Arbuscular Mycorrhiza–Speciﬁc Signaling in Rice …Arbuscular Mycorrhiza–Speciﬁc Signaling...

Arbuscular Mycorrhiza–Specific Signaling in Rice Transcendsthe Common Symbiosis Signaling Pathway W

CarolineGutjahr,aMari Banba,b VincentCroset,a KyungsookAn,c AkioMiyao,dGynheungAn,cHirohikoHirochika,d

Haruko Imaizumi-Anraku,b and Uta Paszkowskia,1

a Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerlandb Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japanc National Research Laboratory of Plant Functional Genomics, Pohang University of Science and Technology, Pohang 790-784,

Koread Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602,

Japan

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis

(SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both

AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and

although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify

and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene

expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-

dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted

to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-

spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns,

therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential

gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and

furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

INTRODUCTION

The arbuscular mycorrhizal (AM) symbiosis occurs in all plant

lineages, including angiosperm dicotyledons and monocotyle-

dons, and thus also in important crop grasses such asmaize (Zea

mays), wheat (Triticum aestivum), and rice (Oryza sativa). The first

step in the development of AM symbiosis (reviewed in Smith and

Read, 1997; Harrison, 2005; Paszkowski, 2006) is a molecular

dialogue prior to contact. At the root surface, the fungal hypha

differentiates into a hyphopodium from which a penetration peg

enters the rhizodermis. The fungus progresses through the outer

into the inner root cortex and spreads intercellularly along the

longitudinal axis of the root, forming highly ramified structures,

termed arbuscules, inside cortex cells. At the whole root level,

development of the AM symbiosis is asynchronous, with various

stages of colonization being present simultaneously. Thus, sig-

naling between the symbiotic partners occurs, at least partly, cell

autonomously with fine-tuned stage specificity.

Despite the progress made in past years, knowledge of the

molecular events and components involved in these signaling

processes is still limited and mostly derived from studies in

legumes, in which forward genetic screens uncovered a major

signaling pathway, the common symbiosis (SYM) pathway,

which is nonspecifically required for the formation of both rhizo-

bial and AM symbioses (for a recent review, see Parniske, 2008).

To date, the components identified are a symbiosis Leu-rich

repeat receptor kinase (SYMRK), DOES NOT MAKE INFEC-

TION2 (DMI2); two predicted cation channels, CASTOR and

POLLUX (DMI1); two nuclear porins, NUP85 and NUP133, which

are all necessary for the induction of Ca2+ spiking (Figure 1)

(Kosuta et al., 2008); and the calcium/calmodulin-dependent

protein kinase CCAMK (DMI3), which acts downstream of Ca2+

spiking (Figure 1) and is thought to transduce the calcium signals.

CCAMK physically interacts with and phosphorylates CYCLOPS

(INTERACTING PROTEIN OF DMI3 [IPD3]), a protein of unknown

function (Messinese et al., 2007; Parniske, 2008).

A better understanding of the molecular events governing the

development and function of the AM symbiosis is needed,

particularly in grasses, including major crops, to aid agricultural

exploitation of the symbiosis. Rice is an attractive model crop for

studying AM signaling because the genome sequence is avail-

able and there are extensive mutant collections for reverse-

genetics screens (Hirochika et al., 2004). Candidates for ortho-

logs of all seven commonSYM genes have been computationally

predicted to be present in the genomes of a number of dicotyl-

edons and monocotyledons (Zhu et al., 2006; Messinese et al.,

2007). Although roots perform equivalent functions in both

1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Uta Paszkowski([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.062414

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angiosperm classes and the cytological appearance of AM

symbioses is similar, the architecture of the root system and

cellular patterning differ considerably (Hochholdinger and

Zimmermann, 2008). The extent to which cereal roots utilize

the same SYM signaling pathway for the interaction with AM

fungi has been addressed for CCAMK (Chen et al., 2007) and

CYCLOPS (IPD3) (Chen et al., 2008), the two signaling compo-

nents operating downstream of the Ca2+ response. In rice,

mutation of either the CCAMK or the CYCLOPS gene leads to

a mycorrhizal mutant phenotype similar to that described for

legumes, and rice CCAMK was able to restore full mycorrhizal

and rhizobial colonization in the Medicago truncatula (barrel

medic) dmi3 mutant (Godfroy et al., 2006; Chen et al., 2007,

2008). The function of CCAMK and CYCLOPS, therefore, ap-

pears to be conserved between rice and legumes.

Transcriptional outputs associated with specific physiological

processes can be used as diagnostic tools to identify the

signaling pathways involved (Glazebrook et al., 2003; Bari

et al., 2006; Sato et al., 2007). To unravel the signaling cues

that uniquely affect the AM symbiosis demands the careful

selection of AM-specific indicator transcripts. Over the past

years, a number of transcriptome analyses have illustrated the

dramatic changes that occur in plant roots upon mycorrhizal

colonization (Liu et al., 2003; Guimil et al., 2005; Hohnjec et al.,

2005). A set of genes specifically expressed during the AM

symbiosis have been identified from M. truncatula (reviewed in

Krajinski and Frenzel, 2007), but their utility as diagnostic tools for

signaling has not been assessed. The dependence of transcrip-

tional induction on common SYM signaling components has

been demonstrated in Lotus japonicus andM. truncatula, and the

existence of additional AM signaling cues has been suggested

(Weidmann et al., 2004; Kistner et al., 2005; Massoumou et al.,

2007; Siciliano et al., 2007). However, to date, it has been difficult

to exclude symbiosis-independent influences on the induction of

these gene sets, since developmental, nutritional, and patho-

genic responses were not characterized. Thus, the relevance of

the common SYM pathway and the existence of additional

signaling cues for AM-specific processes are currently not

known for legumes or other mycorrhizal plant species.

Here, we have used rice to investigate functional conservation

of the SYM pathway and to determine additional signaling cues

for the AM symbiosis. We selected rice lines (rice sym mutants)

with insertions into signaling components upstream (CASTOR

and POLLUX) and downstream (CCAMK andCYCLOPS) of Ca2+

spiking and demonstrated that all of these genes are required for

AM colonization of rice, thus illustrating broad evolutionary

conservation of symbiotic signaling. Genome-wide transcrip-

tome profiling of mycorrhizal rice roots had previously predicted

a group of genes to be exclusively AM-induced (Guimil et al.,

2005). Here, we characterized and subsequently used these

genes as molecular indicators of AM-specific signaling. Their

application to the study of rice symmutants reveals that although

the common SYM signaling pathway is of central importance for

successful AM symbiosis in rice, it is supported by additional

symbiotic signaling cues that act in parallel to, or depart from,

common SYM signaling.

RESULTS

AM-Specific Marker Genes in Rice

Gene transcripts that accumulate exclusively in mycorrhizal

roots provide valuable readouts for the analysis of specificity in

AM signaling. We selected 18 rice genes previously identified by

whole genome transcriptome analysis as being strongly induced

during the development of AMsymbiosis but silent in response to

mock treatments, root pathogen inoculation, or the application of

different phosphate regimes (Guimil et al., 2005) (see Supple-

mental Table 1 online).

Corroborating the Specificity of Marker Gene Induction

To further corroborate the AM-specific expression of these

genes, we examined the effect of root colonization by another

beneficial fungus, Piriformospora indica, on marker gene expres-

sion. P. indica has been reported to promote the growth of a wide

variety of plant species, including cereal crops (Varmaet al., 1999;

Waller et al., 2005). Similar to AM fungi, root colonization by P.

indica is strictly confined to the cortex,where the fungusdevelops

intracellular coils that are different from the arbuscules formed by

AM fungi (Varma et al., 1999). Root colonization by P. indica is

accompanied by the production of pear-shaped chlamydospores

that are easy to detect (see Supplemental Figures 1A and 1B

online) (Varma et al., 1999; Waller et al., 2005). At 5 weeks

postinoculation (wpi), we found 50 6 8% (SE) of rice root length

colonized by P. indica; however, none of the 18 gene transcripts

was detected (see Supplemental Figures 2A and 2B online).

Figure 1. Components of the Legume SYM Signaling Pathway.

The SYM pathway shared between AM and rhizobial symbioses consists

of seven proteins: a receptor-like kinase encoded by SYMRK/DMI2

(Endre et al., 2002; Stracke et al., 2002); putative cation channels

encoded by the highly homologous genes CASTOR and POLLUX/

DMI1 (Ane et al., 2004; Imaizumi-Anraku et al., 2005); nuclear porins,

encoded by NUP85 and NUP133 (Kanamori et al., 2006; Saito et al.,

2007); a calcium/calmodulin-dependent kinase encoded by CCAMK/

DMI3 (Levy et al., 2004; Mitra et al., 2004); and a protein encoded by

CYCLOPS (IPD3) (Chen et al., 2008; Parniske, 2008). SYMRK, CASTOR,

POLLUX, NUP85, and NUP133 are required for Ca2+ spiking, an early

response of root hairs to Nod factor application and to approaching AM

fungi (Kosuta et al., 2008). The calcium/calmodulin-dependent kinase

CCAMK/DMI3 is thought to decipher the calcium signals (DMI3) (Levy

et al., 2004; Mitra et al., 2004; Kosuta et al., 2008). CYCLOPS (IPD3)

interacts with CCAMK, serves as a phosphorylation substrate for

CCAMK (Messinese et al., 2007; Parniske, 2008), and is required for

mycorrhizal colonization (Chen et al., 2008). The proteins shown in

boldface were investigated in this study.

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To investigate the expression of the 18 genes in other parts of

the plant, RT-PCRwas performed on cDNA from rice root, shoot,

stem, leaf, panicle, embryo, and callus. Since transcripts of four

genes (ARBUSCULAR MYCORRHIZA18 [AM18], AM20, AM29,

and AM42) were too low to be detected by RT-PCR, their

expression was analyzed by real-time RT-PCR. All 18 genes

were expressed in AM roots but not in other plant tissue or callus

cultures (see Supplemental Figures 2A and 2B online). We

conclude, therefore, that expression of this particular set of

genes is, most likely, AM-specific.

Temporal Expression of Marker Genes

Coordinated signaling pathways must underlie the consecutive

steps leading to the establishment of an AM symbiosis. Although

mycorrhizal colonization at the whole root level is asynchronous,

temporal expression studies permit enrichment for early (pre-

symbiotic molecular crosstalk, hyphopodia formation, and rhi-

zodermal penetration) and late (cortex invasion, cortical spread,

and arbuscule formation) stages of the interaction. To monitor

possible transcriptional responses at particular stages, we ex-

amined the temporal expression of our gene set at 3, 5, 7, and 9

wpi with Glomus intraradices. A low titer of fungal inoculum was

used to enhance resolution at early time points. Fungal coloni-

zation was microscopically inspected (Figure 2A) and quantified

(Figure 2B). At 3 wpi, no intraradical colonization was found, and

colonization was low at 5 wpi, with 1.5 6 0.2% of the total root

length containing arbuscules. The amount of intraradical hyphae

and arbuscules rose sharply between 5 and 7 wpi and remained

highwithout significant further elevation at 9wpi. By contrast, the

number of vesicles increased continuously throughout the time

course.

In mycorrhizal rice roots, activation of the mycorrhiza-specific

phosphate transporter PT11 (Paszkowski et al., 2002) was

detected at 7 and 9 wpi (Figure 2C), indicating arbuscule forma-

tion, in a manner similar to that observed for the putatively

orthologous genes Mt PT4, St PT4, and Le PT4 from M.

truncatula, potato (Solanum tuberosum), and tomato (Solanum

lycopersicum), respectively (Harrison et al., 2002; Nagy et al.,

2005). Transcripts of four rice genes (AM1, AM2, AM3, and

AM11) appeared very early (at 3 wpi) and their expression

increased sharply between 5 and 7 wpi, remaining high through-

out the course of the experiments (Figure 2C). Transcripts of the

other 14 genes were first detected at 7 wpi, in parallel with PT11

(Figure 2C). While some transcript levels reached maximal ex-

pression at 7 wpi, others increased further at 9 wpi. None of the

marker genes was expressed in mock-inoculated roots at any

time point. In summary, we observed two distinct expression

patterns that reflect signaling operating at early or later devel-

opmental stages of the AM association.

It has been well documented that expression of mycorrhiza-

specific phosphate transporter genes is restricted to arbuscu-

lated cells (Harrison et al., 2002;Nagy et al., 2005). According to a

recent report, such transporters are induced by lysophosphati-

dylcholine (LPC) in potato hairy roots and tomato cell cultures in

the absence of AM colonization (Drissner et al., 2007). Therefore,

use of LPC could distinguish arbuscule-expressed late genes

that are induced along with PT11. However, PT11 was not

expressed in roots treated with LPC, although CYCLOPHILIN2

transcripts could be visualized, thus indicating live roots after

LPC infiltration (see Supplemental Figure 3 online).

Robustness of the Marker Genes

Different AM isolates have been reported to stimulate different

transcriptional responses in their hosts (Hohnjec et al., 2005). To

determine the suitability of our gene set as universal AM-specific

markers, we characterized transcript accumulation in rice roots

inoculated with Gigaspora rosea, an AM fungus phylogenetically

distant fromG. intraradices (Schussler et al., 2001). Interestingly,

whileG. intraradices produced finely branched arbuscules in rice

(Figure 2A), Gi. rosea formed predominantly coils and coarse

arbuscular coils (Figure 2D). At 7 wpi, total root length coloniza-

tion had reached 32 6 14%, with frequent occurrence of

arbuscular coils (Figure 2E). Vesicles were absent from Gi.

rosea–colonized roots, as is typical for members of Gigaspor-

aceae. All marker gene transcripts were detected in roots col-

onized by Gi. rosea (Figure 2F), exhibiting expression levels

comparable to roots colonized byG. intraradices (Figure 2C), but

were absent in mock controls. Taken together, the transcripts of

all 18 genes could be correlated with the degree of fungal root

invasion by two distantly related AM fungi, corroborating the

robustness and universal character of the marker gene set for

monitoring AM symbioses.

Systemic Induction of Marker Genes

Transcriptional plant responses to interaction with AM fungi can

be cell autonomous, restricted to specific cell types, or systemic

(Chabaud et al., 2002; Harrison et al., 2002; Kosuta et al., 2003;

Liu et al., 2003, 2007; Nagy et al., 2005). To examine whether any

of the 18 ricemarker genes can be activated by systemic signals,

we studied their transcription in split-root experiments. In this

experimental setup, only half of the root system was inoculated

(mycorrhizal part). At 6 wpi, G. intraradices had colonized the

mycorrhizal part to 33 6 10% and was absent in the non-

inoculated part, reflected by the absence of fungal structures

(Figure 3A). Transcripts of all 18marker geneswere present in the

mycorrhizal part of the roots and, with the exception of two

genes, absent in the noninoculated part of the roots (Figure 3B).

AM3 and AM34 were expressed in both the inoculated and

noninoculated halves of the root system (Figure 3B), suggesting

activation by systemic signals elicited by and/or transmitted from

themycorrhizal half of the root system.AM3 belongs to the group

of early and continuously highly expressed genes (Figure 2C). By

contrast, transcripts of AM34 were only detected at later stages

of the interaction (Figure 2C). None of the 18 genes was system-

ically induced in shoots of mycorrhizal plants (see Supplemental

Figures 2A and 2B, online).

Spatial Expression of Rice AM Marker Genes

To investigate gene expression at cellular resolution, we selected

a subset of strongly inducedmarker genes from rice representing

particular expression profiles and examined the accumulation of

their transcripts using in situ hybridization. AM1 was chosen as

AM-Specific Signaling in Rice 2991

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Figure 2. Expression Analysis of Rice Marker Genes during Colonization by G. intraradices and Gi. rosea.

(A) Trypan blue staining of wild-type roots at 6 wpi withG. intraradices.G. intraradices formed highly branched arbuscules. A, arbuscule; IH, intraradical

hypha. Bar = 50 mm.

(B) Percentage root length colonization by G. intraradices during a time-course experiment determined by a modified grid-line intersect method.

Harvest time points are indicated (wpi). Means6 SE of five plants represented by two replicate samples are shown. ext hyphae, extraradical hyphae; int

hyphae, intraradical hyphae.

(C) Real-time RT-PCR–based expression analysis of marker genes during a time-course experiment of rice roots inoculated with G. intraradices.

Expression levels are shown relative to the constitutively expressed CYCLOPHILIN2 gene. Error bars indicate SD from three technical replicates.

(D) Trypan blue staining of wild-type roots at 7 wpi withGi. rosea.Gi. rosea formed arbuscular coils but no arbuscules or vesicles. AC, arbuscular coil; C,

coil; EC, epidermal coil. Bar = 50 mm; bar in inset = 20 mm.

(E) Percentage root length colonization by Gi. rosea (Gr) at 7 wpi determined by a modified grid-line intersect method. Means 6 SE of five plants

represented by two replicate samples are shown.

(F) Real-time RT-PCR–based expression analysis of mock-inoculated and Gi. rosea–inoculated rice roots at 7 wpi. The experiment was repeated four

times with similar results. Expression levels are shown relative to the constitutively expressed rice CYCLOPHILIN2 gene. Error bars indicate SD from

three technical replicates.

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an early expressed gene, AM3 as an early and systemically

expressed gene, and PT11 as a late expressed gene. The

digoxigenin-labeled PT11 antisense probe revealed signals re-

stricted to arbusculated cells (Figure 4A). No signal was detected

when the sense probe was applied to sections from AM roots

(Figure 4B) or when antisense probe was applied to tissue

sections from mock-inoculated roots (Figure 4C). Thus, the

localization of PT11 expression corresponds to that of its

predicted orthologs Mt PT4 (Harrison et al., 2002), Le PT4, and

St PT4 (Nagy et al., 2005).

Expression of AM1 was detected in rice cells colonized by

small arbuscules and in cortex cells flanking intercellularly grow-

ing hyphae (Figure 4D). Due to resolution constraints, it was not

possible to distinguish the small arbuscules from developing or

senescing arbuscules. However, the early expression of AM1 is

consistent with transcript accumulation in developing arbus-

cules, and this is further supported by the observation that cells

with fully developed arbuscules did not produce signals with the

same AM1 antisense probe (Figure 4E). Sections from mycor-

rhizal roots hybridized to the AM1 sense probe (Figure 4F), and

sections from mock-inoculated roots treated with the AM1

antisense probe (Figure 4G) had no signals.

AM3 transcripts were visualized in arbusculated cells only

(Figure 4H). According to the results of the split-root experiment

(Figure 3B), a systemically induced AM3 expression was ex-

pected; but it was not detected. This, however, may result from

transcript accumulation below the limit of detection, but in a high

number of cells. No signal was observed using the sense probe

(Figure 4I) or hybridizing mock-inoculated root sections with

antisense probe (Figure 4J).

In summary, the variety of transcript accumulation patterns

found suggested the existence of cell-autonomous signaling

events operating selectively in certain cell types during the

colonization of rice by G. intraradices. As in M. truncatula, the

rice mycorrhiza-specific phosphate transporter PT11 is strictly

induced in arbusculated cells (Figure 4A). By contrast, AM1 is

transcribed in two types of cortex cells, those adjacent to

intercellularly growing hyphae and those containing small

Figure 3. Analysis of the Systemic Expression of Rice Marker Genes.

(A) Percentage root length colonization by G. intraradices of the inocu-

lated halves of the split roots at 6 wpi, determined by a modified grid-line

intersect method. ext hyphae, extraradical hyphae; int hyphae, intra-

radical hyphae.

(B) Real-time RT-PCR–based expression of marker genes in the mycor-

rhizal half (+G.i.) and the noninoculated half (�G.i.) of a split root and a

mock-inoculated control. Gene expression levels are shown relative to

the expression of the constitutive rice CYCLOPHILIN2 gene. Error bars

represent SD for three technical replicates. The experiment was repeated

twice with similar results.

Figure 4. Spatial Expression of Three AM Marker Genes.

In situ hybridization of PT11 ([A] to [C]), AM1 ([D] to [G]), and AM3 ([H]

to [J]).

(A), (D), (E), and (H) Sections from rice roots colonized by G. intraradices

and probed with antisense probes.

(B), (F), and (I) Sections from rice roots colonized by G. intraradices and

probed with sense probes.

(C), (G), and (J) Sections from mock-inoculated rice roots probed with

antisense probes.

In situ hybridizations were repeated at least twice with equivalent results.

Black arrowheads, fully grown arbuscules; red arrowheads, small arbus-

cules; blue arrowheads, apoplastic intraradical hyphae. Bars = 30 mm.


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arbuscules (Figure 4D). The spatial expression is consistent with

the observed early and progressive gene induction associated

with fungal spread within the host root (Figure 2C). In addition

to being expressed systemically (Figure 3B), AM3 showed

arbuscule-specific transcript accumulation (Figure 4H).

Identification of AM-Specific Signaling in Rice

To examine the dependence of marker gene expression on the

nonspecific common SYM signaling pathway in rice, we first

determined the functional conservation of the pathway between

legumes and rice. Putative rice orthologs of legume SYMRK,

CASTOR, POLLUX, NUP85, NUP133, CCAMK, and CYCLOPS

have previously been identified computationally (Kanamori et al.,

2006; Zhu et al., 2006; Messinese et al., 2007; Saito et al., 2007),

and the requirement of CCAMKandCYCLOPS (the two signaling

proteins downstream of the Ca2+ response) for AM development

in rice was recently reported (Chen et al., 2007, 2008).

Expression of Common SYM Genes in Rice

To compare expression patterns of the common SYM genes

between rice and legumes, RT-PCR was performed on cDNA

from different organs of the rice plant as well as frommycorrhizal

roots and callus (see Supplemental Figure 4 online). SYMRK,

CASTOR, and POLLUX were expressed in almost all of the

tissues tested. High transcript levels of both genes encoding

nucleoporins were found in all samples tested. As reported

previously (Chen et al., 2007, 2008), RNA levels of rice CCAMK

(DMI3) were highest in roots and panicles (see Supplemental

Figure 4 online) and those of CYCLOPS (IPD3) (Messinese et al.,

2007) were highest in roots; however, only low levels could be

detected in other plant organs and callus. Interestingly, none of

the genes was differentially expressed in roots upon colonization

byG. intraradices. In general, all putative rice orthologs of legume

SYM pathway genes exhibited root expression, and where

available data permit comparison, expression patterns were

similar to those in legumes (Imaizumi-Anraku et al., 2005;

Kanamori et al., 2006; Saito et al., 2007). Exceptions were (1)

CCAMK, which was only weakly expressed in M. truncatula

flowers (Levy et al., 2004) but highly expressed in rice panicles,

and (2) POLLUX, which was expressed mainly in roots, but not in

any other organ, of M. truncatula (Ane et al., 2004) but in rice

showed broad expression with high RNA levels in panicles.

Identification and Characterization of Rice Common

SYM Mutations

To study the conservation of common SYM signaling in rice, we

screened for mutant lines with insertions in putative SYM

orthologs encoding signaling components upstream and down-

stream of Ca2+ spiking. Rice lines with insertions into CASTOR

andPOLLUX (upstream) and intoCCAMK andCYCLOPS (down-

stream) (Figure 1) were selected from public databases (http://

orygenesdb.cirad.fr/, http://signal.salk.edu/) and from PCR-

based screens of the retrotransposon Tos17-induced rice

mutant collection (Hirochika et al., 2004) (Figure 5; see Supple-

mental Table 2 online).

For each of the rice SYM genes, a full-length cDNA is available

that allows determination of the gene structure (http://cdna01.

dna.affrc.go.jp/cDNA/). Analogous to L. japonicus (Imaizumi-

Anraku et al., 2005), rice CASTOR and POLLUX consist of 12

exons and 11 introns, with CASTOR having longer introns than

POLLUX (Figure 5A). Rice CCAMK consists of seven exons and

six introns and is similar in structure to CCAMK of M. truncatula

(Chen et al., 2007). Rice CYCLOPS (IPD3) has 11 exons and a

similar structure to M. truncatula IPD3 (Figure 5A) (Messinese

et al., 2007; Chen et al., 2008). Thus, gene structure is highly

conserved.

For CASTOR, two lines predicted to contain T-DNA insertions

in the second exon (1B-08643 and 3D-50377) were retrieved

frommutant collections. PCR analysis confirmed the presence of

the T-DNA for line 1B-08643 (castor-1) (Figure 5A; see Supple-

mental Table 2 online) but not for 3D-50377. In the case of

POLLUX, three lines were identified: 1C-03411 (pollux-1) con-

tained a T-DNA insertion in the first intron; NC6423 (pollux-2) and

ND5050 (pollux-3) had Tos17 insertions in the third and fourth

exons, respectively (Figure 5A; see Supplemental Table 2 online).

Four insertion lines were identified for CCAMK: the three lines

NF8513, NG2508, and NE1115 carry Tos17 insertions, and line

2B-50404 contains a T-DNA insertion. While NF8513 and

NG2508 have been characterized previously (Chen et al.,

2007), NE1115 represents a novel allele containing a Tos17

insertion within the fourth exon. NE1115 and NF8513 were used

in our studies as rice ccamk-1 and ccamk-2, respectively (Figure

5A; see Supplemental Table 2 online). The fourth line, 2B-50404,

was not considered due to reduced germination frequency. We

identified 54 insertions in the region of CYCLOPS. We chose

three Tos17 insertion lines, NG0782, NC2415, and NC2713, for

characterization. In each of these lines, the retrotransposon is

inserted in the sixth exon. We refer to these lines as cyclops-1,

cyclops-2, and cyclops-3, respectively (Figure 5A; see Supple-

mental Table 2 online). To confirm the location of each insertion

relative to the given gene structure, sequencing across both the

59 and 39 gene-insertion boundaries was performed. The posi-

tions of all insertions were as reported in the databases of the

respective mutant collection (Figure 5A). Therefore, this study

included multiple mutant alleles of POLLUX, CCAMK, and CY-

CLOPS and one of CASTOR.

To quantify transcript accumulation in the mutant lines,

RT-PCR was performed using one primer pair flanking the

insertion and two other primer pairs targeting the regions up-

stream and downstream of the insertions corresponding to the

59 and the 39 ends of the cDNA, respectively (Figure 5A; see

Supplemental Table 3 online). CASTOR transcripts were not

detected in castor-1, suggesting that this is a null allele (Figure

5B). No RT-PCR products were obtained using primers spanning

the transposon insertion site for any of the three pollux, the

ccamk-2, and the three cyclops alleles; however, transcripts of

the respective genes were detected in each of the lines with

primer pairs targeting the 59 end of the transcript (Figure 5B). For

ccamk-2 and the three cyclopsmutants, transcripts correspond-

ing to the 59 and 39 regions of the insertions were monitored

(Figure 5). Although still partially transcribed, the genes are likely

compromised due to the several kilobases of T-DNA or retro-

transposon inserted within central exons (Figure 5A). In line

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NF8513 (ccamk-2), retrotransposon insertion into the third intron

of CCAMK led to missplicing of the third exon and an in-frame

fusion of the second and fourth exons (Chen et al., 2007). The

deletion of the third exon removes a putative Ca2+-dependent

autophosphorylation domain (Gleason et al., 2006; Tirichine

et al., 2006) and is predicted to disrupt CCAMK function.

CASTOR, POLLUX, CCAMK, and CYCLOPS Are Required

for AM Colonization in Rice

To define and compare the roles of CASTOR, POLLUX, CCAMK,

and CYCLOPS in AM colonization, siblings from segregating T1

families of each mutant line were used for phenotypic charac-

terization, enabling the direct comparison of mutant and wild-

type alleles in the heterozygous and homozygous state for each

locus under the same experimental setting. The sizes of the T1

families depended on seed availability and varied between 30

and 60 siblings. The genotypes of individual plants were deter-

mined, and at least seven plants from each genotype were

selected for microscopic inspection at 6 wpi.

Since the insertion mutants were generated in three different

rice japonica cultivars, Dongjin, Hwayoung, and Nipponbare, we

quantitatively and qualitatively examined colonization by G.

intraradices of each variety. Heterozygous and homozygous

Figure 5. Rice Common SYM Genes and Characterization of SYM Loci.

(A) Gene structures and positions of insertions in rice CASTOR, POLLUX, CCAMK, and CYCLOPS drawn to scale. The A of each ATG designates

nucleotide 1. Black boxes represent exons separated by introns (solid lines). Mutant line names and positions (in nucleotides) of the respective

insertions are indicated. The mutants castor-1 and pollux-1 carry T-DNA (LB, left border; RB, right border); the other mutants carry Tos17

retrotransposon insertions. Arrows indicate primers used in (B).

(B) Analysis of CASTOR, POLLUX, CCAMK, and CYCLOPS transcripts in mutant and wild-type rice by RT-PCR using primer pairs located 59 (black

arrows) or 39 (light gray arrows) of the insertion and flanking the insertion (IF, insertion flank; dark gray arrows). For castor-1 and pollux-1, cultivars

Dongjin and Hwayoung were used as wild-type controls, respectively. For all other mutants, Nipponbare was employed as the wild type. Amplification

of the wild-type control band corresponding to the 59 and 39 flanks of the pollux-2 and pollux-3 alleles was performed with the same primer pairs.


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wild-type segregants aswell as nonsegregating individuals of the

parental varieties exhibited 69 6 9% of total root length coloni-

zation, which was accompanied by abundant production of

arbuscules and vesicles (Figures 6A to 6C and 7A). Root colo-

nization of the sym mutants exhibited a patchy pattern of

hyphopodia clusters accompanied by attempted penetration of

rhizodermal cells (Figures 6D to 6O). Each of the homozygous

mutants lacked cortex colonization and, therefore, arbuscules

and vesicles (Figures 6D to 6O; seeSupplemental Table 2 online).

Total root length colonization was low and reached 4 to 14%

(Figure 7B), including 1 to 10% of hyphopodia colonization of the

different mutants, respectively.

Fungal morphology in colonized homozygous mutant plants

was similar for mutant alleles of the same gene and across all

mutant lines, with occasional formation of hyphopodia (Figures

6D to 6L) or groups of twisted hyphae on the root surface (Figures

6D, 6I, 6L, and 6O). The fungus regularly penetrated individual

rhizodermal cells, accompanied by either hyphal branching

(Figures 6E to 6K) or coil formation (Figure 6E). Sometimes

extensive hyphal growth into neighboring rhizodermal cells was

observed, with cells almost entirely filled with deformed fungal

structures (Figure 6M). Rarely, fungal hyphae were observed to

grow through several cells of a file, branching inside cells, as

shown for pollux-1 (Figure 6N).

In summary, the morphological AM phenotypes were very

similar for all mutant lines. The phenotypes of the rice castor,

pollux, ccamk, and cyclops mutants resembled those of legume

mutants (Kistner et al., 2005; Parniske, 2008), thus suggesting

evolutionary conservation of these signaling components across

distant angiosperms.

Molecular Phenotyping of the Rice SYM Pathway Mutants

To account for the irregular distribution of hyphopodial clusters

on symmutants, we performedmolecular quantification of fungal

DNA to precisely estimate the total amount of fungus present in

each sample that could contribute to the elicitation of the host

root. For this purpose, we first confirmed the previously reported

(Isayenkov et al., 2005; Alkan et al., 2006) correlation between

microscopically and molecularly quantified colonization levels

(see Supplemental Figure 5 online). In a time-course experiment,

the kinetics of the relative amounts of fungal DNAmatched those

of the microscopically determined degree of fungal root coloni-

zation (i.e., both Gi ITS1 DNA levels and the amount of fungal

colonization structures were low at 3 wpi, increased slightly at 5

wpi, rose dramatically between 5 and 7 wpi, and were further

enhanced at 9 wpi) (see Supplemental Figure 5 online). In wild-

type and sym mutant rice, molecular determination of coloniza-

tion levels mirrored the microscopic data, with comparably high

amounts of fungal DNA for the wild type (Figures 7A and 7C) and

much lower amounts for the sym mutants (Figures 7B and 7D).

A subset of nine marker genes was selected for real-time

RT-PCR–based molecular phenotyping of rice sym mutants

inoculated with G. intraradices. The castor-1, pollux-1 and

pollux-2, ccamk-1 and ccamk-2, and cyclops-1 and cyclops-2

mutant alleles were analyzed. The selected marker genes reflect

particular expression patterns: four early expressed genes (AM1,

AM2, AM3, and AM11), the systemically expressed genes (AM3

and AM34), and the three highest accumulating, late expressed

genes (AM10, AM14, and AM15). Rice PT11 was used as a

marker for arbusculated cells. To test the suitability of the real-

timeRT-PCR primers for all cultivars used, transcript levels of the

nine marker genes were compared. All nine marker genes were

monitored reliably and at equivalent levels in mycorrhizal roots of

each of the three cultivars (Figure 7E). Expression of the nine

marker genes was determined in mutants (Figure 7F). Transcript

levels were normalized against fungal DNA present in identical

samples to account for possible variation in inoculum strength

(Figures 7G and 7H).

Despite indistinguishable morphological phenotypes of AM

interaction in all four mutants, the expression patterns of marker

genes differed among the mutants (Figures 7G and 7H). The

profile of gene expression was equivalent in castor-1 and the two

pollux alleles, both proteins acting upstream of Ca2+ spiking. The

patterns were different in the two lines mutated inCYCLOPS and

therefore perturbed downstream of the Ca2+ response. Such

profile specificity could not be attributed to differences in fungal

presence (Figures 7B and 7D). Interestingly, while ccamk-1 dis-

played an expression profile identical to those of castor-1 and the

pollux alleles, which is consistent with a loss of Ca2+ response,

ccamk-2 exhibited a pattern equivalent to those of the cyclops

mutants, which is in accordance with a defect in the interaction

with CYCLOPS. Although mutations in CASTOR, POLLUX,

CCAMK, and CYCLOPS blocked colonization of the root cortex,

the two early marker genes, AM1 and AM2, were expressed in all

seven mutants (Figures 7G and 7H). Elicitation of gene activity,

therefore, occurred in the absence of functional common SYM

signaling. Three additional genes were detected in ccamk-2 and

the two cyclops lines: the two systemically induced genes, AM3

and AM34, and the late induced gene, AM14 (Figures 7G and

7H). AM11 was the only one of the four genes induced at early

symbiotic stages not to be expressed in any mutant (Figures 7G

and 7H), indicating a dependence of gene activation on the intact

common SYM signaling pathway. Transcripts of the other late

and nonsystemically induced genes, such as AM10, AM15, and

PT11, were absent in the mutants. Whereas the spatial expres-

sion patterns of AM10, AM11, and AM15 are not known, PT11

expression depends on arbuscule formation. Since arbuscules

were not formed in the mutants, the absence of expression of

late induced genes could be due to either the absence of the

particular symbiotic structures or deficiencies in symbiotic sig-

naling. It would have been informative to determine gene ex-

pression at the cytological level in the mutant background, but

in situ hybridization was not sensitive enough to detect low-level

transcripts in inoculated mutant roots.

The mutant-specific gene expression patterns, such as the

lack of detectable mRNA encoding the early induced geneAM11

in all mutants, together with their deficient symbiotic phenotypes

are consistent with a crucial role of the common SYM pathway in

rice. By contrast, the detection of transcripts of early activated

genes in all mutants suggests the existence of AM signaling

operating independently of the common SYM pathway. Expres-

sion of three additional genes, AM3, AM14, and AM34, required

Ca2+ spiking but was independent of CYCLOPS, suggesting

signaling diverging from the common SYM pathway at the Ca2+

response.

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Figure 6. AM Phenotypes of Rice sym Mutants.

Trypan blue staining of mutant and wild-type rice roots at 6 wpi with G. intraradices is shown.

(A) to (C) Roots of the three cultivars, Dongjin (A), Hwayoung (B), and Nipponbare (C), show abundant arbuscules and vesicles.

(D) castor-1. The fungus formed a hyphopodium in a groove between two rhizodermal cells. Extraradical hyphae branching off the hyphopodium

showed septa formation.

(E) pollux-1. Arising from a hyphopodium, several penetration pegs entered a rhizodermal cell, but the fungus did not progress into the cortex.

(F) pollux-2. The fungus formed a hyphopodium from which two penetration pegs entered two rhizodermal cells, where hyphae branched and formed

septa.

(G) pollux-3. Fungal hyphae branched inside a rhizodermal cell and formed septa.

(H) ccamk-1. Several fungal hyphae entered a rhizodermal cell, branched, and formed finger-like swellings.

(I) ccamk-2. Penetration pegs from two small hyphopodia entered a rhizodermal cell and branched. Cortex colonization was not observed.

(J) cyclops-1. Penetration pegs starting from a hyphopodium entered into different neighboring rhizodermal cells, branched inside the cells, and formed

septa. The fungus did not colonize the cortex.

(K) cyclops-2. A hypha branched inside a rhizodermal cell, growing back and forward inside the cell and forming septa.

(L) cyclops-3. A hypha formed a swollen hyphopodium and entered a rhizodermal cell, filling it with a large swelling.

(M) castor-1. The fungus occupied several neighboring rhizodermal cells and formed branches and swellings.

(N) pollux-1. A hypha traveled intracellularly through rhizodermal cells, forming branches in the rhizodermis.

(O) cyclops-3. Fungal hyphae formed several swellings on the rhizodermal surface.

A, arbuscule; EH, extraradical hypha; HP, hyphopodium; HS, hyphal swelling; RB, rhizodermal branch (intracellular); RC, rhizodermal coil (intracellular);

RS, rhizodermal hyphal swelling (intracellular); V, vesicle. Arrowheads indicate septa. Bars = 50 mm.


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Figure 7. Molecular Phenotypes of sym Mutants.

(A) and (B) Percentage root length colonization of wild-type cultivars Donjin (DJ), Hwayoung (HY), and Nipponbare (N) (A) and common symmutants (B)

by G. intraradices at 6 wpi, determined by the grid-line intersect method. Means 6 SE of five plants represented by two replicate samples are shown.

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DISCUSSION

Rice is the staple food for half of the global population; in addition

to being an important crop, it is also a powerful model system for

cereals. An improved understanding of the establishment and

functioning of AM symbiosis in rice has the potential to directly

impact management strategies for the optimization of sustain-

able agricultural systems. The goal of this study has been to pave

the way for investigations of AM-specific signaling in rice. In

contrast with previous work, we studied a comprehensive list of

genes specifically activated during AM symbiosis. This specific-

ity was concluded from the absence of their mRNAs in non-

mycorrhizal organs of the plant or in roots invaded by other

beneficial (see Supplemental Figure 2 online) or pathogenic fungi

or in response to varying phosphate regimes (Guimil et al., 2005).

Thus, these genes are an invaluable tool to molecularly dissect

signaling cascades specific to the AM symbiosis.

Temporal expression analysis of rice roots colonized by G.

intraradices distinguished early and late induced genes. Expres-

sion of the previously characterized rice mycorrhiza-specific

phosphate transporter PT11 (Paszkowski et al., 2002) served as

a marker for arbuscule formation, and in situ hybridization

confirmed specific expression in arbusculated cells, consistent

with a role in symbiotic phosphate uptake at the periarbuscular

interface (Harrison et al., 2002; Javot et al., 2007).

Activation of the genes AM1, AM2, AM3, and AM11 prior to

arbuscule development defined markers for early induction and

the existence of a signal acting at initial stages of the interaction

corresponding to presymbiotic recognition, hyphopodia forma-

tion, and early penetration. Transcription of these four genes

remained high at later time points, as a result of systemic root

elicitation, of continuously high but local induction, or of activa-

tion by additional AM-specific signals. Evidence for the existence

of all three signaling alternatives was obtained from split-root

experiments (Figure 3) and in situ hybridization (Figure 4). Ex-

pression of AM3 in both the colonized and noncolonized parts of

a split root indicated systemic induction (Figure 3B). Interest-

ingly, in situ hybridization revealed that mRNA of AM3 accumu-

lated in cells with fully developed arbuscules (Figure 4H). Thus,

activation of this gene is possibly regulated by two distinct

signaling pathways, the first operating systemically and the

second operating arbuscule-specifically. Although it was to be

expected that systemic induction would lead to hybridization

signals in nonarbusculated cells, systemic expression might

correspond to the relatively low amounts of mRNA per cell that

are undetectable by in situ hybridization but, when induced in

most cells of the root, are detectable by real-time RT-PCR.

Further support for a dual induction of AM3 is provided by

expression values during split-root experiments. Consistent with

the additive expression of systemic and arbuscule-associated

induction, mRNA levels in mycorrhizal roots were higher than

those in nonmycorrhizal roots, corresponding to only systemic

values (Figure 3B). Interestingly, AM3 encodes a small secreted

protein of only 98 amino acids containing a Lys motif (LysM)

domain. LysM domains are known to bindN-acetylglucosamine,

which is a component of rhizobial Nod factors, as well as of the

fungal cell wall constituent chitin (reviewed in Buist et al., 2008).

Intriguingly, legume receptor-like kinases that mediate percep-

tion of the bacterial Nod factors contain LysM domains that

physically interact with Nod factors (Radutoiu et al., 2007;

reviewed in Buist et al., 2008).

None of the other early induced genes exhibited systemic

induction, but AM1 was found to have a distinct spatial expres-

sion pattern. AM1 was expressed most strongly in cells neigh-

boring intercellularly growing hyphae and in cells containing

small arbuscules (Figure 4D) but was not detected in cells

hosting fully developed arbuscules (Figure 4E). Arbuscules

have a lifespan of only 4 to 10 d (reviewed in Hause and Fester,

2005), with different developmental stages simultaneously pres-

ent in each root system. Although microscopic resolution of the

in situ hybridization did not distinguish between developing

and collapsing arbuscules, the early induction of AM1 argues

for arbuscule formation rather than senescence. AM1 encodes

the putative type III peroxidase PRX53 (Passardi et al., 2004),

a secreted protein potentially involved in either the production

or the scavenging of reactive oxygen species. Hydrogen perox-

ide has been associated with mycorrhizal structures (Salzer

et al., 1999; Fester and Hause, 2005), but as AM fungi them-

selves produce hydrogen peroxide (Lanfranco et al., 2005),

these results are difficult to interpret. Interestingly, peroxidase-

generated hydroxyl radicals are involved in plant cell growth

by nonenzymatic loosening of the cell wall (Liszkay et al.,

2003, 2004).

The remaining 14 genes exhibited the second major expres-

sion pattern, typified by the absence ofmRNAat early time points

but having elevated transcript levels at later time points. The

presence of PT11 within this group suggests that some genes

followed arbuscule-specific induction and thus might be in-

volved in arbuscule development and/or function. LPC was

applied exogenously to determine which of the late induced

genes are activated in parallel to PT11 and thus are related to the

Figure 7. (continued).

(C) and (D) Real-time RT-PCR–based measurement of G. intraradices ITS DNA relative to rice CYCLOPHILIN2 DNA in G. intraradices–inoculated (Gi)

and mock-inoculated (M) wild-type roots of the three wild-type rice cultivars (C) and the common sym mutants (D) at 6 wpi.

(E) and (F) Quantitative PCR-based expression analysis of nine marker genes relative to expression of the constitutive rice CYCLOPHILIN2 gene in the

three mock-inoculated and G. intraradices–inoculated wild-type cultivars (E) and the common sym mutants (F). RNA for expression analysis was

extracted from the same root samples used for the experiments shown in (C) and (D).

(G) and (H) Expression of nine marker genes inG. intraradices–inoculated wild-type cultivars (G) and common symmutants (H) relative toG. intraradices

ITS DNA level (shown in [C] and [D] above).

All experiment were repeated at least twice with similar results. For all real-time RT-PCR experiments, mean values of three technical replicates are

displayed. Error bars indicate SD.


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same signaling pathway. LPC did not induce PT11 (see Supple-

mental Figure 3 online), which may be due to technical short-

comings or to the absence of this signaling pathway in rice.

However, alternative expression patterns associated, for exam-

ple, with progressive cortex colonization or late systemically

activated genes may also be a feature of this group of genes.

Indeed, 1 of the 14 genes, AM34, was expressed in both halves

of the split-root system, suggesting systemic induction (Figure

3B). In contrast with the early and systemically induced AM3, the

relative expression of AM34 was similar in the colonized and

noncolonized parts of the root system, reflecting a single signal

governing the late systemic transcription ofAM34. The low levels

of AM34 mRNA did not permit in situ hybridization. This gene is

predicted to encode a UDP-glycosyltransferase, a class of

enzymes that catalyze the transfer of glucosemoieties to diverse

substrates, including plant hormones, secondary metabolites,

and xenobiotics, for their inactivation (Lim and Bowles, 2004). In

transcript-profiling experiments, various glucosyltransferases

were upregulated upon mycorrhizal colonization (Liu et al.,

2003, 2007; Manthey et al., 2004; Hohnjec et al., 2005; Deguchi

et al., 2007), consistent with the increased production of glyco-

sylated secondary metabolites in mycorrhizal roots (Schliemann

et al., 2008).

The 18 selected genes proved robust as a universal marker set

for AM symbioses in rice, since levels of their AM-induced

transcripts were comparable during interaction with the phylo-

genetically distant AM fungus Gi. rosea. This is particularly

striking considering that the intracellular infection structures of

Gi. rosea are rather coarse and simple arbusculated coils,

compared with the highly ramified, finely branched arbuscules

of G. intraradices. Moreover, the two fungi vary in symbiotic

functions, since some plant species benefit considerably more

from an association with G. intraradices than with Gi. rosea

(Burleigh et al., 2002; Smith et al., 2003, 2004). Notably, a core

set of plant genes was also found in M. truncatula to exhibit

related expression patterns during interactions with three dis-

tantly related AM fungi (Liu et al., 2007). Thus, AM signaling

routes appear to be common to different AM symbioses despite

morphological and nutritional differences.

AM-specific signaling might in part occur dependently or

independently of the unspecific commonSYMsignaling pathway

discovered in legumes. We analyzed a set of rice mutants

affected in genes coding for putative orthologs of legume pro-

teins representing components of the common SYM pathway

upstream and downstream of the Ca2+ response. Inability of the

mutants to establish mycorrhizal symbiosis demonstrated func-

tional conservation of the encoded proteins and of the SYM

signaling cascade across distant plant species. This finding is

supported by previous reports that two individual proteins,

CCAMK and IPD3 (CYCLOPS), are required for AM symbiosis

in rice (Chen et al., 2007, 2008). Functional conservation of

symbiotic signaling factors among dicotyledons and monocot-

yledons is consistent with an evolutionary origin of AM symbiosis

predating the divergence of the two main angiosperm classes.

This evolutionary conservation possibly also extends beyond the

angiosperms, but this remains to be demonstrated. Although our

rice mutant selection did not include putative orthologs of all

currently known legume SYM genes, it is likely that the complete

signaling pathway is conserved. This view is supported by the

recent observation that rice SYMRK restores AM colonization in

the L. japonicus symrk mutant (Markmann et al., 2008).

Confirmation of the relevance of the common SYM pathway

for AM development in rice led us to investigate the possibility of

signaling routes operating independently of the known pathway.

The similarly strong morphological and quantitative phenotypes

of the rice common sym mutants (Figure 6) allowed for their

dissection at the molecular level and the search for signaling

routes in addition to the commonSYMpathway in the absence of

secondary effects perturbing the comparison. Nine markers

corresponding to discrete signaling events reflected by their

different spatial and temporal expression patterns were selected

for molecular phenotyping. The absence of AM11 mRNA places

the activation of this gene downstream of the common SYM

pathway (Figure 8). Also, for the late markers AM10, AM15, and

OsPT11, no transcript was detected in any of themutants (Figure

8). However, PT11 expression was strictly dependent on arbus-

cule formation, consistent with its absence in themutants (Figure

8). The determination of whether induction of the two late genes

AM10 and AM15 depends on arbuscules or intact common SYM

proteins awaits spatial expression analysis. The activation of two

early genes, AM1 and AM2, in all mutants and three additional

genes, the early systemic AM3, the late systemic AM34, and the

late inducedAM14, in a subset of mutants indicated an induction

independent of cortex colonization and arbuscule formation.

While this was expected for the early systemic marker AM3, it

Figure 8. AM-Specific Signaling Pathways Including Bifurcation at the

Ca2+ Response.

The common SYM pathway displaying the components analyzed within

this study consists of CASTOR and POLLUX upstream and CCAMK and

CYCLOPS downstream of Ca2+ spiking. Expression of AM11 is induced

early and relies on an intact SYM pathway. Induction of systemically (S;

gray arrows) induced genes, AM3 and AM34, and the late (L; black

arrows) induced gene, AM14, relies on an intact Ca2+ response and thus

requires CASTOR, POLLUX, and CCAMK but is independent of CY-

CLOPS. Expression of PT11 is dependent on arbuscule formation. The

dependence of AM10 and AM15 on SYM pathway signaling or on

arbuscule formation is not known (dashed arrows with question marks).

AM1 and AM2 are induced early but do not require functional CASTOR,

POLLUX, CCAMK, or CYCLOPS, demonstrating the existence of an

alternative AM signaling pathway (AP).

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suggested that late systemic induction of AM34 in wild-type

roots, as well as late activation of AM14, does not require fungal

penetration of the cortex. Interestingly, the seven mutants

displayed two classes of expression patterns. The first pattern

is defined by the expression of two of the four early genes, AM1

and AM2, in all alleles of the four mutants, suggesting that their

induction is independent of the common SYM pathway, but

triggered by alternative signaling acting in parallel, either during

presymbiotic recognition or hyphopodium formation, or both

(Figure 8). The second pattern is characterized by the induction

of three further genes, AM3, AM14, and AM34, reliant on a

functional Ca2+ response but independent of CYCLOPS, thus

suggesting deviation from the common SYM pathway down-

stream of Ca2+ spiking (Figure 8). Intriguingly, the expression

pattern of the two mutant alleles of CCAMK differed, with

ccamk-1 showing a similar pattern to that of the castor and

polluxmutants and ccamk-2 having a pattern similar to that of the

cyclops mutants (Figures 7F and 7H). The expression pattern of

ccamk-1 can plausibly be explained by the lack of functional

perception of Ca2+ oscillations, which phenocopies the absence

of Ca2+ spiking. This is consistent with the transcriptional allele

characterization that predicts CCAMK function to be compro-

mised due to Tos17 insertion between the calmodulin binding

domain and the three EF hands. By contrast, the transcription

profile of ccamk-2 can be interpreted in different ways. In the first

scenario, low amounts of wild-type transcript might still be

produced for the induction of a subset of marker genes but

might not be sufficient to restore symbiosis. Alternatively, since

the ccamk-2 allele is predicted to encode a CCAMK derivative

mutated within the kinase domain (Chen et al., 2007), expression

of AM3, AM14, and AM34 appears reliant on CCAMK but might

be independent of its kinase function. A recent publication

reported that CCAMK phosphorylates CYCLOPS (Parniske,

2008). It is tempting, therefore, to speculate that the mutation

in the kinase domain affected CYCLOPS phosphorylation in

ccamk-2. The expression profile of the ccamk-2 mutant is

consistent with this interpretation, as it mimicked that of the

three cyclops mutants. Further experimental evidence is re-

quired to confirm the lack of kinase activity and thus CYCLOPS

phosphorylation in ccamk-2. In summary, the transcription

of AM3, AM14, and AM34 depended on functional CASTOR,

POLLUX, and CCAMK but not on CYCLOPS, thus indicating

the presence of novel AM-specific signaling components that

mediate the induction of the three marker genes downstream of

the Ca2+ response (Figure 8).

We conclude that AM-specific signaling in rice involves a

complex network, including the evolutionarily conserved, and

currently best defined, common SYM pathway. However, our

results also suggest alternative signaling routes that either de-

viate from, or are independent of, the common SYM pathway.

METHODS

Plant Material

Rice (Oryza sativa cv Nipponbare) was used for the characterization of

rice marker genes. Rice lines carrying T-DNA insertions in CASTOR and

POLLUX (lines 1B-08643 and 1C-03411, respectively) arose in the

japonica cultivars Dongjin and Hwayoung, respectively (Jeong et al.,

2002, 2006). In the Nipponbare background, Tos17 (Miyao et al., 2003)

insertions were identified in POLLUX (lines NC6423 and ND5050 for

pollux-2 and pollux-3, respectively), inCCAMK (lines NE1115 for ccamk-1

and NF8513 for ccamk-2 [Chen et al., 2007], respectively), and in

CYCLOPS (IPD3) (lines NG0782, NC2415, and NC2713 for cyclops-1,

cyclops-2, and cyclops-3, respectively). Segregating T1 individuals were

used from each line in this study. T1 corresponds to progeny from the

self-pollinated hemizygous or heterozygous primary T0 transformants

(T-DNA–induced alleles) or regenerants (Tos17-induced alleles), respec-

tively.

Plant Growth and Inoculation Conditions

Plants were grown in a phytochamber with a 12-h/12-h day/night cycle at

28/228C and 60% humidity. They were watered every 2nd d for the first

2 wpi and thereafter fertilized every 2nd d with a mix of 0.005% (w/v)

Hauert-Flory Typ A (Hauert) and 0.01% (w/v) Sequestren Rapid (Syngenta).

These growth conditions were shown previously to maintain efficient and

equal colonization.

Inoculation by AM Fungi

Rice seeds were surface-sterilized twice with 3.5% sodium hypochloride

solution for 15 min, washed extensively with sterile water, and pregermi-

nated in sterile water for 5 d at 258C in the dark. Germlings were

transplanted into washed and autoclaved quartz sand and inoculated

upon planting with ;1000 Glomus intraradices spores per plant from a

commercially available inoculum (Biorize). Plants were harvested at 6 wpi

or as indicated in the text. For time-course experiments, a low titer (100

spores per plant) of aseptically grown G. intraradices (Becard and Fortin,

1988) was used. Gigaspora rosea BEG9 inoculum was obtained from

Biorize. Gi. rosea spores were collected with forceps and immersed in

water, and 100 spores per plant were injected into the vicinity of the

seedling root with a Pasteur pipette. Mock-inoculated plants received

fungal growth medium or water.

Inoculation by Piriformospora indica

Cultures of P. indica were kindly provided by Phillip Franken (Institute for

Ornamental and Vegetable Crops), and rice plants were inoculated

according to Waller et al. (2005). P. indica–inoculated plants were

harvested at 5 wpi.

Split-Root Experiment

Plants were pregerminated for 2 weeks on 0.4% water agar until the

crown roots had reached a length of;10 cm. The rootswere then divided

into two pots, one of which was inoculated with G. intraradices and the

other mock-inoculated. At 6 wpi, the inoculated and noninoculated parts

of the root system were harvested separately.

Root Staining and Mycorrhizal Quantification

Roots were stained with trypan blue and mycorrhizal colonization was

quantified with a modified grid-line intersect procedure as described

previously (Paszkowski et al., 2006). Images were prepared with a Leica

DM 5000 B microscope equipped with a Leica DFC420 camera. Briefly,

total root length colonization, determined by the total amount of fungus

and by the presence of specific structures, was scoredmicroscopically at

100 random points per root sample. For reliable representation of root

length colonization by specific mycorrhizal structures, all structures

present at one random point were recorded separately.


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LPC Infiltration of Rice Roots

Plants were germinated for 10 d on 0.4% water agar. Roots were then

infiltrated with LPC as described by Drissner et al. (2007) with minor

modifications: roots were separated from shoots and immersed in 100

mMLPC (stock, 100mM in methanol) in 10mM sodium phosphate buffer,

pH 6. The same buffer with equal amounts of methanol served as a

control. Roots were infiltrated three times for 2min followed by incubation

in nutrient solution (0.005% [w/v] Hauert-Flory Typ A and 0.01% [w/v]

Sequestren Rapid) for 3 h at 308C. Roots were then shock-frozen in liquid

nitrogen.

Identification of Homozygous Mutant Lines

DNA was extracted following Berendzen et al. (2005). Genotyping of

segregating seedling populations was performed by PCR. Wild-type and

mutated loci were distinguished by the use of specific primer pairs:

combining an insertion-specific primer with a gene-specific primer iden-

tified the mutant allele, while two gene-specific primers spanning the

insertion amplified the wild-type allele (see Supplemental Table 4 online).

The amplicon diagnostic of the mutant allele was subsequently se-

quenced following standard protocols.

RNA Extraction, cDNA Synthesis, RT-PCR, and Real-Time RT-PCR

RNA was extracted from 100 mg of ground root tissue using the

NucleoSpin Plant RNA extraction kit according to the manufacturer’s

instructions (Macherey-Nagel). cDNA synthesis and real-time RT-PCR

were performed as described earlier (Guimil et al., 2005). Primer se-

quences for real-time RT-PCRwere adopted fromGuimil et al. (2005) (see

Supplemental Table 5 online). The absence of contaminating genomic

DNA was confirmed by performing a control PCR on RNA not reverse-

transcribed (2RT). PCR amplification efficiencies were calculated with

the program LinRegPCR (Ramakers et al., 2003). Expression values were

calculated according to Guimil et al. (2005) and normalized to the

geometric mean of amplification of four nearly constitutively expressed

genes: ACTIN1 (The Institute for Genomic Research [TIGR] identifier,

LOC_Os03g50890),CYCLOPHILIN2 (TIGR identifier, LOC_Os02g02890),

GAPDH (TIGR identifier, LOC_Os08g03290), and POLYUBIQUITIN (TIGR

identifier, LOC_Os06g46770). Normalized expression values were dis-

played as a function of CYCLOPHILIN2 expression.

Fungal colonization was quantified by real-time RT-PCR according to

Isayenkov et al. (2004). Fungal DNA was extracted from the root homog-

enate used for RNA extraction employing the DNeasy Plant DNA extrac-

tion kit (Qiagen). Primer sequences to quantify G. intraradices were

retrieved from Alkan et al. (2006) (see Supplemental Table 5 online). RT-

PCR was performed according to standard PCR protocols using 35 PCR

cycles and the indicated primers (see Supplemental Table 6 online). The

rice CYCLOPHILIN2 transcript served as an internal constitutive control,

and genomic DNA served as a technical control for amplification.

Amplicons were visualized on a 1% agarose gel.

In Situ Hybridization

To generate probes for in situ hybridization, the primer pairs indicated

(see Supplemental Table 4 online) were used to amplify a stretch of;200

bp corresponding to gene-specific regions of PT11, AM1, and AM3. The

amplicons were sequenced and cloned in the sense and antisense

orientations into the pGEM-T Easy vector (Promega) with respect to the

T7 promoter. Digoxigenin-labeled RNA probes were synthesized from

linearized plasmids with T7 RNA polymerase (Promega) as described

(Langdale, 1993).

Root segments of;1 cm in length were fixed in 4% paraformaldehyde

in PBS overnight at 48C. The tissue was then dehydrated in a graded

ethanol series and subsequently in Rotihistol (Roth) in successive steps of

60-min duration. Finally, root pieces were embedded in Paraplast extra

(Kendall), sectioned to 8-mm thickness on a rotary microtome (Leitz), and

mounted on Superfrost Plus (Menzel) slides.

In situ hybridization was performed as described (Langdale, 1993) with

a modified washing buffer referred to as 0.23 SSC (13 SSC is 0.15 M

NaCl and 0.015 M sodium citrate). Alkaline phosphatase activity

was detected with Western Blue (Promega) supplemented with 1 mM

Levamisole (Sigma-Aldrich).

Accession Numbers

Sequence data from this article can be found in theOryza sativaGenome

Initiative (TIGR; http://rice.plantbiology.msu.edu/) database with the

following accession numbers: Os07g38070 (SYMRK), Os03g62650

(CASTOR), Os01g64980 (POLLUX), Os01g54240 (NUP85), Os03g12450

(NUP133), Os05g41090 (CCAMK), Os06g02520 (CYCLOPS),

Os01g46860 (PT11), Os04g04750 (AM1), Os08g34249 (AM2),

Os01g57400 (AM3), Os05g22300 (AM10), Os06g20120 (AM11),

Os11g26140 (Os AM14), Os01g57390 (Os AM15), Os03g40080 (AM18),



Os10g18510 (AM34), Os04g13090 (AM39), Os03g38600 (AM42).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Colonization of Rice Roots by P. indica.

Supplemental Figure 2. Expression Analysis of Marker Genes.

Supplemental Figure 3. Absence of PT11 Induction upon LPC

Treatment of Rice Roots.

Supplemental Figure 4. Expression of Rice Common SYM Genes.

Supplemental Figure 5. Quantification of AM Colonization by Mi-

croscopy and Real-Time RT-PCR.

Supplemental Table 1. Putative Functions of Proteins Encoded by

Genes Exclusively Expressed in Mycorrhizal Roots.

Supplemental Table 2. Insertion Lines of Rice Common SYM Genes

and Their Mycorrhizal Phenotypes.

Supplemental Table 3. Primers Used for SYM Transcript Analysis in

sym Mutants.

Supplemental Table 4. Primers Used for Genotyping of Rice Inser-

tion Lines.

Supplemental Table 5. Primers Used for Quantification of Fungal

DNA and Gene Expression by Real-Time RT-PCR.

Supplemental Table 6. Primers Used for Detection of Rice Gene

Expression by RT-PCR.

ACKNOWLEDGMENTS

We thank Patrick King and Ruairidh Sawers for editing the manuscript

and also Jerzy Paszkowski for valuable comments on the manuscript.

We are grateful to Manuel Bueno for his assistance with real-time RT-

PCR, Jaqueline Gheyselinck and Roman Zimmermann for advice on in

situ hybridization, Ivan Felipe Acosta for assistance with computational

programs, and Marcel Bucher for help with LPC infiltration. We thank

Phillip Franken for providing P. indica inoculum and Martin Parniske for

sharing unpublished information on CYCLOPS. This study was supported

by Swiss National Foundation Grant 3100AD-104132, by a National

3002 The Plant Cell

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Centres of Competence in Research grant (Plant Survival), by a Ph.D.

scholarship of the German Academic Merit Foundation to C.G., and by

Swiss National Foundation Professeur Boursier Grant PP00A–110874 to

U.P. H.I.-A. was supported by the Ministry of Agriculture, Forestry, and

Fisheries of Japan (Rice Genome Project Grant PMI-0001), and G.A.

was partly supported by the Biogreen 21 Program (Grant 20070401-

034-001) of the Rural Development Administration, Korea.

Received August 5, 2008; revised November 4, 2008; accepted Novem-

ber 11, 2008; published November 25, 2008.

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NOTE ADDED IN PROOF

During review of this article, confirmation of the functional conservation

between legume and rice of common SYM signaling components oper-

ating upstream of calcium spiking (CASTOR and POLUX) was provided

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in press.


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