ACCESSING MULTI-TIERED AND CELL-SPECIFIC REGULATION OF RESPONSES TO WATER EXTREMES IN RICE

Germain Pauluzzi1*, Mauricio A. Reynoso1*, Kaisa Kajala2,3, Sigrid Heuer4, Neelima Sinha2, Roger Deal5, Siobhan M. Brady2,3 and Julia Bailey-Serres1

1 UC Riverside, Center for Plant Cell Biology, Botany and Plant Sciences Department 2 Department of Plant Biology, UC Davis, Davis, California, 95616 USA 3 Genome Center, UC Davis, Davis, California, 95616 USA 4 Australian Centre for Plant Functional Genomics 5 Department of Biology, Emory University, Atlanta, GA, 30322 *Germain Pauluzzi and Mauricio A. Reynoso contributed equally to this work

References & Acknowledgements 1) Bailey Serres et al (2012). Trends in Plant Sci 17, 130. 2) Deal et al (2010). Nature Prot 6, 56. 3) Zanetti et al (2005). Plant Phys 138, 624 This work was supported by NSF DBI – 1238243 of USA. The authors would like to thank members of each lab for valuable help.

Translating Ribosome Affinity Purification (TRAP)

Root cell-type specific promoters

Isolation of Nuclei TAgged in specific Cell Types (INTACT)

FUTURE WORK

Floods and droughts are increasingly experienced in agricultural systems worldwide. In rice (Oryza sativa L.), these extremes in water availability invoke responses at the organ-, tissue- and cell-specific levels that enable metabolic to developmental responses relevant to survival. For example, the SUB1A gene is induced by submergence in meristematic regions. It confers tolerance to complete submergence by limiting growth, reducing cell damage and, protecting axillary shoot meristem viability (1). In the root system, specific cell types play varied roles under different water availability conditions. As an example, several layers of cortex differentiate in lysigenous cortical aerenchyma. These large lacunae serve as gas conduits from aerial tissue to the root and alleviate oxygen deprivation in waterlogged and compact soils. Furthermore, a layer of exodermis and sclerenchyma situated between the epidermis and the cortex are critical in limiting radial oxygen loss and water stress. Our project goal is to assess different layers of gene regulation of specific cell-types during development and in response to extremes in water availability. To achieve this goal we have established technologies that allow access to the epigenetics modifications (epigenome), nuclear transcriptome and ribosome-loaded transcripts (translatome) of target cell-types in rice. Isolation of Nuclei TAgged in specific Cell Types (INTACT) (2) has been transferred to rice to affinity purify nuclei, survey chromatin (i.e., histone modifications) and quantify nuclear RNA populations. Translating Ribosome Affinity Purification (TRAP) (3) has been established in rice to immunopurify ribosomes and associated mRNAs. As a proof-of-concept we employed both technologies with near-constitutive and cell-type specific promoters to drive expression of the fusion proteins. For INTACT we used the WPP1 nuclear envelope targeting sequence-GFP-biotin ligase recognition peptide fusion construct developed for Arabidopsis. The biotin ligase protein was codon optimized for rice. For TRAP we used a His-FLAG-GFP-tagged version of a rice ribosomal protein L18 (RPL18). The effectiveness of these reagents in transgenic rice was compared to Agrobacterium rhizogenes-transformed tomato roots and transgenic Arabidopsis seedlings. We have characterized a number of root cell-specific and meristematic promoters from rice for our collaborative cross-species analysis of water stress responses in rice, tomato and Medicago.

Pre-mRNA

Chromatin TRAP Yields:

Polysomal mRNA (polymRNA-seq) Ribosome footprints (Ribo-seq)

INTACT Yields:

Chromatin for histone analyses (i.e. H3K27me3, H3k4me3) Nuclear RNA for transcriptome (nucRNA-seq)

mRNA

Polysomal RNA AAAA…

ER

AAAA…

smRNAs

FLAG-tagged ribosomes

Biotin-tagged protein associated to nuclear envelope (NTF)

promoter GFP OsRPL18 pUBI (maize) HygR HF

RB Tnos Tnos

HF-GFP-RPL18

Promoter GFP BLRP BirA pOsACT2 pUBI (maize) HygR

(Os optimized)

Nuclear tagging fusion (NTF)

WPP Domain RB Tnos T35S LB Tnos 3X myc (from AtRANGAP1)

Proof-of-concept using 35S Promoter

35S:NTF transgenic roots

Agrobacterium tumefaciens transformation of rice calli Optimized Biotin Ligase BirA is

expressed in transgenic rice

BirA 3X myc

40

35

35

S:TR

AP

-

con

tro

l

Lin

e 2

.3

Lin

e 3

.2

Lin

e 4

.3

Western blot α-myc

kDA

Biotin Ligase Recognition

Peptide

Transgenic tissue promoter:NTF ACT2:BirA

Crude Nuclei preparation Binding to Streptavidin Dynabeads

Nuclei Quantification Extraction of DNA, RNA

or Proteins

Nuclei visualization

Propidium iodide staining Bright field

70 55 40 35

S10

00

P1

00

0

UB

BEA

DS

S10

00

P1

00

0

UB

BEA

DS

p35S:NTF/ pACT2:BirA

Wildtype

NTF ~41 kDA

Streptavidin Western Blot

15

10

Wild

typ

e

p3

5S:

NTF

/ p

AC

T2:B

irA

Histone H3

~15 kDA

Histone H3 is present in affinity purified nuclear fractions

No

DN

A

Wild

typ

e

GADPH transcript

p3

5S:

NTF

/pA

CT2

:Bir

A

RT-PCR for GADPH

kDA

kDA

Western blot α-Histone H3

NTF is present in crude and affinity purified nuclear fractions

Nuclear tagging fusion (NTF)

mRNA is detected on nuclear fractions

BEADS BEADS

Agrobacterium tumefaciens transformation of rice calli

Sucrose Gradient Sedimentation

Ab

sorb

ance

(2

54

nm

)

40S

60S

80S

polysomes

70

55

40

35

25

Western α-FLAG

α-RPS6

kDa

Proof-of-concept using 35S Promoter

35S:HF-GFP-RPL18 transgenic roots

nucleolus

cytosol

HF-GFP-RPL18 is incorporated into polysomes

TRAP is feasible in O. sativa

+

53 kDa

Unbound Total

Wild

typ

e

35

:HF-

GFP

- R

PL1

8

Wild

typ

e

35

:HF-

GFP

- R

PL1

8

Wild

typ

e

35

:HF-

GFP

- R

PL1

8

Western

a-FLAG

TRAP

RT-PCR ACTIN1

TRAP samples yield HF-GFP-RPL18, which is incorporated into polysomes, ribosomal RNA (rRNAs) and mRNAs associated with these complexes involved in translation

Nuclei yield is estimated in about 350,000 nuclei per gram of leaves tissue

Chromatin Immunopurification (ChIP) is feasible using INTACT purified rice nuclei

S1000 Supernatant of the crude nuclei preparation P1000 Crude nuclei extract UB Unbound fraction BEADS INTACT fraction bound to Streptavidin Dynabeads

35S:NTF ACT2:BirA leaves treated with formaldehyde

α- H3K4me3 0.5 ug

α- GFP 0.6 ug (neg. control)

INTACT ~100,000 nuclei as input

Inp

ut

DN

A

Ch

IP G

FP

No

DN

A

Ch

IP H

3K

4m

e3

GADPH PCR Amplification

Can nuclei yield be improved for rice?

-Generation of rice transgenic lines expressing NTF and HF-GFP-RPL18 under cell-type specific promoters -Drought and submergence treatments on TRAP and INTACT lines - Translatome, epigenome and nuclear transcriptome for systematic comparison with tomato and Medicago - Identification of genetic components that specify exodermis, sclerenchyma and Cortex

GFP BLRP

(from OsRANGAP1) WPP Domain

Replacement of WPP domain with O. sativa homologue to AtRANGAP1

GFP BLRP WPP Interacting Protein Design of GFP fusion protein containing O. sativa homologue to AtWIP1 (WPP interacting protein) containing a transmembrane domain

SUB1A is expressed in shoot basal meristem.

Epidermis Exodermis Sclerenchyma Cortex Endodermis

Pericycle

Xylem

Phloem

Cell-type Systems Approach

Total TRAP

25S

18S

Nuclear

5.8S 5S

* Plastid rRNA?

Agilent 2100 Analysis of RNA samples. Total RNA and TRAP polysomal RNA from leaves of Rice wild type and expressing HF-GFP-RPL18.

Root Meristem

4: Root Tip, Stele specific

3 Root System, OsSHR1 expressed in stele and lateral root primordia 2 Shoot Base, OsSHR1 expressed in nodal root primordia

1 OsSHR1 expressed in leaf lamina joint not leaf blade. TRAP done with leaf blade only.

Total TRAP Total TRAP Total TRAP Total TRAP

ACTIN1 OsSCR1

OsSHR1 OSH1

Cell Type specific polysomes immunopurification shown by ACTIN1, SHR1, SCR1 and OSH1 mRNA amplification by endpoint PCR. The absence of detection of OsSCR1 in TRAP fraction of root tips and of OSH1 in TRAP fraction of shoot base confirm the cell-specificity of the mRNAs

Cell-type specific TRAP using OsSHR1 promoter

Size Marker

Wildtype 35S:HF-GFP-RPL18

Total TRAP

1 mm

1 mm

1

2

3 4

1 mm

1 mm

Leaf blade Shoot base Roots Root Tips

Total TRAP Total TRAP Total TRAP Total TRAP

Leaf blade Shoot base Roots Root Tips

25 μm 5 μm 5 μm

LB

FLAG-tagged ribosomes can be immunopurified HF-GFP-RPL18 is present in TRAP sample

TRAP yields rRNAs and mRNAs

ACTIN1 mRNA is detected in TRAP sample

nucleus

p35S:NTF/ pACT2:BirA

INTACT yields nuclei in O. sativa

10 μm 10 μm

GADPH gene is amplified from ChIP DNA

AAAA…

AAAA…

Root Stele

GUS assay on T0 regenerated seedlings of cell-type specific promoter candidates.

Exodermis / Endodermis

0,5 mm 25 µm

0,5 mm 0,5 mm 25 µm

25 μm

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