological replicates, each composed of 4 technical repeats, were

used for each gene. Bars = SD.

(H) The correlation among the three internal controls in (E)-(G).

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Figure S6 The transcript levels of ABA response related genes in various lines. (A),

(B): RAB18. (C), (D): RD29B. (E), (F): MYC2. TIP4;1-like was used as an internal

control in (A), (C), and (E). AP2M was used as an internal control in (B), (D), and (F).

At least three biological replicates, each composed of four technical repeats, were

performed for each gene. Bars = SD.

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Table S1 Proteins preyed from yeast two-hybrid screening using NRP as bait. FyPP3 (AT3G19980) in this list was shown as bold.

Accession Number Description CopiesAT3G24120 Homeodomain-like superfamily protein 11AT4G13640 unfertilized embryo sac 16 (UNE16) 10AT3G53870 Ribosomal protein S3 family protein 6AT3G11540 Encodes an N-acetyl glucosamine

transferase that may glycosylate other molecules involved in GA signaling.

4

AT5G12130 PIGMENT DEFECTIVE 149 (PDE149) 2AT5G14740 Encodes a beta carbonic anhydrase likely

to be localized in the cytoplasm. 2

AT1G78460 SOUL heme-binding family protein 2AT3G19980 Encodes catalytic subunit of

serine/threonine protein phosphatase 2A.

1

AT2G37460 nodulin MtN21-like transporter family protein

1

AT3G11470 4'-phosphopantetheinyl transferase superfamily

1

AT1G75270 Dehydroascorbate reductase 2 (DHAR2) 1AT5G16510 REVERSIBLY GLYCOSYLATED PROTEIN

5, RGP5 is a member of the reversably glycosylated family of proteins.

1

AT5G56370 F-box/RNI-like/FBD-like domains-containing protein

1

AT1G06760 Winged-helix DNA-binding transcription factor family protein

1

AT5G05000 Outer membrane GTPase protein that may function in import of nuclear encoded proteins into the chloroplast.

1

AT4G25260 Plant invertase/pectin methylesterase inhibitor superfamily protein

1

AT1G67280 Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily protein

1

AT3G20480 Tetraacyldisaccharide 4'-kinase family protein

1

ATCG00490 large subunit of RUBISCO. 1

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Table S2 Primers used in this study.

Name Sequence (5’ to 3’)Primers used for FyPP3DN and FyPP3/FyPP1 RNAi generation

FyPP3DN-F ATGGGGGACTTCGTGAATAGAGGTTACAACAGCFyPP3DN-R GCTGTTGTAACCTCTATTCACGAAGTCCCCCAT

FyPP3/FyPP1 RNAi-LIC1

CGACGACAAGACCCTGATCTTATGTGGAGTGATCC

FyPP3/FyPP1 RNAi-LIC2

GAGGAGAAGAGCCCTGTTGTCATTGAAACTCAATATAG

FyPP3/FyPP1 RNAi-LIC3-TT-

LIC2CCAGCACGGAACCCTTGAGGAGAAGAGCCCT

FyPP3/FyPP1 RNAi-LIC4-TT-

LIC1AGAGCACACGACCCTTCGACGACAAGACCCT

Primers used for real-time PCRFyPP1-1 ACCTGAACCTCTTCTGGATGTFyPP1-2 TGGTCTTGCTTACAGAACAAAGATGFyPP3-1 CCCTTGTGAAAGTTACAAGTCAAFyPP3-2 TGGGGGAAAGAAAAGAGAGCANRP-1 CTGGCGAATCTCGTTTCCCTNRP-2 ACCGTCGTAGTGGTGCAAAAEF1a-1 GATGACTCCAACCAAGCCCAEF1a-2 CACAGCGAAACGTCCAAGTG

RD29B-1 TGGAGTCACAGTTGACACGED29B-2 CCTTCTCATGATGCTCTTCTTCTTCRAB18-1 AGCTCGGAGGATGATGGACARAB18-2 TCGCTTGAGCTTGACCAGACMYC2-1 GGAATGACTGATTACCGGCTACAMYC2-2 TCCATCATAGAAGCGTTGTCGT

Primers used for ProNRP:GFP and ProFyPP3:GFP generationProNRP:GFP-

FGACCTGCAGGCATGCAAGCTTGCTCTTTCTCTATGAATTT

ProNRP:GFP-R

TACTAGTCAGATCTACCATGGTCTCTAACTCTCTGATTGATCT

ProFyPP3:GFP-F

GACCTGCAGGCATGCAAGCTTGCGACTTCCACAAGTGTTGTTG

ProFyPP3:GFP-R

TACTAGTCAGATCTACCATGGTAACTCTCTCTCTATATATATA

1302Pro-CEXU-R-GFP

GCCCATTAACATCACCATCTAATTC

Primers used for the verification of hybrid plants and transgenic plants

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Salk041306F TTCTCCCTATTTTTGGGGTTGSalk041306R CAGAGGAACGAAGACGACAAG

LBb1.3 ATTTTGCCGATTTCGGAAC35S-F CTCCACTGACGTAAGGGATGACGABI5-R TACAAAGCGGAGCTGGAAGTGTC

salk041306RT-F

CAAAGGGGTTTACACGTCGATGAAG

salk041306RT-R

TCAAGGGTTTTGGTCAGCAAAAATG

ProFyPP3:GFP-F

GACCTGCAGGCATGCAAGCTTGCGACTTCCACAAGTGTTGTTG

GFP-R TTTCCGTATGTTGCATCACCTTCProNRP:GFP-

FCTATCGGCACAAATAAATCTCCTC

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