Distinct roles of Lotus japonicus SYMRK and SYM15 in root colonization and arbuscule formation

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New Phytologist

(2004)

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: 381–392

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Research

Blackwell Publishing, Ltd.

Distinct roles of

Lotus japonicus SYMRK and SYM15

in

root colonization and arbuscule formation

Kirill Demchenko

1,2

, Thilo Winzer

3

, Jens Stougaard

4

, Martin Parniske

3

and Katharina Pawlowski

1

1

Albrecht von Haller Institute for Plant Sciences, Plant Biochemistry, Göttingen University, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany;

2

Komarov

Botanical Institute RAS, Prof. Popov St. 2, 197376, St.-Petersburg, Russia;

3

The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH,

UK;

4

Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark

Summary

• The colonization of

Lotus japonicus

roots by the arbuscular mycorrhizal fungus

Glomus intraradices

was analysed in plant mutants affected in the symbiosisgenes,

SYM15

or

SYMRK. SYMRK

encodes an LRR receptor-like kinase that is, likethe

SYM15

gene, essential for both mycorrhizal and rhizobial symbioses.• Different colonization patterns were observed in growing vs meristematicallyarrested roots.• Three steps in the interaction were differentially impaired in the mutants: surfaceopening, where the anticlinal cell walls of two adjacent epidermal cells separate fromeach other in the vicinity of fungal hyphae; intracellular passage of hyphae throughan exodermal cell and an adjacent cell of the outermost cortical layer; and arbusculeformation in cells of the two innermost cortical layers.• The combined results indicate that LjSYMRK is required for the intracellularpassage through exodermis and outermost cortical cell layer whereas LjSYM15 isrequired for surface opening and arbuscule formation.

Key words:

Lotus japonicus

,

Glomus intraradices

, arbuscular mycorrhiza,arbuscule, root meristem, signal transduction, saprophytic growth.

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New Phytologist

(2004)

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: 381–392

Author for correspondence:

Martin ParniskeTel. +44 1603 450249Fax: +44 1603 450111Email: [email protected]

Received:

12 January 2004

Accepted:

19 March 2004

doi: 10.1111/j.1469-8137.2004.01123.x

Introduction

Arbuscular mycorrhizae (AM) are symbioses formed betweenfungi of the order Glomales and the roots of > 80% of theextant terrestrial plant taxa (Newman & Reddell, 1987). Theassociations are considered mutually beneficial in that the plantsupplies the fungus with carbon sources, while the fungusassists the plant with the uptake of phosphate and other mineralnutrients (Smith & Smith, 1997). Plant mutants unable toform AM represent useful tools for the identification ofsteps in the development of the symbiosis, and a subset ofnodulation mutants, affected in their root nodule symbiosiswith rhizobia, are also affected in the AM symbiosis (Duc

et al

., 1989).A large number of Nod mutants from the model legume

Lotus japonicus

(Handberg & Stougaard, 1992) has beenanalysed for their AM phenotype and six common

SYM

geneshave been identified, mutation of which affects both rhizobial

and AM symbioses. The phenotype of common

SYM

mutantsindicates that the two symbiotic interactions overlap in theirearly steps (Kistner & Parniske, 2002). One of the common

SYM

genes is

SYMRK

whose product is involved in rhizobialNod factor-dependent signal transduction as well as beingrequired for signal exchange with AM fungi (Stracke

et al

., 2002)

.

However, accumulating evidence also suggests that signallingindependent of the common

SYM

genes contributes to thecommunication between the symbionts (Kosuta

et al

., 2003).Another common

SYM

gene is SYM15 (Stougaard, 2001);represented in this study by two alleles; the weak

Ljsym15-1

(Schauser

et al

., 1998) and the strong

Ljsym15-2

( J. Stougaard,unpublished data).

In mutants affected in the common

SYM

genes

SYMRK

,

SYM3

and

SYM4

, intraradical colonization of root patches bythe fungal symbiont are observed at a low frequency (Wegel

et al

., 1998). The analysis of such events in

Ljsym4

mutantsby Novero

et al

. (2002) has identified three successive steps of

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fungal colonization of the plant root. They have shown thatthe colonization of

Lotus

roots by AM fungi is controlled atthree points: (i) at the intercellular penetration betweenepidermal cells; (ii) at the intracellular passage through theroot exodermis (see also Senoo

et al

., 2000); and (iii) at theformation of arbuscules in the two innermost cortical celllayers. Novero

et al

. (2002) concluded that

LjSYM4

is necessaryfor the accommodation of AM fungi in both epidermaland cortical cells. We have used a detailed cytological analysisto study the position of two common

SYM

genes,

LjSYMRK

(Stracke

et al

., 2002) and

LjSYM15

(Schauser

et al

., 1998, J.Stougaard, unpublished data) in this conceptual frameworkand identified significant differences between the symbioticperformance of the corresponding mutants. We conclude thatthe

SYMRK

and

SYM15

genes have distinct functions duringthe establishment of the arbuscular mycorrhizal symbiosis.

Materials and Methods

Plant growth conditions

To maximize colonization pressure,

L. japonicus

seedlingswere grown in a chive nurse plant system with

G. intraradices

as microsymbiont. For this, seeds of

Allium schoenoprassum

(chive) were surface-sterilised in 1.2% NaOCl for 3–5 min,washed with sterile dd H

2

O, and placed in a pot containingAbsodan (Damolin, Fur, Denmark) in 1/2 strength Hoagland’ssolution (Hoagland & Arnon, 1938) around a chive nurseplant with roots colonized by

G. intraradices

at 22

°

C in 16 hlight, 8 h dark. After 4–6 week, mycorrhization was analysedafter ink staining of single roots, and plants with mycorrhizedroots were transferred to individual pots and grown for4–6 wk.

L. japonicus

seeds were scarified for 10 min inconcentrated H

2

SO

4

, washed extensively in sterile dd H

2

Oand germinated on 1% water agar in Petri dishes, first forone day at 24

°

C in the dark, then for two days at 16 h lowlight intensity, 8 h dark. Ca. 20 seedlings were planted arounda chive nurse plant, grown at 22

°

C and 16 h light, 8 h darkand watered once per week with 1/2 strength Hoagland’ssolution.

Cytological methods

Ink staining for visualization of intra- and extraradical fungalstructures was performed according to Vierheilig

et al

. (1998).Root systems, or parts of roots, were incubated for 7 min in10% KOH at 95

°

C in small Petri dishes on a hot plate,washed 3

×

with dd H

2

O and 2

×

in 1% acetic acid, stainedfor 3 min at 96

°

C with 5% ink (Sheaffer Jet Black) in 8%acetic acid. To remove excessive dye, roots were rinsed with1% acetic acid once for 5 and once for 20 min. To remove airbubbles, roots were infiltrated in vacuum for 15 min. Rootswere mounted on microscope slides in glycerol and observedunder a microscope.

For Trypan Blue staining, roots were either autoclaved for10–15 min or incubated at 90

°

C in 10% KOH for up to2 h. After rinsing with water, they were soaked in 1% HCl atroom temperature for 30 min. Then, HCl was replaced witha Trypan Blue solution (0.05% (w/v) trypan blue, 50% (v/v)glycerol, 0.2% (v/v) HCl) and the roots were incubated at60

°

C for at least 1 h. After rinsing with water, excessive dyewas removed by repeated incubations in acidified glycerol(50% (v/v) glycerol, 0.5% (v/v) (HCl) at room temperature.

To visualize fungal structures in root systems before embed-ding for microscopy, root systems were fixed with Navashin’sfixative (1% chromic acid/40% formalin/100% acetic acid;10 : 4 : 1 (v/v/v); Sass, 1958), or with 4% paraformaldehydeand 0.25% glutaraldehyde overnight at 4

°

C. Roots were washedwith dd H

2

O for 3–4 h. For Feulgen staining, roots wereincubated in 5 N HCl for 40 min and stained with Schiff ’sreagent for 1 h. Schiff ’s reagent was prepared by dissolving5 g of basic Fuchsin (Fluka, Buchs, Switzerland) in 900 mlboiling dd H

2

O, letting the solution cool down, and adding20 ml of conc. HCl at 50

°

C and 20 g of K

2

S

2

O

5

dissolvedin 80 ml H

2

O at 25

°

C. Afterwards, root systems were washed4

×

for 10 min in SO

3

-water (20 g K

2

S

2

O

5

and 20 ml conc.HCl in 1 l dd H

2

O) and for 2

×

20 min in dd H

2

O and observedunder a stereo microscope. Colonized root sections were cutin

c

. 0.5 cm long pieces, dehydrated in a graded ethanol seriesand embedded in Paraplast plus (Sigma, St. Louis, MO, USA).Serial sections (8 µm) were cut with a rotation microtome(RM2125RT; Leica Microsystems, Wetzlar, Germany), attachedto SuperFrost® Plus slides (Roth, Karlsruhe, Germany) anddeparaffinized in xylene. For Feulgen/Alcian Blue staining,sections on slides were rehydrated in a graded ethanol series,equilibrated in dd H

2

O, and incubated in Schiff ’s reagentfor 1–2 h. Then, sections were washed for 3

×

15 min withSO

3

-water and for 2

×

5 min in dd H

2

O before beingincubated for 1–2 h in 1% Alcian Blue 8GX (Sigma) in 3%acetic acid. Sections were washed in dd H

2

O for 3

×

5 min,dehydrated in a graded ethanol series, equilibrated in xyleneand mounted with Eukitt™ (Kindler, Freiburg, Germany).

Microscopy

Microscope BX51 and stereo microscope SZX12 (OlympusOptical GmbH, Hamburg, Germany) were used. Results weredocumented using a ColorView II digital camera set and SISanalySIS® software (Soft Imaging System GmbH, Münster,Germany).

Results

Colonization of

Lotus japonicus

roots by

Glomus intraradices

The cytology of

L. japonicus

ecotype Gifu roots infected by

G. intraradices

was examined in detail. Roots of

L. japonicus

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wild type were readily colonized by G. intraradices hyphae,leading to the formation of arbuscules after 1 wk of co-cultivation(WOC), and vesicles after 2 wk. In a set of typical experi-ments, after 3 wk, root length colonization was 32.4 ± 7.8%,and increased to 84 ± 7.5% after 6 wk, while arbuscularcolonization increased from 29.1 ± 10.7% to 77 ± 6.8%.Arbuscules were present in c. 90% of the colonized roots.

The observed structures are depicted schematically inFig. 1a. Colonization started with a fungal hypha formingan appressorium at the longitudinal border between twoepidermal cells (Fig. 1b,c). Sometimes, fungal outgrowths wereformed at the appressorium (Fig. 1b). From the appressorium,a hypha grew into an exodermal cell (Fig. 1b,c). The hyphagrew through the exodermal cell and a cell of the outermost

Fig. 1 Colonization of Lotus japonicus wild type roots by Glomus intraradices after 2 wk of co-cultivation (WOC). (a) A schematic overview of the colonization of wild type L. japonicus roots by G. intraradices. Fungal hyphae (blue) grow through epidermis and exodermis to form arbuscules in the inner cortex. Plant genes involved in different steps of root mycorrhization are also shown. Signal exchange at the root surface leads to separation of the anticlinal cell walls of two adjacent epidermal cells, requiring SYM15. A fungal hypha enters through this gap into an exodermal cell, requiring SYMRK (and SYM4, see Bonfante et al., 2000; Novero et al., 2002), passes through an adjacent cell of the outermost cortical layer and enters the apoplast again. The hypha can form side branches that enter cells of the two innermost cortical layers, to form arbuscules, a process requiring SYM15. (b, c, e) Cleared roots with fungal structures stained with ink. (b, c) The root is seen from the side; (e) is in top view. (d, f, g) 8-µm thick sections of paraffin-embedded roots stained with Feulgen/Alcian Blue. (b) A fungal appressorium (white asterisk) is visible between two epidermal cells. A hypha proceeds from the appressorium through an exodermal and subexodermal cell. The branching point within the exodermal cell is marked by an arrow. Sometimes, intercellular lobe-like fungal structures grow out from the appressorium (arrowheads). (c) A fungal appressorium (arrowhead) without lobe-like structures is shown. Again, a hypha grows from the appressorium into an exodermal cell, branches into a subexodermal cell (branching point marked by arrow) and further into the root cortex. Root hairs are labelled by black asterisks. (d) Cross section of a fully colonized root. Arrows point at intercellular hyphae, arrowheads point at intracellular hyphae. L. japonicus roots contain idioblasts whose vacuoles are filled with red material. (e) Fully colonized root. Fungal hyphae, arbuscules and vesicles can be seen in the inner cortex. (f) Longitudinal sections of a fully colonized root. Nearly all cells of the innermost cortical cell layer contain arbuscules. An intercellular vesicle can be seen between the second and third innermost cortical cell layer. (g) Detail of an arbuscule in the innermost cortical cell layer. An arrow points at the plant cell nucleus. A, arbuscule; EN, endodermis; EP, epidermis; EX, exodermis; I, idioblast; IH, intercellular hypha; V, vesicle. Bars; (b, d-f) 50 µm (g) 20 µm.

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cortical layer and re-enters the root apoplast (Fig. 1d,e). Ahyphal network was established in the apoplast of the innerroot cortex. Typically, intercellular hyphae grew longitudinallyalong the root, forming branches that entered the inner cor-tical cells leading to arbuscule formation (Fig. 1g,f ), first inthe innermost cortical cell layer (Fig. 1f ) and later in thepenultimate cortical cell layer (Fig. 1d). After arbuscules haddeveloped, vesicles are formed intercellularly between cellsof the three innermost cortical layers (Fig. 1d–f ). The intra-cellular passage occurs through living plant cells that preventcontact between fungi and plant cell cytoplasm by the synthesisof a perifungal membrane that is in continuum with the plantcell plasma membrane (Parniske, 2000).

Early colonization of LjsymRK mutant roots by Glomus intraradices

With LjsymRK mutant lines, even after 4–6 WOC, extensivegrowth of extraradical hyphae but no fungal growth beyondthe exodermis could be observed (Fig. 2a). Typically, hyphaeexhibited balloon-like cell expansions after formation ofappressoria (Fig. 2b,c) and such swollen hyphae were oftenoriented in longitudinal direction of the root, between twofiles of root epidermal cells, over a distance of several cells(Fig. 2b). Occasionally, swollen hyphae were located between

epidermal and exodermal cells (Fig. 2b,c,e,f ). In rare cases,swollen hyphae were found within epi- or exodermal cells(Fig. 2d–f ). Numbers of appressoria were c. 4–10 timeshigher in all LjsymRK mutant lines compared to the wild type(Fig. 3a), but 90–98% of colonization attempts were abortedwithin the epidermis/outer cortex. The number of colonizationattempts that successfully grew longitudinally 1–3 cells pastthe entry point (Fig. 2e–f ) increased substantially betweenthe third and the sixth WOC but these remained confined tothe epidermis/outer cortex and the formation of arbuscules orvesicles was never observed. We could not establish qualitativeor quantitative differences in colonization structures betweenthe tested LjsymRK alleles. This is consistent with all of thembeing probably loss-of-function alleles (Stracke et al., 2002).

LjSYM15 is required for the epidermal opening response

Ljsym15-1 is a weak allele that leads to delayed nodulation(Schauser et al., 1998), whereas plants carrying Ljsym15-2 donot nodulate (data not shown). This difference in allelestrength is also reflected by their mycorrhiza phenotype.On plants carrying the strong allele Ljsym15-2, no successfulcolonizations were observed after up to 8 WOC. Extensivehyphal growth was observed around the roots, typically on the

Fig. 2 Colonization of LjSYMRK mutant roots by Glomus intraradices after 4–6 wk of co-cultivation (WOC) with the chive nurse plant. (a–c, e–g) cleared roots with fungal structures stained with ink. (d) An 8-µm thick cross section of a root embedded in paraffin, stained with Feulgen/Alcian Blue. (a, b, e–g) are in top view, in (c) the root is seen from the side. (a) After 4 WOC, extensive hyphal growth was observed around the roots. (b–g) Structures found after 6 WOC. (b) Hyphae on the root surface, precisely at the border between two files of epidermal cells (arrow). Fungal hypha entered the roots between two epidermal cells (asterisks in b, c) and form swellings between epidermal and exodermal cells (arrowhead in (c)) or within epidermal cells (arrowheads in (b)). (d) An arrow points at a fungal hypha that has entered an exodermal cell but does not proceed further. (e, f) Two different focal levels of a fungal appressorium (arrowhead) formed between two epidermal cells (arrows) that has grown an extension between epidermal and exodermal cells which has expanded into an adjacent epidermal cell (asterisk). (g) A fungal hypha has entered between two epidermal cells (arrows), grown between epidermis and endodermis and entered and filled an epidermal cell forming an intracellular vesicle. EP, epidermis; EX, exodermis; IV, intracellular vesicle. Bars; (a) 100 µm (b–d) 50 µm (e–g) 12.5 µm.

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root surface in a longitudinal direction, precisely at the borderbetween two files of epidermal cells (Fig. 4a). By contrast toLjsymRK mutants, longitudinal growth between epidermalfiles or slight swelling of hyphae was only exceptionally observed(Fig. 4a). This is an important difference, which suggestsSYM15 is required for the entry of fungal hyphae betweentwo epidermal cells, whereas SYMRK is not.

LjSYM15 is required for arbuscule formation

On roots of plants carrying the weaker allele Ljsym15-1,occasional colonization events were observed after 4–6 WOC(Fig. 4e–f ). In these cases, a fungal hypha entered the rootsbetween epidermal cells, penetrated an exodermal cell andpassed into the inner cortex (Fig. 4b–c) where it grew andbranched. Often, a slight swelling of the appressorium wasobserved, but never balloon-like structures like on LjsymRKroots (Fig. 2g). In the inner cortex, arbuscules and intercellularvesicles were formed (Fig. 4d–f ). However, in contrast withwild type roots, in which arbuscule formation preceded vesicleformation, vesicles were observed before or simultaneouslywith arbuscule formation in Ljsym15-1 roots (Fig. 4e) andextensive intraradical, extracellular hyphal growth beforearbuscule formation was always observed (Fig. 4b), indicatingan impairment in arbuscule formation in this mutant. In crosssections, most cells of the inner cortical layer of wild type rootscontained arbuscules (Fig. 1d), while in Ljsym15-1 sections,typically no or only one to three arbuscule-containing cellscould be seen (Fig. 4d). In longitudinal sections of wild typeroots, arbuscules were found in adjacent cells, forminglong files (Fig. 1e,f ). In Ljsym15-1 roots, only short files ofarbuscule-containing cells could be observed (Fig. 4f ). Thisevidence points to a role of LjSYM15 in arbuscule formation,which was further supported by observations on late colonizationevents on plants carrying the stronger Ljsym15-2 allele.

Late colonization of mutant roots

Despite their complete resistance to intraradical colonizationfor up to six (LjSYMRK ) or up to 8 WOC (Ljsym15-2 ), rootsystems of LjsymRK and Ljsym15-2 mutants contained a few

Fig. 3 Quantification of Lotus japonicus root colonization by Glomus intraradices. After 3 months of co-cultivation mycorrhizal colonization was assessed by Trypan Blue-staining in two independent experiments using the line-intersect-method of McGonigle et al. (1990). (a) Numbers of appressoria per cm or root after 3 wk of co-cultivation (WOC; 3w) and 6 WOC (6w) are given. Successful colonization attempts that led to intraradical colonization are given in black. After 3 wk, root length colonization of the wild type was 32.4 ± 7.8%, arbuscular colonization was 29.1 ± 10.7%. After 6 wk, root length colonization of the wild type was 84 ± 7.5%, arbuscular colonization was 77 ± 6.8%. Whole roots of 5–8 plants per line were analysed. (b) Extraradical colonization of roots from the wild type and three LjSymRK mutant alleles after 3 months of co-cultivation with G. intraradices. Y-axis values represent percent of root length colonization. Results of two independent experiments are given (one in white, the other in black). Whole root systems of 5–9 plants per line and experiment were analysed. (c) Intraradical colonization of roots from the wild type and three LjSYMRK mutant alleles after 3 months of co-cultivation. Total colonization, hyphal colonization, arbuscular and vesicular colonization, respectively, are given in percentage of root length colonization. Results of two independent experiments are presented. Whole roots of 5–9 plants per line and experiment were analysed.

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lateral roots that showed high densities of AM colonizationafter a minimum of 8 WOC for LjsymRK (Fig. 5) and 10 WOCfor Ljsym15-2 (Fig. 6). Only on LjsymRK roots and at a lowfrequency were hyphae, vesicles and arbuscules observed inthe inner cortex (Fig. 5f ). Arbuscular colonization occurredin all LjsymRK mutants, but was generally very low (themaximum was 12.8% root length colonization for LjsymRK-3).Arbuscular colonization occurred in distinct patches of limited

size, suggesting each of these patches resulted from a singlecolonization event. To obtain a measure for the colonizationpressure on these roots, we quantified the extraradical rootlength colonization and found it to range around 45% on thewild type and around 80–85% on LjsymRK mutants (Fig. 3b).However, this was accompanied by an intraradical colonizationof up to 97% of wild type root length, while only a fractionof the root length was internally colonized in LjsymRK mutant

Fig. 4 Colonization of LjSYM15 mutant roots by Glomus intraradices after 4–8 wk of co-cultivation (WOC). (a, b, e, f) Cleared roots with fungal structures stained with ink. (c, d) 8 µm thick Feulgen/Alcian Blue-stained cross sections of a root embedded in paraffin. (a) Top view (b–f) the roots are seen from the side. (a) For Ljsym15-2, after 8 WOC, extensive hyphal growth was observed around the roots; typically fungal hyphae were growing on the root surface in longitudinal direction, precisely at the border between two files of epidermal cells. (b) For Ljsym15-1, occasional colonization was observed after 4–6 WOC (arrow points at appressorium). (c) In these cases, a fungal hypha entered the roots between two epidermal cells (arrow points at appressorium) and passed through an exodermal cell into the inner cortex where it grew and branched (see b). An arrowhead points to an intercellular hypha. In the inner cortex of Ljsym15-1 roots, G. intraradices could form arbuscules (d–f) and vesicles (d, e), but the frequency of arbuscules (d, f) was much lower than in wild type roots (compare Fig. 1). An arrow in (e) points at a very young arbuscule at a colonization patch where one mature and two younger vesicles can be seen. A, arbuscule; EP, epidermis; EX, exodermis; I, idioblast; V, vesicle. Bars; (a) 100 µm (b, e, f) 50 µm (c, d) 25 µm.

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lines (Fig. 3c). By far the greatest proportion of overall rootlength colonization in the mutants was colonization by hyphaeand vesicles, not arbuscules. Hence, an effect of SYMRK onarbuscule initiation cannot be excluded.

Using light microscopy on paraffin-embedded plantmaterial stained with Feulgen/Alcian Blue, no difference could

be seen between arbuscules formed in growing wild type rootsand roots of LjsymRK mutant lines (Fig. 1d,f,g vs Fig. 5f,k).Like in the wild type, vesicles were formed near arbuscules inroots of LjsymRK mutant lines (Fig. 5f ). On roots of Ljsym15-2plants, however, no arbuscules were ever found. We concludethat SYMRK is not required for the formation of structurally

Fig. 5 Colonization of meristematically arrested roots of LjsymRK-2 mutants. (a–c) A root system stained with Feulgen (d) shows a cleared root stained with ink in top view (e–k) show 8 µm thick root sections stained with Feulgen/Alcian Blue. (a–c) Root system after 13 wk of co-cultivation (WOC) with the chive nurse plant. Colonization by Glomus intraradices as indicated by spore/vesicle formation (see (c) for details) can be seen in roots without active meristems (b) that show a yellowish colour after staining with Feulgen. (d) Hyphae and intracellular vesicles can be seen in a cleared root stained with ink. Spores have been formed by hyphae at the root surface. (e) In the outer cortex of some arrested roots, hyphae grew longitudinally down to the root tip (arrow) and formed vesicles. (f) When hyphae reached the inner cortex, arbuscules that could not be distinguished cytologically from those in wild type roots (arrows; for details see a longitudinal section in (k)) and vesicles were formed. (g) Fungal structures were found even in the vascular tissue, here a vesicle between endodermis and pericycle. (h) A fungal spore was formed between epidermal cells (arrow). (i) To enter the root, infecting hyphae entered an exodermal cell (arrow) to pass to the inner cortex. (j) In a longitudinal root section, an intracellular vesicle is shown inside an exodermal cell. (k) Arbuscules formed in an arrested LjsymRK-2 root shown in a longitudinal section. EP, epidermis; I, idioblast; S, spore; V, vesicle; IV, intracellular vesicle. Bars; (a) 2 mm (b) 500 µm (c) 300 µm (d, e) 50 µm (f-k) 25 µm.

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intact arbuscules, while SYM15 is required for the initiationand progression of arbuscule development as indicated bythe complete loss of arbuscule formation in the strong alleleand the delayed development of arbuscules in the weakallele.

Late cortical colonization of plant mutants is limited to meristematically arrested or damaged roots

After 10–13 WOC, one or more lateral roots of LjsymRK andLjsym15-2 plants were densely colonized by G. intraradices.

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By using the Feulgen stain applied to entire root systems, wediscovered that late successful colonization patches wereconfined to meristematically arrested roots as evidenced byfull cell differentiation down to the root cap, or to damaged rootsthat had their tips torn off. Meristem arrest was confirmed byFeulgen/Alcian Blue staining of longitudinal sections (Figs 5eand 6d). Most lateral roots with arrested meristems, andall roots whose younger parts had been torn off, showed ayellowish colour after staining with Feulgen (Figs 5a–c and6a–c). We suspect that the yellow Feulgen stain is a functionof root ageing which occurs after meristem arrest. Aging wouldalso provide an explanation for the brittle nature of these rootsthat leads to frequent loss of the tip during uprooting. Onlyyellowish lateral roots, but not all of them, were colonized byG. intraradices. These observations suggest that meristem arrestand the physiological alteration of the root that leads to yellowFeulgen stain preceded late successful fungal colonizationevents. Many meristematically arrested LjsymRK and Ljsym15roots showed extensive hyphal and vesicular colonization(Figs 5e–f and 6b–e), also in the outer cortex (Figs 5e,g and6f; data not shown for Ljsym15-1) proceeding as far as into theroot tip (Figs 5e and 6d) and in LjsymRK roots occasionallyentering the vascular tissue (Fig. 5e,g).

Vesicles were often formed ectopically during late colonizationof mutant roots, e.g. in the outer cortex of LjsymRK and Ljsym15roots (Figs 5e,g and 6f; data not shown for Ljsym15-1) and pro-ceeding as far as into the root tip (Figs 5e and 6d). In LjsymRKroots fungi occasionally entered the vascular tissue (Fig, 5e,g).By contrast, in wild type roots, vesicles were only found betweenthe cells of the three innermost cortical cell layers (Fig. 1e,f ).

A mutant-specific feature was the occurrence of intracellularhyphal swellings and vesicles; arrested LjsymRK-2, -7 andLjsym15-2 roots harboured vesicles within epidermal or exo-dermal cells. We suspect that they developed in already deadplant cell wall ‘ghosts’. In sections, intracellular vesicles dis-played a similar cytoplasmic structure but seemed to possessthinner cell walls than extracellular vesicles (Figs 5d,j and6g–i). By contrast, in wild type roots, vesicles were alwaysintercellular and only found between the cells of the threeinnermost cortical cell layers (Fig. 1e–f ). Intracellular swellingsof hyphae were found exclusively in mutant roots, while extra-cellular swellings occurred at increased frequency comparedto the wild type (Fig. 2b,e–f ). Swollen hyphae were notnecessarily terminal organs, and if intracellular, never filled

the entire cell. In sections, their cytoplasmic structure resembledthat of hyphae, not of vesicles (data not shown).

Fungal entry into meristematically arrested lateral roots

Fungal hyphae entered LjsymRK roots between epidermalcells and reached the inner cortex of LjsymRK roots via passagethrough an exodermal cell (Fig. 5i). While the entry throughan exodermal cell superficially resembled the process of normalwild type colonization (Fig. 1a–c), fungal hyphae entered theapoplast again below the exodermis instead of proceedingintracellularly through the next cell layer. Furthermore, theexodermal cells used for passage always showed very strongstaining with Alcian Blue and collapsed cytoplasm, suggestingcell death (Fig. 5i), but it is unclear whether this was cause orconsequence of fungal penetration.

In Ljsym15-2 roots, entry of fungal hyphae was different fromall other interactions analysed. Entry of fungal hyphae wasfound mostly in parts of the root system where the cortex wasexposed (Fig. 6e). It is not clear whether this damage of epi- andexodermis occurred during root harvesting. However, the strik-ing correlation between entry point and absence of outer celllayers suggests that this damage occurred during co-cultivation.Very few cases were found where entry occurred intercellularlythrough what based on suberization of walls of adjacent cells,appeared to be dead regions of the outer cell layers (Fig. 6f; dataon suberization not shown). Hyphae proceeded from suchdamaged parts of the root, and grew longitudinally betweencell files into nondamaged parts of the root (Fig. 6b,d–f).

The effect of SYM15 on the intracellular passage throughthe epi-/exodermis could not be determined with certainty,since in plants carrying the strong allele, Ljsym15-2,fungal hyphae never entered an exodermal cell. The unper-turbed intracellular passage in roots carrying the weak allele,Ljsym15-1, suggests that SYM15 plays little or no role in thisstep.

Discussion

Different functions of SYMRK and SYM15 in root symbiosis

The common SYM genes are required for both nodulationand arbuscular mycorrhiza, and probably act in a pathway

Fig. 6 Colonization of meristematically arrested roots or dead roots of Lotus japonicus sym15–2 mutants. (a–c) Root systems stained with Feulgen (d, e, g, h) show cleared root systems stained with ink (f, i) show 8 µm thick root sections stained with Feulgen/Alcian Blue. (d, e, g, h) are in top view. (a–c) Ljsym15-2 root system after 13 wk of co-cultivation (WOC) with the chive nurse plant. Colonization by Glomus intraradices as indicated by vesicle/spore formation (b, c; arrows) can be seen. These roots showed a yellowish colour after staining with Feulgen. (d) In some infected roots, hyphae and spores (arrow) were found in the outer cortex down to the root tip. The root shows full differentiation down to the root cap. (e) Sometimes, colonization sites were found at points where root epidermis and outer cortex were destroyed. (f) Fungal hyphae have entered the root and reached the inner cortex (arrows). A vesicle has been formed in the outer cortex. (g) An infected root section shows extensive hyphal growth, as well as intracellular vesicles. (h, i) Intracellular vesicles were formed inside cells of the epidermis. A, arbuscule; EP, epidermis; EX, exodermis; H, hypha; S, spore; V, vesicle; IV, intracellular vesicle. Bars; (a) 1 mm (b, c) 250 µm (d, e, g) 100 µm (f) 50 µm (h, i) 25 µm.

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leading from the perception of microbial signalling moleculesto the activation of symbiotic plant responses, includingsymbiosis-related gene activation and the induction of anintracellular accommodation programme ( Kistner & Parniske,2002). However, up to now, positioning of these genes relativeto each other was hampered by the lack of differentiatingphenotypic symptoms. In the interaction with rhizobia, theonly phenotypic marker that discriminates between theirfunction, is the calcium spiking response of root hair cellsto the application of Nod factors (Oldroyd et al., 2001a,b).Here we show that the steps involved in the development ofthe arbuscular mycorrhiza symbiosis are differentially impairedin mutants affected in the SYMRK or SYM15 genes.

Three steps for root colonization by AM fungi

Arbuscular mycorrhiza development involves cell type-specificresponses to fungal invasion. Because fungal colonizationcan at low but detectable frequencies bypass symbiotic blocksin outer cell layers, the effects of a symbiotic mutation on innercell layers can be studied. This potential to break through outerblocks makes fungal colonization a sensitive assay system tostudy the role of common SYM genes in cell types other thanthe epidermis. A synthesis between the results of Novero et al.(2002) and those presented here points to three successiveresponses of different cell layers that are required for fungalcolonization (Fig. 1a). First, the epidermis response allowsfungal penetration between two epidermal cells. In the vicinityof the fungus, the anticlinal cell walls of two adjacent epidermalcells separate from each other and a fungal hypha entersthrough this gap. The second and the third step both requirethe activation of an intracellular accommodation programmein different cell types and with different outcomes. The secondresponse is by epidermal (Wegel et al., 1998; Novero et al.,2002) or exodermal (this study) cells, and allows intracellularpassage through these layers. The third step, arbuscule formation,requires a more elaborate response of the host inner corticalcells to host a more complex fungal structure.

Infection of meristematically arrested roots

Ageing led to changes of LjsymRK and Ljsym15-2 roots thatmade them accessible for fungal hyphae as fungal colonizationonly occurred in meristematically arrested roots that stainedyellow with Feulgen. The responsible physiological changes seemto affect epidermal and exodermal cells. However, meristematicarrest did not alleviate the requirement of SYM15 for arbusculeformation by cortical cells. Older roots of nonmycorrhizalplants can be colonized by arbuscular-mycorrhizal fungiwith intercellular hyphae and inter or intracellular vesicles(reviewed by Brundrett, 1991; Giovannetti & Sbrana, 1998).These interactions lack appressoria and arbuscules and areconsidered as saprophytic growth of the fungus on the plantroots (Brundrett, 1991). The question arises whether the

infection of meristematically arrested roots represents a bonafide symbiotic interaction, or should be regarded as saprophyticfungal growth. On LjsymRK roots, arbuscules could be formed,indicating the late formation of a structurally intact symbiosis.For Ljsym15-2 roots where no arbuscules were formed, it wasnot possible to distinguish between late symbiosis and saprophyticgrowth.

The role of common SYM genes at different steps of AM development

The effect on LjSYMRK and LjSYM15 on the three steps inroot colonization by G. intraradices as found in this study issummarized in Fig. 1a.

Surface opening In the vicinity of the fungus, the anticlinalcell walls of two adjacent epidermal cells separate from eachother. This response is impaired in Ljsym15-2 mutants. Aconsequence of this is that the fungus never proceeds beyondthe cleft between epidermal cells, in contrast to symRK mutantswhere hyphae grow further and form balloon-like structureswhich is never observed in Ljsym15-2 mutants. We concludethat it is an active host response that requires the activity ofthe SYM15 but not of the SYMRK gene. The cytologicalanalysis of the infection process of Ljsym4 mutants by Noveroet al. (2002) showed that surface opening also occurs there,hence like SYMRK, SYM4 is not required for this process.

Intracellular passage through outer cell layers The next stepis the intracellular passage of fungal hypha through plant cellsinto the inner cortex. Here, two different ports of intracellularentry appear to be possible. Novero et al. (2002) and Wegelet al. (1998) presented evidence for fungal entry into epidermalcells, and subsequent progression of the hypha into theadjacent exodermal cell. In this study, we regularly observedfungal passage between two epidermal cells and entry into anexodermal cell. After intracellular passage through exodermisand the outermost cortical cell layer, fungal hyphae grewmostly intercellularly to the two inner cortical cell layerswhere arbuscule formation can take place.

Our observations of wild type roots suggest that a minimumof two cell layers have to be crossed intracellularly before releaseof hyphae and intercellular colonization of the cortex (see e.g.Fig. 1d). Both epidermis and exodermis seem to have thepotential to initialise the intracellular phase of hyphal growth,and both exodermis and the outermost cortical cell layer areable to support their further intracellular passage. Intracellularfungal accommodation by the outer cell layers was affectedin roots of LjsymRK (this study) and Ljsym4 plants (Bonfanteet al., 2000; Novero et al., 2002).

Hyphae were observed within epidermal and exodermalcells during failed colonization attempts of LjsymRK mutantroots, very similar to the situation observed in Ljsym4 rootsby Bonfante et al. (2000). For the passage through plant cell

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walls fungal turgor pressure has been shown to be sufficient(Talbot, 2003). The swelling of fungal hyphae frequently observedin such cases of forced entry may be a result of changes infungal cell wall composition that are induced in course of theinteraction. It has been shown for Ljsym4-1, the cytoplasm ofboth interacting cells collapses, if the plant does not activelysupport the internalization (Bonfante et al., 2000).

Arbuscule formation During arbuscule formation, a sidebranch of an intercellular hypha enters a cell of one of thetwo innermost cortical layers and branches profusely. Inthe wild type, hyphae grow longitudinally along the rootand arbuscules form in every cell that is passed. This processwas disturbed in Ljsym15 mutants. Ljsym15-1 plants showeda delay and reduction in arbuscule formation (Fig. 4e,f ), andin Ljsym15-2 roots arbuscule formation was never observed.Novero et al. (2002) reported a change in the ratio of hyphal toarbuscular colonization of Ljsym4-1 roots and concludedthat SYM4 has a role in arbuscule initiation. SYM4 is thereforedistinct from both SYMRK and SYM15: like SYMRK it is notrequired for epidermal opening, but like SYM15 it has a rolein arbuscule formation.

Signalling during mycorrhizal colonization of plant roots

LjSYMRK, encoding an LRR receptor-like kinase (Strackeet al., 2002), seemed to affect only the intracellular passagethrough exodermis and outermost cortical cell layer. The resultspresented by Wegel et al. (1998), Bonfante et al. (2000) andNovero et al. (2002) indicate that LjSYM4 is probably essentialfor the same step. LjSYM15 is required for full surface openingand for arbuscule formation, both processes independent ofLjSYMRK. In congruence with these results, Ljsym4 (Harriset al., 2003) and by deduction from results obtained in Medicagotruncatula and pea (Endre et al., 2002), LjsymRK mutants areaffected in the calcium spiking response to rhizobial Nodfactors. LjSYMRK and LjSYM4 have therefore been placedat a similar hierarchical level in a genetic pathway ( Kistner &Parniske, 2002). LjSYM15, on the other hand, may be part ofa signal transduction pathway independent of LjSYMRK andLjSYM4.

The most parsimonious interpretations of the geneticdata are linear signalling pathways in plant-microbe symbioses(Stougaard, 2001). However, refined analysis of common SYMmutants showed that signal transduction networks are likelyto be involved (Kosuta et al., 2003). Our results clearly showthat it is crucial to consider the structural context whendrawing such models. Different plant cell types are involvedin the AM interaction, and these produce cell type-specificresponses. Our results suggest the existence of a signallingnetwork, of which individual components are required foran overlapping but noncongruent set of cell-type specificresponses. The detailed cytological analysis of the colonization

process in plant mutants carrying weak alleles, as well as theanalysis of late colonization of arrested roots, will continue toprove valuable in revealing the function of genes in symbioticsignalling in different cell types.

Acknowledgements

We would like to thank Anke Sirrenberg and Catherine Kistnerfor helpful discussions and Uwe Wedemeyer for taking careof the plants in Göttingen. This work was supported bythe German Research Council (Focus Program Mycorrhiza,SP1084). KD acknowledges additional support by RussianScience Support Foundation, the Russian Foundation forBasic Research and the Focus Program of the Russian Academyof Sciences. Research at The Sainsbury Laboratory is fundedby the Gatsby Charitable Foundation.

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