Dynamics of Periarbuscular Membranes

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In arbuscular mycorrhizal (AM) symbiosis, host plants

supply photosynthates to AM fungi and, in return, they

receive inorganic nutrients such as phosphate from finely

branched fungal arbuscules. Plant cortical cells envelope

arbuscules with periarbuscular membranes that are

continuous with the plant plasma membranes. We prepared

transgenic rice (Oryza sativa) plants that express a fusion of

green fluorescent protein with rice AM-inducible phosphatetransporter, OsPT11GFP, and grew them with AM fungi.

The fluorescence of the fusion transporter was observed

in the arbuscule branch domain, where active nutrient

exchange seems to occur. In contrast, a signal was not

detected around intracellular hyphal coils on colonization

by either Glomus mosseae or Gigaspora rosea, making

the difference between Arum- and Paris-type mycorrhizae

ambiguous. We also invented a simple device involving

glass-bottomed Petri dishes for in planta observation of

fluorescent proteins in living AM roots with an inverted

fluorescence microscope. The plant bodies remain

completely intact, avoiding any stressful procedure such as

cutting, staining, etc. Since rice roots exhibit a very low levelof autofluorescence, the device enabled clear time-lapse

imaging to analyze the formation, function and degeneration

of arbuscules. In cortical cells, arbuscules seemed to be

functional for only 23 d. Suddenly, the arbuscular branches

became fragile and they shrank. At this stage, however,

the periarbuscular membranes appeared intact. Then, the

fluorescence of the transporter disappeared within only

2.55.5 h. The collapse of arbuscules occurred in the

subsequent several days. Thus, our device has a great

advantage for investigation of dynamic features of AM

symbiosis.

Keywords: Arbuscule

In planta imaging

Mycorrhizalsymbiosis Oryza sativaPhosphate transporter.

Abbreviations: AM, arbuscular mycorrhiza; dpi, days post-

inoculation; GFP, green fluorescent protein.

Introduction

The majority of terrestrial plant species establish symbiotic

relationships with arbuscular mycorrhizal (AM) fungi (Smith

and Read 2008). The main benefit for the plants is improved

acquisition of mineral nutrients, particularly phosphate (Pi),

from the soil. Plants colonized by AM fungi obtain scarcely

diffusible Pi exclusively via externally spread fungal extraradicalmycelium (Smith et al. 2003). Two anatomical types of arbuscu-

lar mycorrhizae based on the morphology of the symbiotic

interfaces have been described;Arum-type and Paris-type myc-

orrhizae, which are named after the plant species on which they

were first described (Smith and Smith 1997, Cavagnaro et al.

2001a). In the case ofArum-type mycorrhizae, fungal intraradi-

cal hyphae spread in the apoplast of root cortical cells and

form intracellular symbiotic structures, arbuscules, where the

fungal hyphae are highly branched to increase their surface

area. In Paris-type mycorrhizae, fungal hyphae form extensive

intracellular coils and arbusculated coils in the cortex with no

intercellular growth. In addition, a variety of intermediate types

have also been recorded (Dickson 2004).In colonized cortical cells, hyphal branches are enveloped

by a plant-derived membrane called the periarbuscular mem-

brane, which is the site of major Pi transfer to the plants

(Harrison 2005). The Medicago truncatulaPi transporter MtPT4

is specifically localized on the periarbuscular membrane but

not on either the plasma membrane or the membrane sur-

rounding arbuscule trunks (Harrison et al. 2002, Pumplin and

Harrison 2009). Importantly, accumulation of MtPT4 protein

and arbuscule formation are closely linked. Immunodetection

(Harrison et al. 2002) and detailed observation of transgenic

roots expressing MtPT4GFP (green fluorescent protein)

chimeric protein under the control of the MtPT4 promoter

(Pumplin and Harrison 2009) showed that MtPT4 is localizedaround finely branched mature arbuscules but not around

very young arbuscules with only one or two dichotomous

branches, becomes punctate in the degenerating arbuscules

and then is absent in the collapsed arbuscules. Therefore, it is

Dynamics of Periarbuscular Membranes Visualized witha Fluorescent Phosphate Transporter in ArbuscularMycorrhizal Roots of Rice

Yoshihiro Kobae and Shingo HataLaboratory of Crop Science, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 JapanCorresponding author: E-mail, [email protected]; Fax, +81-52-789-5558(Received December 4, 2009; Accepted January 18, 2010)

Plant Cell Physiol.51(3): 341353 (2010) doi:10.1093/pcp/pcq013, available online at www.pcp.oxfordjournals.org The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

341Plant Cell Physiol.51(3): 341353 (2010) doi:10.1093/pcp/pcq013 The Author 2010.

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probable that expression of the MtPT4 protein is coordinated

with arbuscule development and degeneration. AM-inducible

Pi transporter genes have been found in diverse plant species:

Lotus japonicus(Maeda et al. 2006, Guether et al. 2009, Takeda

et al. 2009), M. truncatula(Harrison et al. 2002, Grunwald et al.

2009), tomato (Solanum lycopersicum) (Nagy et al. 2005,

Xu et al. 2007), potato (Solanum tuberosum) (Nagy et al. 2005),tobacco (Nicotiana tabacum) (Chen et al. 2007a), barley

(Hordeum vulgare) (Glassop et al. 2005), wheat (Triticum

aestivum) (Glassop et al. 2005), maize (Glassop et al. 2005,

Nagy et al. 2006) and rice (Oryza sativa) (Paszkowski et al. 2002,

Gimil et al. 2005, Glassop et al. 2007). However, with the

exception of MtPT4 (Harrison et al. 2002, Pumplin and Harrison

2009), the intracellular localization of transporter proteins has

not been determined.

Much less is known about the morphology and the interface

for Pi transfer in Paris-type mycorrhizae than in the Arum

type. According to general explanations, fungal penetration

into epidermal cell layers is followed by: (i) production of

intracellular hyphal coils in the outer cortical cells; (ii) direct cellto cell spreading and extensive production of hyphal coils in

the inner cortical cells; and (iii) development of hyphal coils

into arbusculated coils predominantly in the inner cortical cells

(Cavagnaro et al. 2001b, Karandashov et al. 2004). Some AM-

inducible Pi transporter genes, i.e. potato StPT3(SOLtu;Pht1;3)

(Karandashov et al. 2004), rice OsPT11(ORYsa;Pht1;11) (Glassop

et al. 2007, Gutjahr et al. 2008) and OsPT13 (ORYsa;Pht1;13)

(Glassop et al. 2007), were reported to be up-regulated in the

infected cells in both Arum- and Paris-type mycorrhizae.

However, there is no direct evidence as to whether or not Pi

transporters accumulate on the membranes surrounding the

hyphal coils.

The rice OsPT11gene is specifically induced in cortical cells

on colonization by AM fungi (Paszkowski et al. 2002, Glassop

et al. 2007, Gutjahr et al. 2008). Its transcription level is not

up-regulated on the association of three pathogenic fungi

with wild-type rice (Paszkowski et al. 2002, Gimil et al. 2005).

In dmi3mutant rice, the level is not up-regulated even by AM

fungi because hyphal entry is blocked at the epidermal surface

and the fungi are unable to develop arbuscules. Occasionally,

nevertheless, fungi penetrate the cortical cells, and form

intracellular coils but are unable to induce the OsPT11 gene

(Chen et al. 2007b), suggesting that OsPT11expression is strictly

regulated by successful arbuscule formation for localization to

the functional interfaces for Pi transfer in the AM symbiosis.

Thus, it will be crucial to confirm the OsPT11 localization in

hyphal coil-containing cells.

To investigate plantAM fungi interactions, mycorrhizal

roots must be pulled out from the soil to analyze symbiosis

because the major symbiotic events always occur in the opaque

rhizosphere. Thus, in most experiments, the plantAM fungi

interactions may have already lost the feature of symbiosis

when samples are examined by microscopy. This is the most

serious problem for clarifying protein localization in vivo in

relation to AM symbiosis.

In order to reveal the exact symbiotic interface for Pi

transfer, we generated pOsPT11:OsPT11-GFP transgenic rice

plants that express OsPT11GFP fusion protein in AM roots

and investigated the OsPT11 localization in two morphologi-

cally distinct symbiotic riceAM fungi combinations. Here we

also report a novel and simple device that enables the in planta

imaging of AM roots in the rhizosphere without interferencewith the symbiosis. Using this device, we unveiled the dynamics

of periarbuscular membranes with the aid of OsPT11GFP

fluorescence.

Results

OsPT11 is localized in the branch region ofperiarbuscular membranes

The rice phosphate transporter gene OsPT11 is expressed in

AM roots (Paszkowski et al. 2002, Glassop et al. 2007), but the

localization of the encoded protein has not been determined.

To investigate the intracellular localization of the OsPT11protein, we generated transgenic rice plants expressing the

OsPT11GFP fusion protein. A genomic DNA fragment

containing a 2.6 kb promoter and the 1.8 kb coding region of

OsPT11 was connected to the GFP gene on Gateway binary

vector pGWB204 (Nakagawa et al. 2007). The construct was

introduced into rice calli byAgrobacterium-mediated transfor-

mation, and the resulting primary transgenic plants (T0) were

selfed to obtain T1seeds. Although the T0generation is enough

to investigate the protein localization, all experiments in this

study were performed using the T1generation for convenience.

After germination, T1seeds were sown on soil containing AM

fungal inoculants, the symbiotic morphology and OsPT11

expression being subsequently investigated. Four independent

transgenic lines were examined in each experiment, and

identical patterns of GFP fluorescence were observed. The

figures shown in this article are representative images. Rice

roots colonized by Glomus mosseaeshowed the typical Arum-

type symbiotic morphology. Intercellular hyphae extended

longitudinally between the root cortical cells (Fig. 1A) and

arbuscules grew from well-developed intercellular hyphae

(Fig. 1B). Arbuscules were formed approximately 7 days post-

inoculation (dpi) (data not shown), and a transgenic line

expressing OsPT11-GFP showed that the OsPT11 protein was

expressed specifically in arbuscule-containing cells, no other

signal being observed in the root system (Fig. 1C). Because

the roots of rice seedlings contain less autofluorescent

material compared with those of other model plants, e.g.

L. japonicus and M. truncatula, whole-tissue observation of

GFP by fluorescent microscopy could be easily carried out. To

obtain clearer images of OsPT11 localization, roots expressing

GFP were excised and longitudinally sectioned quickly with

a vibratome, and then the GFP-positive undisrupted cells

were imaged by confocal microscopy (Fig. 1DL). OsPT11

was specifically localized around the branched arbuscules,

but there was no GFP signal on the membranes surrounding

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the arbuscular trunks or plasma membranes (Fig. 1DF).

In heavily branched mature arbuscule-containing cells, most of

the cell volume was occupied by arbuscule branches and

trunks (Fig. 1H). All GFP signals were observed at the periphery

of arbuscular branches in young to mature arbuscules,

apparently indicating the localization in a specific region of

the periarbuscular membrane (Fig. 1I). This localization of

OsPT11 is similar to that of MtPT4 (Harrison et al. 2002,

Pumplin and Harrison 2009). GFP signals were occasionally

observed in the outer cortical cells when they contained

intracellular hyphae (Fig. 1JL). Although the riceG. mosseae

symbiosis apparently showed a typicalArum-type morphology,

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Fig. 1 Localization of OsPT11GFP in a typical Arum-type mycorrhiza.pOsPT11:OsPT11-GFPtransgenic plants were inoculated with Glomus

mosseae. (A and B) Trypan blue staining of colonized roots, 10 and 20 dpi, respectively. (C) A whole tissue image of OsPT11GFP accumulation,

20 dpi. Note that autofluorescent material is low in rice roots. (D) GFP signal, 8 dpi. (E) Differential interference contrast (DIC) bright-field image.

(F) Merged image of D and E. (G) Confocal GFP image of relatively young arbuscules, 20 dpi. (H) GFP image of densely branched mature arbuscules,

20 dpi. (I) GFP signal of arbuscular branches. Arrowheads indicate periarbuscular membranes, 8 dpi. (J) DIC image of hyphal coils that had spread

into outer cortical cells, 8 dpi. (K) GFP image of J. Some cells formed arbusculated coils and others formed hyphal coils. (L) Close-up and merged

image of the yellow-dotted area in J and K. DL are portions of a single confocal section image. a, arbuscules; h, intercellular hyphae; at, arbusculetrunks; hc, hyphal coils; ep, epidermal cells; ac, arbusculated coils. Bars = 100 m (A, C), 20 m (B, DH, JL), 5 m (I).

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fungal hyphae first penetrated the outer cortical cells and then

spread intracellularly in the outer cortex layers, eventually

forming arbusculated coils. The OsPT11GFP signal was

observed in arbuscules but was undetectable on the membrane

surrounding the coils (Fig. 1JL).

Gigaspora roseacolonization yields Arumand Paris-type mycorrhiza in rice

It is generally assumed that hyphal coils and arbusculated coils

are involved in Pi transfer to plants in Paris-type mycorrhizae

(Karandashov et al. 2004, Glassop et al. 2007, Smith and

Read 2008). The above description of the outer cortex being

colonized by G. mosseae prompted us to examine Paris-type

symbiosis. To determine whether or not OsPT11 is expressed

around Paris-type hyphal coils, a transgenic line expressing

OsPT11-GFP was inoculated with G. rosea, which was

reported to form intracellular coils but no arbuscules in rice

(Gutjahr et al. 2008). Trypan blue staining showed that

G. rosea predominantly forms intracellular coils in cortical

cells and usually forms not only small ramified structures butalso fine branched arbusculated coils (Fig. 2A). Although the

hyphae spread cell to cell intracellularly to form hyphal coils

(Fig. 2B), intercellular hyphae also developed (Fig. 2C). This

suggested that the G. roseaaccession used in this study forms

Arum- and Paris-type mycorrhizae (Dickson 2004) in rice roots.

Confocal microscopy revealed that the OsPT11 protein is

localized around highly branched arbuscules (data not shown)

and small arbuscules that emerged from thick coils (Fig. 2DF),

but not on the plasma membrane or the membrane surround-

ing the coils.

To date, arbusculated coils have rarely been observed in

outer cortical cells (Smith and Smith 1997, Cavagnaro et al.

2001b, Karandashov et al. 2004). However, OsPT11GFP fluo-

rescence indicated the presence of arbuscule structures in

rice outer cortical cells colonized by G. mosseae (Fig. 1JL)

and G. rosea (Fig. 2G, H). To address this inconsistency,

we performed trypan blue staining after fluorescence imaging.

A GFP fluorescence image (Fig. 2G) and a bright-field image

(Fig. 2H) indicated that OsPT11GFP is actually localized

around the arbuscules adjoining coiled hyphae. Surprisingly,

the trypan blue image did not show the arbuscules at the

corresponding positions (Fig. 2IL). As a consequence, the

colonized outer cortical cells looked as if they did not form

arbuscules. In the sample preparation, trypan blue staining

was performed immediately after capture of the GFP image

(within 1 min of the beginning of clearing with KOH). Thus, it is

unlikely that the arbuscules had been collapsed during the

sample processing in Fig. 2GI.In contrast, inactive arbuscules

exhibiting autofluorescence on G. mosseae colonization, but

not a GFP signal on inner cortical cells, were well stained by

trypan blue (Supplementary Fig. S1). Therefore, it appears

that arbuscules in outer cortical cells are scarcely stained

by trypan blue for an unknown reason. We concluded that

OsPT11 is localized only on periarbuscular membranes, i.e. not

on the membranes surrounding hyphal coils. Occasionally,

a hazy GFP signal was observed in an unknown compartment of

arbuscule-containing cells (Supplementary Fig. S2). Such

images were only obtained for the sectioned roots, never being

seen on real-time imaging with intact roots. This point will be

discussed below.

Transient overexpression of OsPT11GFPin onion epidermal cells

Knowlege of cell polarity and intracellular localization of

transporter proteins is critical to understanding the uptake

of mineral nutrients and signal transduction in plants.

Nevertheless, the default destination of intrinsic proteins of

the periarbuscular membrane is not known. As the first step

to address the mechanism underlying the specific localization

of OsPT11, we carried out bombardment and expressed

OsPT11GFP in onion epidermal cells under the control of

the constitutive 35S promoter. OsPT11GFP accumulated in

the endomembrane system (Supplementary Fig. S3AC). In

contrast, a similar GFP fusion with OsPT2 (LOC_Os03g05640),

another Pht1 transporter that is highly expressed in uninfectedroots of rice (Paszkowski et al. 2002, Ai et al. 2009, Wang

et al. 2009), showed localization on the plasma membrane

(Supplementary Fig. S3DF). Free GFP was observed in the

cytoplasm and nuclei (Supplementary Fig. S3GI).

In planta imaging of OsPT11GFP inAM symbiosis

Fluorescent protein markers are powerful tools for analyzing

the structure and protein composition of periarbuscular

membranes (Pumplin and Harrison 2009). Since arbuscules

are temporal organs and develop mainly in the inner cortical

cells of roots, they are optically and physically inaccessible.

Therefore, if this difficulty could be overcome and fluorescent

marker protein-mediated in planta imaging became possible,

it would be an ideal method for investigating these dynamic

organs. InArabidopsis thaliana, real-time cell imaging enabled

extensive analysis of the cell polarity and functional dynamics

of transporters, as demonstrated for the PIN auxin efflux carrier

family (Dhonukshe et al. 2008, Men et al. 2008). To extend that

technique to research on arbuscules, we established a simple

method for visualizing OsPT11GFP fluorescence in the

rhizosphere (Fig. 3). The T1seeds expressing OsPT11-GFPwere

grown in 35 mm Petri dishes with 27 mm coverslip windows at

the bottom. As the glass bottom was covered with AM fungi

inoculant, roots that extended and reached the bottom were

effectively infected by AM fungi just above the coverslip

window. Consequently, when the viability of the inoculant is

high, real-time OsPT11GFP fluorescence can be successfully

observed within 10 d after seed germination using an inverted

fluorescence microscope. Using this technique, we obtained

time-lapse images of OsPT11GFP expression. The following

interpretations were made by examining reproductive images

of >20 independent colonization units that were observed

in roots of two independent transgenic lines. The figures and

supplementary moviespresented are representative.

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Fig. 2 Localization of OsPT11GFP in an intermediate,Arum- and Paris-type mycorrhiza.pOsPT11:OsPT11-GFPtransgenic plants were inoculated

with Gigaspora rosea. (AC) Trypan blue staining of colonized roots, 12 dpi. (B) Magnified image of the yellow-dotted area in A. The arrow

indicates the intracellularly penetrating hypha. (D) Confocal GFP image of arbusculated coil-containing cells, 12 dpi. (E) DIC image. (F) Merged

image of D and E. (G) GFP image around the outer cortical cells, 9 dpi. (H) Bright-field image of G. (I) Trypan blue staining of the root section used

in G and H. After GFP had been imaged, the root section was immediately and carefully stained. (J, K and L) Magnified images of the yellow-

dotted areas in G, H and I, respectively. Note that the arbuscule structures observed in K with GFP signals (J) were not stained in L. hc, hyphal coils;a, arbuscules; h, intercellular hyphae; ac, arbusculated coils; ep, epidermal cells; cw, cell wall. Bars = 10 m.

In Fig. 4, two distinct roots colonized by G. mosseae

are shown, images being captured for a period of 760 min.

In the root on the left side, newly generated arbuscules

visualized by OsPT11GFP had sequentially and longitudinally

developed (Fig. 4A). In this time-lapse image, the longitudinal

development rate for these arbuscules was estimated to be

0.57 mm d1. The development rate among other mycorrhizal

units (n= 18) varied from 0.42 to 1.68 mm d1(data not shown).

In another root, differentially fluorescent cells were present

within a single colonization unit (Fig. 4B). The first cell

gradually accumulated OsPT11GFP during this period,

the second one retained the fluorescence over time, and the

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Fig. 3 A simple device for in planta imaging. T1seeds ofpOsPT11:OsPT11-

GFPtransgenic plants were grown in 35 mm Petri dishes with 27 mm

coverslip windows at the bottom (A and B). The glass bottom was

covered with AM fungi inoculant (A). Because the condenser of our

microscope would have disturbed the shoot (see also E), the seed was

positioned at the edge of a dish and the shoot came out through the

hole in the cover (C). The cover was necessary in order to prevent

evaporation from the soil. The elongated root system spread just

above the coverslip window, being effectively infected with the AMfungus. OsPT11GFP fluorescence was observed using an inverted

fluorescence microscope (E). The shoots were illuminated with a small

fluorescent lamp to maintain photosynthesis. The roots were

illuminated and observed from the underside.

third one gradually lost the fluorescence (Supplementary

Movie 1).

Imaging of arbuscule degeneration

Arbuscules are transient structures. After arbuscules have

reached the finely branched mature state, they rapidly collapse

(Alexander et al. 1989). To assess the dynamics of arbusculedegeneration, images of collapsing arbuscules were obtained.

Although it is impossible to guess beforehand which arbuscules

will begin to collapse, time-lapse imaging for a long period

enabled the beginning of degeneration to be pin pointed. Roots

of the transgenic line expressing OsPT11-GFP were colonized

by G. roseaand images were captured for a period of 220 min

(Fig. 5). The images revealed a series of early stage degeneration

events, from the early symptom of arbuscules to the disappear-

ance of OsPT11GFP. The initial morphological change of

collapsing arbuscules comprised a rapid shrinkage of arbuscule

branches (Fig. 5and Supplementary Movie 2). It seems likely

that the arbuscule branches suddenly became plastic or fragile,

and gradually but rapidly shrank, and, as a result, the periarbus-cular membranes might become stacked and the OsPT11GFP

signals might become densely accumulated (Fig. 5B, C).

The collapsed state of arbuscules was visible for approximately

2.5 h, and then the GFP fluorescence became undetectable in

this case (Fig. 5B and Supplementary Movie 2). Among

another 10 arbuscules of distinct mycorrhizae, the shortest

collapsed state of arbuscules was visible for 2.5 h and the

longest one lasted for 5.5 h (data not shown). Therefore, it is

probable that the constituents of periarbuscular membrane

proteins were promptly digested or realigned during this short

time period. Notably, the arbuscule shrinkage preceded the

degradation of OsPT11GFP. It seems likely that withdrawal or

autolysis of the fungus induces the degradation event within

the plant cells.

Life cycles of arbuscules

It is important to determine the life cycle of an arbuscule

because the finely branched and transient structure is thought

to reflect the active state in nutrient exchange. We observed

the appearance and disappearance of all visible arbuscules in

one colonization unit in transgenic lines expressing OsPT11-GFP

using the in planta imaging system (Fig. 6A). Photographs were

taken at 0, 10, 23, 30, 36, 45, 51 and 57 h from the beginning of

the experiment. All visible arbuscules in the pictures were

marked with pseudocolors, indicating the time points of their

first appearance. The presence of arbuscules was traced, and

their duration was represented by lines (Fig. 6B). The entire life

cycle of one PT11GFP-positive arbuscule was estimated to be

23 d. This duration is in accord with the earlier estimation by

morphometric techniques (Alexander et al. 1989). Remarkably,

at the end of the observation period (57 h), every arbuscule

seemed to degenerate simultaneously (Fig. 6B). Although the

collapse of arbuscules is controlled cell autonomously (Figs. 4,

5), it is possible that the life cycle of each colonization unit is

regulated by a systemic signal of the plant or controlled by the

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fungal networks. To address this issue, we analyzed the rate of

reduction of arbuscule numbers. Arbuscule numbers in an

infection unit were reduced at similar rates irrespective of

their first appearance (Fig. 6C), confirming that the collapse of

arbuscules is cell autonomous. Next, the numbers of newly

appeared arbuscules in each distinct colonization unit were

counted (Fig. 6D). Extensive generation of arbuscules occurred

during the initial 2 d and only a few arbuscules were formed

on the subsequent day, resulting in their simultaneous degen-

eration in each colonization unit as a consequence. Finally,

we counted the total numbers of visible arbuscules (Fig. 6E).

Each colonization unit showed a single peak in the numbers of

arbuscules, and the numbers gradually decreased. In the last

one-third of the observation period, fresh colonization units

were newly generated (Supplementary Fig. S4), indicating

that the degenerating period of colonization units 1, 2 and 3

(Fig. 6B, D, E) was not caused by artificial effects of the experi-

ments, and also representing the rate of initial development of

arbuscules. Taken these findings together, we estimated the

life cycle of a single colonization unit to be around 3 d (Fig. 6E).

It seems impossible, however, to generalize this duration and

phenomenon observed in rice roots to other plant species

including model legumes, because a single colonization unit of

rice mycorrhiza is smaller and less extended longitudinally than

that of other plant species.

Discussion

Specific localization of OsPT11 on periarbuscularmembranes

Here we demonstrated that rice OsPT11GFP is localized on

periarbuscular membranes, and that there is no detectable

signal on the membranes surrounding the hyphal coils,

intracellular hyphae or any other cells in the whole root

system (Fig. 1C). Thus, we conclude that OsPT11 localization

occurs on periarbuscular membranes with apparent polarity

in colonized cells. Recently, Pumplin and Harrison (2009)

described the periarbuscular membrane as being composed of

at least two distinct domains, the arbuscule branch domain,

which contains MtPT4, and the arbuscule trunk domain, which

contains MtBcp1. In our study, OsPT11 was found not to be

located on the arbuscule trunk domain, supporting their

results (Fig. 1DF). The arbuscule trunk domain is directly con-

tinuous with the plasma membrane, and plasma membrane

aquaporin AtPIP2a is also targeted to this domain but not

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Fig. 4 In planta time-lapse imaging ofpOsPT11:OsPT11-GFPtransgenic plants inoculated with G. mosseae, 12 dpi.T1seeds were grown in 35 mm

Petri dishes with 27 mm coverslip windows at the bottom. OsPT11GFP fluorescence was detected using an inverted fluorescence microscope

without disturbing the symbiotic interaction. (A) Three time points (8, 200 and 760 min after time-lapse started) for two distinct root systems

and at least two colonization units are shown. The arrow indicates the front of arbuscule formation (OsPT11GFP accumulation). Numbers in

the images indicate: 1, an OsPT11GFP-accumulating cell; 2, an OsPT11GFP signal-stable cell; and 3, an OsPT11GFP decreasing and collapsing

arbuscule-containing cell. (B) Time-lapse images of the dotted area in A. A real-time movie is available online. Bars = 100 m (A) and 30 m (B).

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to the arbuscule branch domain (Pumplin and Harrison 2009).

These results suggest that the arbuscule branch domain is

functionally isolated and probably active in nutrient exchange.

It is probable that the protein transport mechanism that

directs AM-inducible Pi transporters to the arbuscule branch

domain is conserved in both dicotyledonous and monocotyle-

donous plants. The cell polarity of transporter proteins has

been well described for the Arabidopsis PINs family. The family

members are initially delivered to the plasma membrane in

a non-polar manner and their polarity is established through

subsequent endocytic recycling (Dhonukshe et al. 2008).

In addition, rice silicon transporters Lsi1 and Lsi2, which are

localized on the distal and proximal sides of casparian strips,

respectively, in the same cells were localized on the plasma

membrane when they were expressed in onion epidermal cells

(Ma et al. 2006, Ma et al. 2007). Interestingly, when we expressed

OsPT11GFP in onion epidermal cells under the control of

the 35S promoter, the protein was not targeted to the plasma

membrane but accumulated in the endomembrane system

(Supplementary Fig. S3). In control experiments, a fusion of

OsPT2, which is located on rice plasma membranes, was

normally localized on onion plasma membranes, and free

GFP accumulated in the cytoplasm and nuclei as expected.

Thus, we think that the delivery mechanism for OsPT11 is

different from that for PINs or rice silicon transporters.

Synchronized with the transcriptional regulation of its gene,

the OsPT11 protein appears to be delivered to periarbuscular

membranes during arbuscule development, with the aid of

unknown interacting proteins or unrevealed membrane sorting

systems.

In addition, we confirmed that coiled hyphae that have

spread to the outer cortex occasionally form small arbuscules

that are hardly stained with trypan blue (Fig. 2JL). Our results

indicate that the visual discrimination of the Paris type and

A

nim022nim001nim4

B

84 min nim022nim651nim231nim068 min

C8 min 84 min 132 min 220 min

Fig. 5 In planta, time-lapse imaging ofpOsPT11:OsPT11-GFPtransgenic plants inoculated with G. rosea, 9 dpi.(A) Three time points (4, 100 and

220 min after the time-lapse started) for a single colonization unit are shown. The arbuscule in the dotted frame showed dynamic collapse.

At 220 min, OsPT11GFP almost completely disappeared. The arrow indicates a newly developing arbuscule. (B) Time-lapse images of the

dotted frame in A. Arrowheads indicate the densely accumulated OsPT11GFP signals. The dotted lines for 60, 84 and 132 min indicate the

outlines of shrinking arbuscule branches. At the time point of approximately 60 min from the beginning of imaging, the arbuscule started toshrink. Note that OsPT11GFP was still localized on collapsing arbuscule branches, indicating that the arbuscule shrinkage precedes the

OsPT11GFP degradation. (C) Three-dimensional graphs of the intensities of pixels in B (8, 84, 132 and 220 min) were created with imageJ

(http://rsb.info.nih.gov/ij/index.html). A movie is available online. Bars = 30 m (A) and 10 m (B).

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A

0 h

10 h

23 h30 h 36 h

45 h51 h

57 h

colonization unit 1

(unit 1)

1 mm

unit1

1 mm

BC

60

80

100

20

40

Time (h)

0

10

23

30

36Formed

arbuscules unit 1Time

u

unit2

D

0

20

10 20 30 40 50 60 (h)

80

120

160

40

unit1

2

3

a

New

arbuscules

unit3

50 arbuscules 100

200

300unit

12345

010 20 30 40 50 60 (h)

E

Total

rbuscules

a

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 (h) 10 20 30 40 50 60 (h)0

a

Fig. 6 Life cycles of arbuscules inpOsPT11:OsPT11-GFPtransgenic plants inoculated with G. mosseae. (A) Classification of arbuscules in a single

colonization unit according to their first appearance. Microscopic observation was started at 10 dpi (0 h); a series of pictures of mycorrhizae

containing GFP-positive arbuscules were obtained, connected with each other, and then all visible arbuscules were marked with pseudocolored

small squares indicating the differential time of their first appearance. (B) Representation of life spans of the observed arbuscules.

All visible arbuscules in the pictures of three distinct colonization units (unit 1, 2 and 3) were marked with the same pseudocolors as in the A,

the presence of active arbuscules was traced, and then the durations were shown by lines. (C) Transition of arbuscule numbers according to theContinued

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Arumtype depending on the morphological type is not critical

for the occurrence of arbuscules, and the difference between

hyphal coils and arbusculated coils is also uncertain.

The active phase of arbuscules

Despite the large numbers of plant species exhibiting AM asso-

ciations, all arbuscules are short lived. Toth and Miller (1984)

described that a mature arbuscule exists for only an instant of

time at the peak of the developmentdegeneration curve.

It was determined by means of a morphometric technique

that the whole arbuscular cycle lasts around 8 d, the active

phase of arbuscules accounting for only 33%of the whole cycle

(Alexander et al. 1989). Consequently, a single colonization unit

includes diverse developmental stages of arbuscules. These

short-lived and temporal characteristics of arbuscules prevent

us from analyzing molecular and cell biological functions.

In order to overcome this difficulty, we established a novel

method that enables us to image a single arbuscule in a

real-time manner without interruption of natural symbiosis

in the rhizosphere. During the development of arbuscules,

the OsPT11GFP signal increased linearly. This suggests that

the expression level of OsPT11 is proportional to the growth

of arbuscule branches. Although we have not yet attempted

real-time imaging with an inverted confocal microscope,

a differential interference contrast bright-field image will

confirm the expected association of OsPT11GFP and the

arbuscule branch structure. Because the level of autofluores-

cence in rice roots is quite low, and arbuscules develop not only

in the inner cortical cells but also in outer cortical cells,

we anticipate that confocal microscopic observation will be

feasible with our system.

The images obtained in the current work indicate that the

OsPT11GFP expression and the protein turnover in the periar-

buscular membrane is a cell-autonomous process. Previous

studies have also shown that a number of mycorrhiza-inducible

genes including those of phosphate transporters (Rausch

et al. 2001, Harrison et al. 2002), a subtilase (Liu et al. 2003,

Takeda et al. 2009), a chitinase (Bonanomi et al. 2001) and a

H+-ATPase (Krajinski et al. 2002) are expressed specifically in

the colonized cells. We estimated the mean of the whole life

cycles of PT11GFP-positive arbuscules to be 23 d, in accord

with the earlier estimation (Alexander et al. 1989). However,

it is notable that some arbuscules lasted for >57 h, but others

were formed and collapsed within 24 h (Fig. 6B). Although

we did not examine the relationship between the arbuscular

life cycles and their sizes, it is possible that the duration of

the arbuscular cycle varies with the root architecture.

One of the characteristics of rice roots is the constitutive

formation of aerenchyma in crown roots and large lateral

roots (Jackson et al. 1985, Drew et al. 2000, Gutjahr et al. 2009).

As fungal structures are not observed in the voids between

cortical cells (Gutjahr et al. 2009), it is probable that AM coloni-

zation would be disturbed by the constitutive programmed

death of cortical cells. Dual-colored real-time imaging of the

OsPT11 transporter and PBZ1 protein, which is expressed in

cells adjacent to expanding aerenchyma (Kim et al. 2008), willprovide new insights into the rice arbuscule formation near

the aerenchyma.

Degradation of arbuscules

In contrast to the gradual arbuscule formation, the collapse is

quite a rapid event. Thus, the process of arbuscule collapse has

never been observed (Toth and Miller 1984). Real-time imaging

showed that arbuscule collapse is completed within 2.55.5 h.

This finding is consistent with the rapid decrease in branch

volume observed on morphometric observation (Alexander

et al. 1989, Toth 1992).

Arbuscule degeneration starts cell-autonomously. In a divi-

sion in Fig. 5, at least five mature arbuscules are included.

Among them, a single arbuscule unit collapsed without

showing any positive or negative effect on the surrounding

cells. This finding supports the observations of Karandashov

et al. (2004), i.e. that potato StPT3 promoter-Fluorescent Timer

is temporally and cell-autonomously activated, and differen-

tially fluorescent cells were present within a single colonization

unit. These results and ours indicate that Pi transporter

expression is probably regulated depending on the functional

state of the plantAM fungi association within a single cell.

On the other hand, it is generally explained that the collapse

of arbuscules is a result of programmed responses within fungal

cells but not within the plant (Peterson and Bonfante 1994,

Smith and Read 2008). The movie of Fig. 5 indicates that

the arbuscular branch suddenly became plastic or fragile at

the beginning of shrinkage. Withdrawal or autolysis of hyphal

masses and simultaneous formation of many septa (Cox and

Sanders 1974, Harrison 2005, Javot et al. 2007) may contribute

to the rapid shrinkage. It is apparent that periarbuscular mem-

branes are intact when the shrinking is underway because

OsPT11GFP is still densely localized on the membranes

without notable degradation (Fig. 5B, C). These results may

indicate that the fungal cells have the priority to determine the

arbuscular cycle. As an arbuscule is linked with others by

intercellular hyphal networks, one arbuscule may be influenced

and synchronously regulated by distant fungal nodes. In other

words, the degeneration of a single arbuscule in Fig. 5might be

affected by that of others. Probably, confocal microscopy

will not reveal these dynamics. We believe that prolonged and

detailed real-time imaging will enable us to gain a new insight

first appearance in A. (D) Numbers of newly generated arbuscules at each time point in the three distinct colonization units. (E) Changes of total

arbuscule numbers. Total arbuscule numbers at each time point in five distinct colonization units were counted. Units 4 and 5 are newly generated

mycorrhizae, indicating that the senescence of units 1, 2 and 3 was not an artificial effect of the experiments, also representing the rate of initial

development of arbuscules.

Fig. 6 (caption continued)

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into such a complicated phenomenon. Unfortunately, the

35 mm dish used in this work is too small to retain the water

supply adequately for a long period. Thus, in order to avoid

any environmental stress to the mycorrhizal symbiosis, the

cultivation system must be developed to a much larger scale.

Although the mechanism underlying OsPT11GFP degrada-

tion has to be elucidated at the cellular level, Pumplin andHarrison (2009) reported that a hazy GFP signal was observed

in collapsing arbuscules inpMtPT4:MtPT4-GFPtransgenic roots.

We observed a similar signal to that of soluble GFP in colonized

cells in root sections (Supplementary Fig. S2), and the fluores-

cent appearance suggested that the cells were still intact

and the GFP was accumulated in unknown intracellular com-

partments including the cytoplasm and vacuoles. However,

a hazy GFP signal was observed even in the mature aubuscule-

containing cells and such images were only obtained for the

sectioned roots, never being seen on our real-time imaging

with intact roots. One possibility explaining this discrepancy

is that periarbuscular membrane proteins are occasionally

digested via stress responses, e.g. defensive or general cleaningup responses of plant cells, when the natural symbiotic rela-

tionship is broken. Thus, real-time imaging is an indispensable

method for studying the degeneration events in arbuscules.

The inactivation mechanism in periarbuscular membranes

would be an important matter, because the protein compo-

nents of periarbuscular membranes determine the periplasmic

environment. Pumplin and Harrison (2009) described that

arbuscule structures appear to be surrounded by a tonoplast

aquaporin-labeled membrane throughout the arbuscule life

cycle. We suggest that real-time imaging can reveal unknown

aspects of the membrane dynamics around arbuscules.

In conclusion, it is noteworthy that real-time imaging of

OsPT11GFP revealed one of the dynamic aspects of AM sym-

biosis, namely a period of nutrient exchange. AM symbiosis

comprises a series of dynamic interactions between plants and

AM fungi, including recognition of each symbiont, penetration

of the epidermis, formation of a pre-penetration apparatus

(Genre et al. 2005, Genre et al. 2008), colonization of cortical

cells and degeneration of arbuscules. Many proteins must be

expressed coordinately in the respective periods of symbiosis;

phosphate transporters of periarbuscular membranes comprise

only a small portion of these. Imaging of these other proteins

with the aid of fluorescence will clarify not only their dynamic

features in plantfungus interactions, which were established

>400 million years ago (Parniske 2008), but also the fundamen-

tal morphology and crucial functions of organelle orientation

and membrane dynamics in the colonized cells.

Materials and Methods

Preparation ofpOsPT11:OsPT11GFP rice

A 4.4 kb genomic fragment of OsPT11 (LOC_Os01g46860)

containing a promoter, 2.6 kb in size, and a coding region,

1.8 kb in size (pOsPT11:OsPT11), was amplified from O. sativa

cv Nipponbare by PCR using a primer pair (5-CACCTTCCAG

CAGCAGTAGAGC-3 and 5-TGCGTGCATGGATGTCTGCC

ATTC-3). The amplified gene was ligated into an entry vector,

pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA), and then

introduced upstream of the promoterless GFP gene in a

binary vector, pGWB204, using a Gateway system (Invitrogen).

The GFP-fused construct was introduced into Agrobacteriumtumefaciens strain EHA105, and O. sativacv Taichung 65 was

transformed as described by Hiei et al. (1994). The transforma-

tion was carried out in the laboratories of Professors Motoyuki

Ashikari and Makoto Matsuoka, Nagoya University. Primary

transgenic plants (T0) were selfed to obtain the T1generation

for colonization by AM fungi and subsequent analysis.

Inoculation of rice with AM fungi

Rice seeds were immersed in deionized water containing 1%

(w/v) Benrate (Sumitomo Chemicals, Tokyo, Japan) for 3 d. The

germinated seeds were rinsed with deionized water three times

and then grown in 35 mm Petri dishes with 27 mm coverslip

windows at the bottom (AGC, Tokyo, Japan) in a greenhousewith a 16 h day/8 h night cycle at 27C. Each dish contained

a soil inoculant of G. mosseae(0.5 g per dish; gift from Shigeki

Chida, Idemitsu Kosan, Tokyo, Japan) or G. rosea (gift from

Professor Joseph B. Morton, West Virginia University) at the

bottom and covered with 4 ml of an autoclaved Kanuma soil/

vermiculite/Kureha soil (Kureha, Tokyo, Japan) mixture (1 : 1 : 1,

by vol.). No other nutrients were added except Kureha soil.

Confocal microscopy

Root segments exhibiting GFP fluorescence were excised,

embedded in 5% agarose without fixing and then sectioned

using a DTK-1000 vibrating-blade microslicer (DSK, Kyoto,

Japan). The sectioned roots (80 m) were imaged using a Zeiss

confocal laser-scanning microscope (LSM 5 Pascal, Zeiss,

Oberkochen, Germany) with a 40 water-immersion objective

(C-APOCHROMAT 40/1.2w). Excised roots were imaged

within 30 min of preparation. Images were processed using an

interface ZEN2008 (Zeiss) and overlaid with Photoshop CS4

Extended (Adobe Systems, CA, USA).

In planta imaging

Twelve- to 15-day-old seedlings (912 dpi) grown in 35 mm

Petri dishes were placed on the inverted platform of a Zeiss

fluorescence microscope (Axiovert 200). The leaves were

kept illuminated at 25C under continuous light with a

portable fluorescent lamp for 412 h. Since frequent near

UV illumination (e.g. once per 1 min) resulted in fade-out of

GFP fluorescence under our conditions, illumination and

photography were performed once per 48 min. Time-lapse

images were obtained with the software AxioVision4.6 (Zeiss).

Trypan blue staining

GFP-positive root segments were excised (23 mm) and cut into

two pieces longitudinally. After imaging the GFP signal with an

epifluorescence microscope ECLIPSE-E600 (Nikon, Tokyo, Japan),

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root pieces were carefully incubated at 99C in 150 l of 3%

KOH for 10 min in a 1.5 ml microcentrifuge tube, and then

soaked in 150 l of 2% acetic acid at room temperature for

5 min. The roots were then stained at 99C for 15 min in 150 l

of lactoglycerol solution containing 0.01% trypan blue.

The trypan blue solution was removed and the roots were

destained in hot water (60C) for 15 min. The stained rootswere examined using the same microscope, the bright-field

image being compared with the corresponding GFP image.

Supplementary data

Supplementary data are available at PCP online.

Funding

This research was supported the Ministry of Agriculture,

Forestry and Fisheries of Japan [Genomics for Agricultural

Innovation, grant No. PMI-0003].

Acknowledgments

We wish to thank Professors M. Ashikari and M. Matsuoka for

their help during the preparation ofpOsPT11:OsPT11-GFPrice.

We also thank Professor J. B. Morton for providing the Gigaspora

rosea and Gigaspora gigantea inoculants, Mr. S. Chida for the

Glomus mosseae inoculant, and Ms. S. Suzuki for technical

assistance in preparation of the OsPT11-GFPconstruct and its

bombardment.

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Dynamics of Periarbuscular Membranes

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