Transient assays for the analysis of miRNA processing and function.

Abstract

Transient assays provide a convenient alternative to stable transformation. For small RNA analysis in plants, the most widely used method, commonly named agroinfiltration, makes use of Agrobacterium tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Agroinfiltration has been used successfully in many different applications, including the analysis of small RNAs. We describe here a protocol for analysis of miRNA processing using agroinfiltration of N. benthamiana leaves.

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Chapter 17
Transient Assays for the Analysis
of miRNA Processing and Function
Felipe F. de Felippes and Detlef Weigel
Abstract
Transient assays provide a convenient alternative to stable transformation. For small RNA analysis in
plants, the most widely used method, commonly named agroinfiltration, makes use of Agrobacterium
tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. Compared to the generation
of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after
inoculation. Agroinfiltration has been used successfully in many different applications, including the
analysis of small RNAs. We describe here a protocol for analysis of miRNA processing using agroinfiltra-
tion of N. benthamiana leaves.
Key words: Agroinfiltration, Transient assay, miRNA processing, Small RNA blot
1. Introduction
The in vivo study of small RNAs requires, in many cases, the use
of transgenic techniques. However, generation of stable transgenics
for many flowering plants is labor-intensive and time-consuming.
Even for species such as Arabidopsis thaliana, for which simple
protocols for plant transformation are available (1, 2), the genera-
tion of stable transgenics can take months. As an alternative to the
generation of stable transformants, transient introduction of
transgenes has been used successfully in a large number of appli-
cations including the analysis of small RNAs, promoters and
suppressors of RNA silencing (3–7) and the study of gene
function (8). The greatest advantage of transient assays over the
generation of stable transformants is its rapidity; transgene
activity/expression can be assayed usually within a few days after
B.C. Meyers and P.J. Green (eds.), Plant MicroRNAs, Methods in Molecular Biology, vol. 592
DOI 10.1007/978-1-60327-005-2_17, © Humana Press, a part of Springer Science + Business Media, LLC 2009
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256 de Felippes and Weigel
transfection (9). The most popular method of transient assay in
plants is the infiltration of Agrobacterium tumefaciens containing
T-DNA in Nicotiana benthamiana leaves, due to the ease of
manipulation and robust transgene expression. In addition, this
method, agroinfiltration, does not require special equipment
(9–11). Finally, the same vectors can often be used for generation
of stable transformants, so that results obtained after agroinfiltra-
tion can be confirmed with stably transformed plants. The tran-
sient expression of the miR319a precursor in N. benthamiana
leaves, RNA isolation, and posterior detection of the mature
miRNA will be described here as an example for the use of tran-
sient assays in miRNA validation. The agroinfiltration protocol
described here is based on technology developed in the labora-
tory of James Carrington, Oregon State University.
2. Materials
1. T-DNA vectors.
2. Oligonucleotide primers.
3. Taq DNA polymerase, restriction enzymes, T4 DNA ligase,
dNTPs, LR ClonaseTM (Invitrogen), agar, agarose.
4. E. coli strain DH5a.
5. A virulent A. tumefaciens strain, e.g., ASE, GV3101,
LBA4404.
6. N. benthamiana plants.
7. Growth medium: Luria-broth (LB) with 50 mg/mL kanamy-
cin (A. tumefaciens ASE selection), 25 mg/mL chloramphen-
icol (A. tumefaciens ASE selection), 100 mg/mL spectomycin
(binary vector selection) and 5 mg/mL tetracycline (pSoup
selection).
8. Infiltration medium: 10 mM MgCl2, 10 mM MES pH 5.7,
150 mM acetosyringone.
9. Hypodermic needle and plastic syringe.
10. Mortar and pestle.
11. TRIZOL® reagent (Invitrogen) (toxic; causes burns).
12. Polyacrylamide gel: Polyacrylamide (neurotoxic while unpo-
lymerized), urea, APS (ammonium persulfate) and TEMED.
13. 10× TBE: 54 g Tris base, 27.5 g boric acid, 20 mL 0.5 M
EDTA, water to 500 mL. Adjust pH to 8.4.
14. Formamide (toxic) and loading dye (RNAse free).
15. Nytran SuPerCharge nylon transfer membrane (Schleicher
& Schuell Bioscience).

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Transient Assays for the Analysis of miRNA Processing and Function 257
16. Extra thick blot paper (BioRad).
17. Trans-Blot® SD semi-dry transfer cell (BioRad).
18. UV Stratalinker® 2400 (Stratagene).
19. Gamma 32P-ATP (10 mCi/mL) (radioactive hazard).
20. Optikinase (USB).
21. Micro Bio-Spin® 6 chromatography columns (BioRad).
22. PerfectHybTM Plus hybridization buffer, 1× (Sigma) (irritant).
23. 20× SSC: 3 M NaCl, 0.3 M sodium acetate. Adjust the pH
to 7.0 with HCl.
24. 10% (w/v) sodium dodecyl sulfate (SDS).
25. X-ray film and X-ray cassette.
26. Photographic film developing solutions.
3. Methods
In this section, we describe (a) the construction of the binary
vector containing the precursor for miR319, (b) the agroinfiltra-
tion of N. benthamiana leaves, and (c) the analysis of miRNA
processing.
The following subsections contain a description of the vectors
used in the transient assay (Subheading 3.1.1) and a description of
the steps for expression of plasmid construction (Subheading
3.1.2).
The molecular basis of A. tumefaciens infection involves the
transfer of a DNA segment called T-DNA (transfer DNA) from
the tumor-inducing (Ti) plasmid of the bacterium to the plant
cell (12, 13). For biotechnological purposes, binary systems are
preferred, in which the trans function for T-DNA transfer are all
encoded on a disarmed Ti plasmid that lacks the tumor-inducing
sequences. For ease of manipulation during the cloning steps, the
T-DNA itself is placed on a separate plasmid, hence the term
“binary.” These plasmids have origins of replication that allow
propagation in both E. coli and A. tumefaciens (13).The native
T-DNA sequences, which cause tumor formation, are replaced by
the sequence of interest, in this case for the expression of miRNAs
(13). In the experiment described in this chapter, a modified
binary plasmid based on pGreenII vectors is used (pFK210; Frank
Küttner and Markus Schmid, pers. communication) (14). pFK210
is Gateway compatible (15), includes a gene conferring spectino-
mycin resistance for selection in E. coli and a T-DNA defined by
the characteristic left and right borders (12, 13). The T-DNA
3.1. Construction
of the Binary Vector
for Agroinfiltration
3.1.1. Description of the
Plasmids

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258 de Felippes and Weigel
region contains the BAR gene conferring resistance to the
herbicide glufosinate (trade name Basta®) for selection of stable
transformants (see Note 1), an expression cassette formed by a
recombination module (attR1-ccdB-attR2, discussed below) (15),
flanked by the strong constitutive promoter CaMV 35S and a
transcriptional terminator from the Pisum sativum ribulose 1,5-bis-
phosphate carboxylase/oxygenase small subunit (rbcS) gene.
To minimize the size of the plasmid and hence increase
the ease of cloning procedures, the pSa replication region in the
pGreenII plasmid lacks the RepA gene, which is necessary for
replication in A. tumefaciens. Therefore, pGreenII plasmids need
to be co-transformed with pSoup (14). pSoup contains, besides
the RepA gene, a tetracycline resistance gene for A. tumefaciens
and E. coli selection.
Before recombination of the sequence of interest into the
expression cassette in pFK210, it is necessary to adapt this mole-
cule to the GATEWAY® technology. This can be done using an
entry vector containing a multiple cloning site (MCS) flanked by
the attL1 and attL2 sites, which can be recombined by a recom-
binase with the attR1 and attR2 sites present in pFK210 (15).
The entry vector normally contains a gene conferring kanamy-
cin resistance for selection in E. coli.
All procedures described here were performed using standard
recombinant DNA methodologies (16). The 400 nucleotide
fragment containing the miR319a precursor (17) was amplified
by PCR from A. thaliana genomic DNA. Restriction sites for
EcoRI and BamHI were included in the sequence of the forward
and reverse primers, respectively. These enzymes were used for
digestion of the PCR product and subsequent ligation into an
entry vector digested with the same enzymes. The resulting plas-
mid was recombined with pFK210, generating the final binary
plasmid, which was introduced in A. tumefaciens (strain ASE)
together with pSoup via electroporation (18). All clones were
confirmed by DNA sequencing (19). Presence of the binary
vector in A. tumefaciens was confirmed by colony PCR, using
specific primers.
In the transient assay described here, wild-type N. benthamiana
plants were used. Seeds were incubated for 3 days in the dark at
4° C soaked in 0.1% agar solution. Plants were grown on soil under
long day condition (23° C, 16 h of light). Two to five-weeks old
plants were used.
1. Inoculate 5 mL of growth medium with a single colony of
transformed A. tumefaciens. Incubate the culture for 20 h at
28–30° C with vigorous shaking.
3.1.2. Generation
of the Expression Vector
3.2. Agroinfiltration
of N. benthamiana
Leaves
3.2.1. Plant Material
3.2.2. Agroinfiltration
of N. benthamiana Leaves

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Transient Assays for the Analysis of miRNA Processing and Function 259
2. Use 2 mL of the culture from step 1 to inoculate 50 mL of
growth medium. Incubate for 16–20 h at 28–30° C with
vigorous shaking.
3. Recover the bacteria by centrifugation of the culture at
2,000 × g (4,000 rpm in a Sorvall SS-34 rotor) for 10 min at
4° C. Remove the supernatant.
4. Resuspend the bacterial pellet in 30 mL of infiltration medium
and incubate 16–20 h at room temperature (21–23° C) with
gentle shaking (see Note 2).
5. Adjust the volume of the culture with the infiltration medium
to a final concentration corresponding to an optical density
(OD) of 0.5 at 600 nm.
6. With a sharp hypodermic needle, make superficial wounds in
the abaxial side of the leaf. Make sure that the wounds do not
perforate the leaf, which could decrease the efficiency of the
infiltration (see Note 3) (Fig. 1).
7. Using a 5 mL syringe without the needle, infiltrate, through
the wounds, the A. tumefaciens solution into the leaf. Use
your fingertip to apply gentle counter pressure to the other
side of the leaf.
8. Mark the leaf by attaching a small tag to the petiole and repeat
steps 6 and 7 on one or two more leaves.
Abaxial
Needle
Infiltrated
area
Adaxial
Fig. 1. Diagram of N. benthamiana leaf and agroinfiltration.