Use of the giant multinucleate plasmodium of Physarum polycephalum to study RNA interference in the myxomycete.

Abstract

The plasmodium of Physarum polycephalum harbors billions of synchronized nuclei in a single cell of complex structure. Due to its synchrony and extreme size, it is used as a model to study events on a single cell level, such as cell cycle and differentiation. We show here for the first time that this model, despite its enormous size and structural complexity, is accessible to RNA interference by simple injection of dsRNA or siRNA. The targeted gene is that of polymalatase, an intracellular adapter of poly(beta-l-malate) involved in the maintenance of the synchrony and functioning as an extracellular hydrolase of this polymer. Real-time reverse transcriptase polymerase chain reaction analysis revealed that the specific mRNA was knocked down to about 10% of the original level. The suppression of a single injection lasted for approximately 14 cell cycles (144 h) and could be prolonged for any time by repeated dsRNA injections. Western blots indicated that the knockdown of RNA was paralleled by a strong reduction in polymalatase synthesis. However, a change in the phenotype of the plasmodium could not be clearly observed. In principle, the plasmodium offers an easy system for studying gene knockdown by RNA interference.

Full text

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ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 342 (2005) 194–199
www.elsevier.com/locate/yabio
0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2005.03.031
Use of the giant multinucleate plasmodium
of Physarum polycephalum to study RNA interference
in the myxomycete
Markus Haindl, Eggehard Holler¤
Institute of Biophysics and Physical Biochemistry, University of Regensburg, D-93053 Regensburg, Germany
Received 14 December 2004
Available online 12 April 2005
Abstract
The plasmodium of Physarum polycephalum harbors billions of synchronized nuclei in a single cell of complex structure. Due to
its synchrony and extreme size, it is used as a model to study events on a single cell level, such as cell cycle and diVerentiation. We
show here for the Wrst time that this model, despite its enormous size and structural complexity, is accessible to RNA interference by
simple injection of dsRNA or siRNA. The targeted gene is that of polymalatase, an intracellular adapter of poly(?-L-malate)
involved in the maintenance of the synchrony and functioning as an extracellular hydrolase of this polymer. Real-time reverse trans-
criptase polymerase chain reaction analysis revealed that the speciWc mRNA was knocked down to about 10% of the original level.
The suppression of a single injection lasted for approximately 14 cell cycles (144h) and could be prolonged for any time by repeated
dsRNA injections. Western blots indicated that the knockdown of RNA was paralleled by a strong reduction in polymalatase syn-
thesis. However, a change in the phenotype of the plasmodium could not be clearly observed. In principle, the plasmodium oVers an
easy system for studying gene knockdown by RNA interference.
 2005 Elsevier Inc. All rights reserved.
Keywords: RNA interference; Polymalatase; Polynucleate cells; Plasmodium; Microinjection
Physarum polycephalum is one of the plasmodium-
forming slime molds [1] of the class Myxomyceteae, a
member of the monophylum Mycetozoa, and is more
closely related to animals and fungi than to plants [2].
The plasmodium is a unique organism, representing a
giant vegetative single cell in the life cycle of this slime
mold. Its nuclei divide every 7–8h in high synchrony,
without dissolution of their nuclear envelope and in the
absence of cytokinesis, thus giving rise to a giant multi-
nucleate cell. The macroplasmodium (routinely in sizes
of 100cm2 or larger) grows easily in the laboratory as a
Xat, disk-like cell on the surface of a solid support.
Because of its large size, the plasmodium has evolved
devices to allow the intracellular equilibration of cellular
consitituents: (1) a complex macroscopic network of
veins consisting of a rhythmically moving endoplasm
surrounded by a resting ectoplasm, which displays a
complicated structure of Wbrils and invaginations of the
plasma membrane [3], and (2) a (putative) molecular
transporter consisting of polymalate and an adapter [4
and references therein]. By adopting a structural similar-
ity to the backbone of nucleic acids, polymalate binds
nuclear proteins, and the adapter guides the polymalate–
cargo complex to the various nuclei. In the extracellular
matrix, secreted adapter functions as hydrolase (poly-
malatase) of extracellular polymalate.
The plasmodium has been found suitable for studying
various cellular and organismic aspects such as cell cycle,
*Corresponding author. Fax: +49 941 943 2813.
E-mail address: eggehard.holler@biologie.uni-regensburg.de
(E. Holler).

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RNA interference in plasmodia of myxomycetes / M. Haindl, E. Holler / Anal. Biochem. 342 (2005) 194–199
195
motility, and diVerentiation. Timely questions are the
molecular basis of the synchrony [5] or the complex
intracellular network recruiting commitment and devel-
opment of sporulation [6,7]. The plasmodium is superior
to any other known model organism by virtue of its
enormous cellular size suitable for injections of materials
such as nucleic acids or other materials [8], the high syn-
chrony of cellular events [9], the fusibility of individual
plasmodia allowing the transfer of molecular informa-
tion [6,10], and the availability of both haploid and dip-
loid species [10] to study gene dominance. However,
attempts to knockout or overexpress genes by conven-
tional techniques were mostly unsuccessful, rendering
P. polycephalum an unsuccessful model system.
To overcome this problem, the goals of our investiga-
tion were (1) to demonstrate that, in contrast to its large
size and complex structure, the plasmodium was accessi-
ble to RNA interference by simple injection, (2) to assess
whether repeated injections give rise to enhanced sup-
pression eVects, (3) to measure the duration of the
achieved RNAi eVect, (4) to test whether the eVect con-
tinued after replating of plasmodia, (5) to examine
whether the eVect was reversible, and (6) to examine
whether, in the present case of the knockdown of poly-
malatase, a change in phenotype was detectable.
Materials and methods
dsRNA and siRNA
The DNA template to be transcribed into dsRNA
was synthesized by PCR using the cDNA derived from
P. polycephalum M3CVII plasmodia. Desalted primers
speciWc for the polymalatase–cDNA (Accession No.
AJ543320) with the sequences 5?-GTG TAA TAC GAC
TCA CTA TAG GGA AAA GGA GGT TCT GAT
CCT AGT-3? (forward primer) and 5?-CAC TAA TAC
GAC TCA CTA TAG GGA TCA CGA TGT CAT
CAG CAA AAC-3? (reverse primer), both containing
the T7-polymerase promoter at their 5? termini, were
custom-made by MWG-Biotech, Germany. The result-
ing 589-bp DNA spanned the nucleotides 1083–1671 of
the gene downstream of the origin of transcription and
was used as template for in vitro dsRNA synthesis as
described by Donze and Picard [11]. The 50-?l reaction
mixture was incubated for 15h and then treated for
30min with RNAse-free DNase (Qiagen, Germany) at
37°C. The mixture was Wnally heated to 95°C for 5min
and cooled to room temperature over a period of 7h.
Double-stranded RNA was precipitated with 2.5 vol-
umes of ethanol/0.2M sodium acetate, pH 4.9, pelleted
at 20,000g for 20 min at 4°C, washed with 70% ethanol,
air-dried, and resuspended in 60?l of diethylpyrocar-
bonate-treated RNase-free water. The integrity of the
dsRNA was validated by agarose gel electrophoresis in
Tris–acetate–EDTA-buVer, pH 8.0, before aliquots of
10?l were stored at ¡80°C. For control injections, non-
speciWc dsRNA was generated by the same method using
a PCR-derived fragment with 592-bp (nucleotides 142–
734) from the vector pGEM(R)-5zf(+) (Technical Ser-
vices, Promega, USA).
To synthesize siRNA, a motif based on the polymala-
tase gene (Accession No. AJ543320) was designed
according to the outlines proposed by Elbashir et al. [12]
employing the advanced RNAi software (oligoengine
internet homepage). The templates 5?-TCG AGT AAG
TAC TAG AGC TCC TAT AGT GAG TCG TAT
TAG T-3? (containing the sense segment) and 5?-AAG
GAG CTC TAG TAC TTA CTC TAT AGT GAG
TCG TAT TAG T-3? (containing the antisense segment)
were annealed with the T7-promotor segment 5?-ACT
AAT ACG ACT CAC TAT AG-3? for RNA synthesis
in the presence of T7-RNA polymerase according to the
method of Donze and Picard [11]. The resulting 19-bp
RNA duplex contained 2nt overhangs essential for the
function of siRNA containing UU in the sense strand
[13]. The template for transcription of nonspeciWc con-
trol siRNA had the sequences 5?-AAG GCG GTA ATA
CGG TTA TCC-3? as sense and 5?-GTG GAT AAC
CGT ATT ACC GCC-3? as antisense strands. All DNA
synthetic work was carried out by MWG, Germany.
Macroplasmodia and injection of dsRNA
Microplasmodia of P. polycephalum strain M3CVII
ATCC 204388 (American Type Culture Collection) were
cultured by the method of Daniel and Baldwin [14] for
48h at 24°C. To start a macroplasmodium, 100mg of
wet microplasmodia were allowed to fuse on 2% agar
(13.5cm petri dish) containing the growth medium and
cultured 22h at 24°C in the dark. The macroplasmo-
dium (5§0.5cm in diameter) was injected into a promi-
nent vein with 10?l of a solution [8] containing 1?g of
dsRNA, siRNA, or control RNAs and allowed to grow
for 24h before the plasmodium was scraped from the
agar surface into liquid nitrogen and stored at ¡80°C
for analysis.
In long-term experiments, macroplasmodia were
grown and injected the same way, but 24h after injection
a 4-cm2 section of the macroplasmodium together with
the agar was cut out and placed as an inoculum upside
down on a fresh agar plate (Wrst generation). The
remainder of the plasmodium was scraped from the agar
plate and analyzed. The inoculum was allowed to grow
for 60h, receiving fresh injections of dsRNA after 24 and
48h. Then a section was transferred for replating, the
remainder of the plasmodium was prepared for analysis
(after 60h from replating) as above, and so on until three
generations had been injected and prepared for analysis.
In a second series, the plasmodium was injected only in
the parental plasmodium, and the following generations

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RNA interference in plasmodia of myxomycetes / M. Haindl, E. Holler / Anal. Biochem. 342 (2005) 194–199
were analyzed without repeating injections. Control
plasmodia were incubated in parallel and treated exactly
as above. One received 10-?l injections of control
dsRNA, and the other did not receive injections. The
method of injection described by Willibald et al. [8]
employed a manually driven microinjector from Leitz
(Wetzlar, Germany), a simple instrument consisting of a
microsyringe and a microdosimeter, and the Binocular
M5 from Wild (Heerbrugg, Switzerland).
Knockdown analysis by real-time RT-PCR
RNA was extracted from plasmodia using the
RNeasy-kit (Qiagen, Germany). One microgram RNA
per sample was copied to cDNA using RevertAid H
Minus M-MuLV Reverse Transcriptase (Fermentas,
Germany) and oligo(dT)18 primer following the stan-
dard protocol of the provider. Realtime RT-PCR was
performed using the Roche-LightCycler (Roche, Ger-
many) with the Quantitect SYBR-Green PCR Kit
(Qiagen) including HotStar-Taq and SYBR Green as
Xuorescent dye for real-time detection of PCR products.
For the analysis by real-time RT-PCR, the primer pairs
were used as given below. Cycling conditions were 95°C
for 15 min, followed by 40 cycles of 95°C for 15s, 58°C
for 20s, and 72°C for 20s. For quantiWcation, the target
gene was normalized to the internal standard gene actin
of P. polycephalum employing the untreated and the
mock-injected samples as controls. As default settings of
the Lightcycler Software Version 3.5.3 were used, mea-
surement of probes in the linear range of the PCR was
ensured. Desalted primer pairs for actin Ppa35 (Acces-
sion No. M21500) and Polymalatase (Accession No.
AJ543320) were synthesized
Germany: (i) actin forward primer, 5?-CAT GTG CAA
GGC TGG ATT TGC TG-3?, (ii) actin reverse primer,
5?-ACC GAC GTA TGA GTC CTT TTG-3?, (iii) poly-
malatase forward primer, 5?-CAA AGG GAT TAT
GAG ACA GCA G-3?, and (iv) polymalatase reverse
primer, 5?-ACT GTG CCA TCC GCC TTC-3?.
by MWG-Biotech,
Analysis by Western blotting
Western blot analysis of polymalatase was performed
using a previously described preparation of a speciWc
antibody [15]. The actin-speciWc antibody was purchased
from Sigma (Germany).
Portions of the same samples analyzed by real time
RT-PCR were homogenized in icecold buVer containing
20 mM Na–morpholinosulfonate, pH 7, 0.5mM CaCl2,
15mM MgCl2, 5mM EGTA, 0.5M hexyleneglycol,
19%(v/v) dextrane, 14mM 2-mercaptoethanol, and pro-
tease inhibitor cocktail (Boehringer–Mannheim Ger-
many). After pelleting at 600g, the supernatant was
centrifuged 10min at 10,000g and represented the cyto-
plasmic fraction.
Portions of 10?g protein were analyzed by Western
blotting on a 10% SDS polyacrylamide gel/Immobilon-P
PVDF membrane (Millipore, Germany) with the anti-
bodies against polymalatase and actin. Immuno-reactive
bands were visualized by chemiluminescence employing
peroxidase-coupled second antibody and the reagents of
NOWA (Mobitec, Germany).
Results
Double-stranded RNA and siRNA induced speciWc
decreases in gene expression at the levels of both mRNA
(Fig. 1A) and protein synthesis (Fig. 1B). However, the
control siRNA and control dsRNA containing unspe-
ciWc sequences were without eVects (Figs. 1A and B).
dsRNA suppressed 89% of the amount of polymalatase-
speciWc mRNA seen for the uninjected control plasmodium.
The eVect of siRNA was an 82% reduction. Injection of
dsRNA in the range 0.2–1.0?g resulted in a constant level
of knockdown eYciency (data not shown). Addition of
speciWc dsRNA (0.4?g/ml) to the culture medium of
Fig. 1. Suppression of mRNA and protein synthesis after the injection
of polymalatase (pma)-speciWc dsRNA or siRNA into macroplasmo-
dia. Plasmodia were injected 22h after inoculation and then grown for
24h before analysis. (A) mRNA quantiWcation by real-time RT-PCR
with reference to actin mRNA. Messenger RNA of the noninjected
control plasmodia was set to 100%. Black column, noninjected control
plasmodia; dark gray columns, control (nonspeciWc) siRNA and spe-
ciWc siRNA; light gray columns, control (nonspeciWc) dsRNA and spe-
ciWc dsRNA. The mRNA suppression was highly signiWcant
(p>0.001). Standard errors of three independent measurements are
indicated. (B) Western blot of cytoplasmic samples of the plasmodia in
(A). The band at 68kDa refers to polymalatase and shows a decrease
in intensity. The band at 42kDa reXects actin as control.

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RNA interference in plasmodia of myxomycetes / M. Haindl, E. Holler / Anal. Biochem. 342 (2005) 194–199
197
microplasmodia did not eVect suppression of mRNA
synthesis, indicating that extrinsic RNA could not pene-
trate into the cytoplasm (data not shown).
The eVect on protein expression was measured by
Western blotting of samples from the cytoplasm. Actin
was used as reference in this case. An inspection of Fig.
1B indicates a suppression for the 68-kDa polymalatase
band in parallel with the suppression of mRNA.
Long-term experiments were carried out to (1) esti-
mate the time of persistence of the RNAi eVect and (2)
to enhance the knockdown level by repeated injections.
After the Wrst injection and growth, 1/20 (by mass) of the
macroplasmodium was replated. The new plasmodium
was injected and replated again, and so on. As shown in
Fig. 2, suppression of polymalatase-speciWc mRNA per-
sisted for approximately 144h after a single dsRNA
injection, corresponding to the duration of 18 cell cycles
and spanning two successive inoculations to fresh agar
plates. In the second “generation” the expression of
polymalatase-speciWc mRNA and protein already began
to recover. However, multiple injections did not enhance
the level of knockdown. The level of protein suppression
did not decrease either, consistent with the absence of a
long-term response such as developing a resistance
against injected dsRNA.
Plasmodia of the Wrst to third generations have been
inspected for changes in the phenotype. No changes in
the overall appearance and growth rate were seen except
that the latter appeared to be insigniWcantly retarded
(p>0.2).
Discussion
Of Myxomycetes, such as P. polycephalum, the plas-
modium represents a cell with an extremely large dimen-
sion and a high structural complexity. Nevertheless,
injection of speciWc dsRNA into the veins proved to
induce an approximately 90% suppression of the poly-
malatase gene. The knockdown was speciWc as control
dsRNA and siRNA were totally ineVective. Because the
knockdown was not complete, this result suggested at
least two explanations. (1) The artiWcially introduced
RNA spread all over the plasmodium except in a few
compartments, which were not accessible and thus
retained their normal mRNA synthesis. However, this
possibility seemed unlikely in face of the results of
repeated injections and maintenance of the very same
eVect for more than 200h. (2) In a survey of more than
2850 cases of various siRNA “gene silencers” it has been
reported that suppression was frequently incomplete
[16]. This may suggest another possibility, namely that
rates of mRNA synthesis and the RNAi-induced cleav-
age were of similar magnitudes, resulting in a steady
Fig. 2. Suppression of mRNA and protein synthesis after the injection of polymalatase (pma)-speciWc dsRNA into macroplasmodia. Plasmodia were
injected with dsRNA 22h after inoculation and then grown for 24h before analysis. The Wrst and following generations were prepared by inocula-
tion with a small amount of each precursor plasmodium on a fresh agar plate and grown for 60h as described under Materials and methods. Black
columns, noninjected control plasmodia; dark gray columns, water-injected control plasmodia; light gray columns, serial experiment with only the
parent plasmodium injected; white columns, serial experiment with injections of the parent plasmodium and of the Wrst to third generations each at
24 and 48h after replating. (A) mRNA quantifcation by real-time RT-PCR with reference to actin mRNA. Messenger RNA of the noninjected con-
trol plasmodia was set to 100%. Standard errors are indicated for three independent measurements. (B) Western blot analysis of the samples in (A).
The band at 68kDa refers to polymalatase and shows a decrease in intensity correlating with the suppression of polymalate-speciWc mRNA in (A).
The band at 42kDa reXects actin as control.

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RNA interference in plasmodia of myxomycetes / M. Haindl, E. Holler / Anal. Biochem. 342 (2005) 194–199
state survival of mRNA. A variety of reasons for an
incomplete break down has been reported, such as
absence of optimal base-pairing between siRNA and tar-
get mRNA [17], limited accessibility of target mRNA
due to stabile secondary structure [17–19], protective
binding of proteins, and speciWc subcellular location of
mRNA [19]. Low-abundance transcripts were less sus-
ceptible to siRNA-mediated degradation than medium-
and high-abundance transcripts [19].
Long-term experiments revealed that a single injec-
tion was quite eYcient, the knockdown lasting through
144h into the second generation (Fig. 2A). For compari-
son, the injected 1?g dsRNA (1.5£1011 molecules) cor-
responded to 3000 molecules dsRNA per nucleus
[5£108 nuclei (g plasmodia)¡1] immediately after the
injection and would have been 7.5 molecules of dsRNA
at the time of analysis in the second generation. The sus-
tentation of the knockdown eVect is in agreement with
the proposed mechanism of RNAi silencing [20,21] and
indicated a high eYciency. Repeated injection of dsRNA
did not induce further decline in mRNA, and the mRNA
returned to its original level after recovery during the
third generation following single injection. Apparently,
the regulation of mRNA production was not aVected by
ongoing RNA interference.
The phenotype of the plasmodium of the injected
parent plasmodium through the third generation was
found to resemble that of the control plasmodium,
except for a slight, however, insigniWcant (p>0.2)
growth retardation. This was referred to the residual
synthesis of polymalatase revealed by Western blotting.
The persisting low level of polymalatase was apparently
suYcient for the maintenance of the physiological sta-
tus of the plasmodium. We have recently shown that an
increase in polymalate content is related to an increase
in growth rate of the plasmodium [22]. Similarly, we had
expected a decrease in growth rate if the polymalatase
(adapter)–polymalate complex were the functional unit
and if the level of polymalatase was reduced by RNAi
interference. A knockout of the protein would have par-
alyzed polymalate function as a molecular carrier of
nuclear proteins. Protein concentrations in diVerent
regions of the plasmodium would have fallen out of
phase, resulting in loss of synchrony and consequently
loss of the normal phenotype of the plasmodium.
Although we could occasionally observe that growth
behavior was slightly diVerent in dsRNA-treated cells
and indeed some regions seemed to have fallen out of
synchrony indicated by retarded growth, the observa-
tion was not statistically signiWcant. A complete knock-
out of the gene expression seems to be required to
registrate a clear change in the phenotype.
The method that we have introduced here for plas-
modia is very simple and eVective. Because of the high
synchronization of plasmodial events and the precision
in timing of an injection, the method oVers the possibil-
ity to knockdown the temporary expression of genes in
a time-resolved fashion. Moreover, the injection can be
precisely placed at particular coordinates of the giant
cell body (plasmodium) to study gene suppression over
intracellular distances. The RNAi technique in conjunc-
tion with the unique properties of the plasmodium of
P. polycephalum should allow with relatively little
experimental expenditure study of versatile cellular phe-
nomena that have hitherto not been accessible.
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