symbiotic fungi control plant root cortex development ... heck et al. cb and suppl..pdfcurrent...

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Symbiotic Fungi Control P
lant Root CortexDevelopment through the Novel GRAS TranscriptionFactor MIG1
Graphical Abstract

Highlights

d Symbiotic AM fungi induce MIG1 in cortical cells with

arbuscules

d MIG1 belongs to a novel clade of GRAS transcription factors

absent in Arabidopsis

d MIG1 intersects with root GA signaling by interacting with

DELLA1 in the cortex

d MIG1 and DELLA1 control cortical radial cell expansion

during arbuscule development

Heck et al., 2016, Current Biology 26, 1–9October 24, 2016 ª 2016 Elsevier Ltd.http://dx.doi.org/10.1016/j.cub.2016.07.059

Authors

Carolin Heck, Hannah Kuhn,

Sven Heidt, Stefanie Walter,

Nina Rieger, Natalia Requena

[email protected]

In Brief

Heck et al. show that arbuscular

mycorrhizal fungi induce in plants the

expression of a novel GRAS transcription

factor, MIG1, that controls root cortical

cell expansion by intersecting with GA

signaling. MIG1 downregulation impairs

symbiosis, demonstrating that microbes

are able to fine-tune plant development

for their own ends.

mailto:[email protected]

http://dx.doi.org/10.1016/j.cub.2016.07.059

Please cite this article in press as: Heck et al., Symbiotic Fungi Control Plant Root Cortex Development through the Novel GRAS Transcription FactorMIG1, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.07.059

Current Biology

Report

Symbiotic Fungi Control Plant RootCortex Development through the NovelGRAS Transcription Factor MIG1Carolin Heck,1 Hannah Kuhn,1 Sven Heidt,1 Stefanie Walter,1 Nina Rieger,1 and Natalia Requena1,2,*1Molecular Phytopathology, Botanical Institute, Karlsruhe Institute of Technology (KIT), Fritz Haber-Weg 4, 76131 Karlsruhe, Germany2Lead Contact

*Correspondence: [email protected]://dx.doi.org/10.1016/j.cub.2016.07.059

SUMMARY

In an approaching scenario of soil nutrient depletion,root association with soil microorganisms can bekey for plant health and sustainability [1–3]. Symbioticarbuscularmycorrhizal (AM) fungi aremajor players inhelping plants growing under nutrient starvation con-ditions. They provide plants with minerals like phos-phate and, furthermore, act as modulators of plantgrowth altering the root developmental program [4,5]. However, the precise mechanisms involved inthis latter process are not well understood. Here, weshow that AM fungi are able to modulate root cortexdevelopment in Medicago truncatula by activatinga novel GRAS-domain transcription factor, MIG1,that determines the size of cortical root cells. MIG1expression peaks in arbuscule-containing cells, sug-gesting a role in cell remodeling during fungal ac-commodation. Roots ectopically expressing MIG1become thicker due to an increase in the numberand width of cortical cells. This phenotype is fullycounteracted by gibberellin (GA) and phenocopiedwith a GA biosynthesis inhibitor or by expressionof a dominant DELLA (D18DELLA1) protein. MIG1downregulation leads to malformed arbuscules, aphenotype rescued by D18DELLA1, suggesting thatMIG1 intersects with the GA signaling to control cellmorphogenesis throughDELLA1.DELLA1wasshownto be a central node controlling arbuscule branching[6–8]. Now we provide evidence that, together withMIG1, DELLA1 is responsible for radial cortical cellexpansion during arbuscule development. Our datapoint towardDELLAproteins being not only longitudi-nal root growth repressors [9] but alsopositive regula-tors of cortical radial cell expansion, extending theknowledge of how DELLAs control root growth.

RESULTS AND DISCUSSION

MIG1, a New GRAS Transcription Factor Induced by AMFungal SignalsRoot colonization by arbuscular mycorrhizal (AM) fungi is limited

to the epidermis and the cortex, where the fungus grows by inter-

and intracellular hyphaeand formsprofusely branchedstructures

called arbuscules in deep layers of the cortex [10]. Arbuscules are

surrounded by a de novo-synthesized plant cell membrane, the

periarbuscular membrane (PAM) that requires a reorganization

of the plant exocytotic machinery [11–15]. It is, therefore,

comprehensible that formation of the mycorrhizal symbiosis re-

quires an extraordinary and regulated developmental adjust-

ment. Plant symbiotic reprogrammingstarts evenbeforephysical

contact, and perception of diffusible fungal signals leads to acti-

vation of the first transcriptional responses [16, 17]. However, the

precise mechanisms linking perception of fungal signals to the

plant developmental changes required for fungal accommoda-

tion in the root cortex are not yet fully elucidated. In recent years,

several GRAS transcription factors (TFs) have been identified as

key components at the core of this process. Thus, theDELLAmu-

tants (della1/della2 in M. truncatula or slr1 in rice) are severely

impaired in arbuscule formation [7, 18], RAM1 is required for ar-

buscule branching [6, 8, 19, 20], and RAD1 is essential for arbus-

cule maintenance [21]. In addition, NSP1, NSP2, and DIP1 were

shown to control the general colonization process [18, 22–24].

For identification of novel GRAS TFs from M. truncatula

featuring in the translation of AM fungal signals into root devel-

opmental changes, reiterative pBLAST analyses using RAM1,

NSP1, and NSP2 were carried out, and 99 putative GRAS TFs

were identified (Table S1). Based on an in silico expression anal-

ysis (http://mtgea.noble.org/v3/) and qRT-PCR data in response

toRhizophagus irregularis spore extract (SE) (Figure S1A), a novel

GRAS TF Medtr2g034280 was selected for further analysis. A

closer look into the genome showed two close homologs located

in tandem (Figures S1B and S1C). Because they were all mycor-

rhiza induced, we named them as MYCORRHIZA-INDUCED

GRAS (MIGs). All MIGs were inducible by R. irregularis extract,

albeit at different time points and with different magnitudes (Fig-

ure 1A). Fractionation of the extract showed the pellet to have the

strongest inducing activity (Figure S1D), suggesting that fungal

cell wall components might be the responsible signals.

Although the expression of all MIGs increased concomitantly

with the colonization of the root (RiTEF) and with the expression

of the arbuscular marker PT4 (Figures 1B and 1C), only MIG1

shows a mycorrhiza-specific expression pattern in the gene

expression ATLAS, while MIG2 and MIG3 are expressed more

ubiquitously. Therefore, we next investigated their promoters

for arbuscule-specific motifs. The analysis showed the presence

of several CTTC-like motifs (each differing from the core motif

TCTTGTTC by a single nucleotide), but only the promoter of

MIG1 also contained the P1BS motif, both in close vicinity to

Current Biology 26, 1–9, October 24, 2016 ª 2016 Elsevier Ltd. 1

mailto:[email protected]


http://mtgea.noble.org/v3/

Figure 1. Expression and Promoter Activity

of MIG1 Are Associated with Mycorrhiza

Establishment

(A–C) Expression levels of genes were analyzed by

qPCR and normalized to the housekeeping gene

MtTEF1a (n = 3). Data represent mean ± SD. Stu-

dent’s t test was used to calculate significance

to mock or non-mycorrhized (nm) control. *p <

0.05; **p < 0.01; ***p < 0.001. rel., relative; dpi, days

post-infection.

(A) Expression of MIG1, MIG2, and MIG3 in

response to SE treatment. Roots of four 10-day-

old wild-type M. truncatula plants were incubated

in SE or mock solution for 15 min, 30 min, 6 hr, or

24 hr. (B and C) Expression levels in response to

R. irregularis inoculation. M. truncatula wild-type

plants were inoculated for 1, 12, or 25 days with

(myc, mycorrhized) or without R. irregularis.

(B) Expression of the arbuscule marker PT4 and

fungal housekeeping gene RiTEF serve as markers

for progression of mycorrhizal colonization.

(C) Expressions of the MIG genes.

(D) In silico analysis of the 2 kb region upstream of

the ATG of MIG1, MIG2, and MIG3. The presence

of the mycorrhiza-inducible cis-regulatory element

CTTC with a single base-pair exchange compared

to the core motif and the phosphate starvation

motif P1BS are shown. The table shows the exact

sequences and location of the identified motifs.

Motifs located on the + strand are depicted in blue,

and motifs on the – strand are depicted in black.

(E–G) Promoter reporter analyses of MIG1 in

M. truncatula composite plants. The 2 kb upstream

region of MIG1 (PMIG12kb) or a truncated version

with 230 bp upstream of the ATG (PMIG1230bp)

were fused to a GUS reporter construct. Trans-

formed plants were harvested after 5 weeks

of growth without (nm, non-mycorrhized) or with

R. irregularis (myc, mycorrhized). Roots were

stained for GUS activity and counterstained with

WGA-FITC (wheat germ agglutinin-fluorescein

isothiocyanate conjugate) to visualize fungal

structures. Scale bars represent 100 mm; lower

panels show single cells containing arbuscules.

(E) Control staining of PMIG12kb roots without

fungal inoculation shows GUS activity restricted to

the central cylinder.

(F) Mycorrhization of PMIG12kb composite plants

resulted in intensive GUS activity in arbusculated

cells.

(G) Mycorrhization of composite plants harboring a

truncation of the promoter sequence with only the

two first CTTC-like motifs (PMIG1230bp) is sufficient

for expression in arbusculated cells.

See also Figure S1 and Tables S1 and S2.


the ATG (Figure 1D). The CTTC motif is sufficient for expression

in cells containing arbuscules, and it is often associated with the

phosphate starvation motif P1BS close to the ATG [25, 26].

Therefore, we next analyzed the spatial expression pattern of

MIG1 in roots using the GUS reporter gene. In contrast to non-

mycorrhizal roots where MIG1 promoter is active in the vascular

cylinder, colonization by R. irregularis redirected MIG1 expres-

sion to cortical cells containing arbuscules (Figures 1E and 1F).

Truncation analyses further revealed that the 230-bp region up-

stream of the ATG containing only the first two CTTC motifs, but

2 Current Biology 26, 1–9, October 24, 2016

no longer the P1BS motif, is sufficient to drive MIG1 expression

in cells containing arbuscules (Figure 1G). These results show

that, despite MIG1 having a lower level of expression than

MIG2 and MIG3, its expression is mainly confined to arbuscu-

lated cells, suggesting a role for MIG1 in those cells.

A Novel Clade of GRAS TFs Not Conserved inArabidopsis

Phylogenetic analyses showed thatMIGs belong to a novel clade

of GRAS TFs absent in Arabidopsis (Figure 2A). Furthermore, no

(legend on next page)

Current Biology 26, 1–9, October 24, 2016 3



members of the clade exist in the monocots, except in the palm

Phoenyx. Members of this clade from Populus trichocarpa were

thought to be genus specific [27]. However, the analyses from

Huang et al. [28] and ours here clearly show that this cluster is

widely distributed in the dicots (Figure S1E). No gene from this

clade has been functionally characterized, possibly because

previous studies did not consider symbiotic conditions, and

expression was often not detected [28]. In contrast, all

M. truncatula and L. japonicus genes from this clade, including

the MIGs reported here, are mycorrhiza induced [21, 29], point-

ing toward a possible symbiotic role for the MIG1 clade.

MIG1 clade members share a conserved amino terminus

comprising three highly conserved regions located directly in

front of the GRAS domain (Figure 2B). In silico secondary struc-

ture prediction of 18 MIG1 clade members (MIG1-3 and 15

randomly selected proteins; Figure S1E) revealed that those

conserved regions correspond to putative alpha helices, one of

them partially overlappingwith predicted DNA-binding sites (Fig-

ure 2B), indicating that MIG1 could directly interact with DNA.

Given that several GRAS TFs are known to have transactivation

activity in their N terminus [30], including the symbiotic proteins

NSP1 and NSP2 [31], as well as the rice SLR1 [32], we tested the

transactivation activity of MIG1 in yeast. Results showed that

MIG1 is able to activate yeast growth when fused to the GAL4

DNA-binding domain, similar to DELLA2 from M. truncatula

and RGA1 from A. thaliana (Figure 2C). Furthermore, we showed

that the amino terminus is sufficient for MIG1 transactivation ac-

tivity. Thus, MIG1 is the first characterized member of a novel

clade of GRAS TFs, highly expressed in arbuscule-containing

cells, with putative DNA-binding sites and transactivation activ-

ity. Taken together, this suggests that MIG1 might act as a

modulator of gene expression changes required for arbuscule

development.

MIG1 Modulates Root Cortex DevelopmentIn order to get insights into the cellular mechanism of MIG1,

we performed overexpression (OE) experiments. Unexpectedly,

MIG1OE (more than 20-fold; Figure S2A) inducedmajor changes

in root cortex development, resulting in a significant enlargement

in root diameter (Figures 3A and 3B; Figure S2B). Such a root-

thickening effect was previously reported in plants treated with

gibberellin (GA) biosynthesis inhibitors [33–36] being fully revers-

ible by application of GA. Likewise, application of paclobutrazol

(PAC), an inhibitor of GA biosynthesis, increased root diameter in

Figure 2. MIG1 Belongs to a Novel Clade of GRAS Proteins and Posses(A) Phylogenetic analysis of the GRAS protein family. The unrooted tree was gene

species A. thaliana,M. truncatula, S. lycopersicum, and O. sativa. Alignment was

neighbor-joining method using MEGA7, bootstrap value of 1,000 (percent va

arrangement was chosen for balanced shape. MIG1 clusters in a new clade with

(B) Amino-acid alignment of the 100 closest relatives to theMIG1 N terminus revea

and III). Consensus secondary structure prediction was performed using 18 rando

MIG1, MIG2, and MIG3 (pink dots). Four predicted a helices (blue) are located

conserved regions (pink). The fourth a helix (light blue) is not as conserved as the o

if located in predicted a helix) were predicted for all proteins within the first cons

(C) Transactivation test of MtMIG1 in S. cerevisiae. MtMIG1, MtDELLA2, and At

versions of MtMIG1 (108 amino acids [aa]; MtMIG1-N) and of MtDELLA2 (81 aa;

transactivation activity in yeast. TRP1 served as transformation markers, and

tryptophan; -WHA, selection media without tryptophan, histidine, and adenine.

See also Figure S1 and Table S2.


control roots and further enhanced the promoting effect ofMIG1

OE in M. truncatula (Figures 3B, S2B, and S2C). Conversely,

treatment with GA or a double DELLA deletion reduced root

diameter (Figures 3B and S2B–S2D), but most interestingly, it

fully abolished the MIG1-promoting effect (Figures 3B and

S2B). Together, these results suggested a link between GA

signaling and MIG1 impacting on root diameter. However, the

synergistic effect observed with PAC indicated that MIG1 could

be acting through an independent pathway or, by contrast, that

the concentration of PAC was not sufficient to fully inhibit GA

signaling. To distinguish between those possibilities, we created

a dominant DELLA non-degradable by GA (D18DELLA1) to fully

block GA signaling in roots [7]. Similar to the application of PAC,

expression of D18DELLA1 increased root diameter. However,

OE of MIG1 in the D18DELLA1 background did not further in-

crease this phenotype, providing genetic evidence that MIG1 in-

tersects with the GA signaling pathway (Figures 3C and S2E).

A larger root diameter might be the consequence of an in-

crease in the number of cortical cell layers, of enlarged cell width,

or both. Although the number of cortical cell layers is usually con-

stant for a plant species, it has been shown that several develop-

mental processes, including interactions with other organisms

(i.e., Rhizobia or root-knot nematodes), can alter their number

[37–39]. In this sense, GA is known to act on the cortex prolifer-

ation by delaying the onset of middle cortex (MC) formation in

A. thaliana, while PAC induces precocious formation of the MC

[40]. This mechanism seems to be conserved in other plants,

as shown in rice, where PAC application increased the number

of cortical cells in fine lateral roots [41]. Therefore, we next

analyzed the number of cortical cell layers in MIG1 OE roots

and observed an increase that was fully reversed by GA treat-

ment (Figures 3D and S2F). GA application also reduced the

number of cell layers in control roots (Figures 3D, S2F, and

S2G), similarly to the double DELLA mutant (Figure S2H). In

contrast, PAC and D18DELLA1 phenocopied the MIG1-medi-

ated cortical cell layer proliferation (Figures 3D, 3E, S2F, and

S2I), further supporting the hypothesis that MIG1 interferes

with the GA signaling impacting on root radial development.

Remarkably,MIG1OE changed not only the number of cortical

cells but also theirmorphology, resulting inwider and longer cells

(Figure 3F). These results were surprising, because ubiquitous

GA signaling inhibition produces wider but shorter cells (Figures

3F and 3G). It is known from A. thaliana that the promoting effect

of GA signaling on root elongation is mediated at the endodermis

ses Transactivation Activity in Its Highly Conserved Amino Terminusrated based on an amino-acid alignment with all GRAS proteins from the plant

performed using ClustalW, and the phylogenetic tree was constructed with the

lue is labeled at each node), p-distance, and pairwise gap deletion. Taxa

out representatives in A. thaliana or O. sativa.

led three highly conserved amino-acid stretches at the end of the N termini (I, II,

mly selected proteins from different plant species of the MIG1 clade, including

in the N terminus of MIG1-related proteins, three of them within the highly

thers and is located in front of them. Several DNA-binding sites (yellow; or green

erved amino-acid stretch, except for RcXP_002519213.1 (Ricinus communis).

RGA1 were fused to the GAL4-binding domain (DBD). In addition N-terminal

MtDELLA2-N) were tested. All GRAS proteins, except MtDELLA2-N, showed

HIS3 and ADE2 were used as reporter genes. -W, selection media without

Figure 3. MIG1 Controls Cortex Development by Intersecting the GA Pathway

(A) Microscopical pictures of empty vector (EV) andMIG1 overexpression (OE) of M. truncatula roots. Longitudinal image (left) and cross-sections of hairy roots

stained with toluidine blue O (right). Scale bars represent 100 mm.

(B, D, and F) Analyses of hairy roots EV and MIG1 OE grown on mock medium or GA3- or PAC-supplemented medium for 2 weeks. Three biological replicates

were analyzed with n (roots) R 90 and n (cells) > 560.

(C, E, andG) Analyses of composite plants EV,MIG1OE,D18DELLA1, andD18DELLA1+MIG1OEgrown onmedia for 6 weeks. n (roots)R 58 and n (cells)R 268.

(B and C) Analyses of root diameter. Data represent mean ± SD.

(D and E) Analyses of number of root cortex layers. Data represent mean ± SD.

(F and G) Analyses of cell width and length. Box-and-whisker plot show first, second, and third quartile, highest and lowest data within a 1.5-interquartile range

(IQR), and outliers (squares). (Mann-Whitney U test was used for calculating the level of significance; different letters indicate significance with p < 0.05.)

(H) DELLA1 and NSP1 are interacting with MIG1 in the nucleus shown by bimolecular fluorescence complementation (BiFC) inN. benthamiana. The C-terminal or

the N-terminal part of Split-YFP (YFPC and YFPN, respectively) was fused N-terminal to the GRAS proteins. Pictures were taken 2 days after co-infiltration with

the A. tumefaciens strain GV3101 into N. benthamiana leaves. Scale bars represent 50 mm.

See also Figures S2 and S3 and Table S2.


by controlling longitudinal cell expansion [9] and that coordina-

tion of DELLA in all root tissues is required for synchronized

growth [42]. However, the role of GA-DELLA controlling radial

cell expansion has not been explicitly addressed so far. Our re-

sults show that ubiquitous inhibition of GA signaling, by ectopic

expression of D18DELLA1 or PAC treatment, significantly in-

creases radial cell expansion in M. truncatula, resulting in an

increased root diameter that resembles MIG1 OE (Figures 3F,


Figure 4. Downregulation of MIG1 Results in a Distorted Arbuscule Phenotype that Is Rescued by Expression of a Dominant DELLA

Composite plants were grown with (myc, mycorrhized) or without (nm, non-mycorrhized) R. irregularis and harvested 5 wpi (weeks post-infection). (A–D) show

analyses of roots transformed with MIG1-RNAi or empty vector (EV) driven by the MtPT4 promoter, the activity of which is restricted to arbuscule-containing

cells. rel., relative. Total number of analyzed root fragments were 349 in PMtPT4:EV and 178 in PMtPT4:MIG1-RNAi. (E–H) show analyses of roots transformed

with EV, dominant DELLA1 under the 35S promoter (D18DELLA1), or P35S:D18DELLA1 together with PMtPT4:MIG1-RNAi (D18DELLA1 + MIG1RNAi). Total

number of analyzed root fragments were 268 in EV, 202 in P35S:D18DELLA1, and 670 in P35S:D18DELLA1+PMtPT4:MIG1-RNAi. Data represent mean ± SD with

n R 3; n denotes the number of biological replicates. A Student’s t test was used to calculate confidence level. Scale bars represent 100 mm, if not otherwise

depicted.

(A and E) Downregulation ofMIG1was confirmed via qPCR. In addition, expression of the duplicationsMIG2 andMIG3was analyzed. PMtPT4:MIG1-RNAi shows

only a significant downregulation of MIG1.

(B and F) Visualization of representative fungal structures stained with WGA-FITC. MIG1-RNAi leads to a higher number of distorted arbuscules.

(C andG)Quantification ofmycorrhizal colonization. Frequency of colonization (F%), abundance ofmature arbuscules (ma%), and distorted arbuscules (da%) are

shown. Expression of the dominant D18DELLA1 rescues the phenotype of MIG1-RNAi in arbuscule development.

(legend continued on next page)




3G, and S2J). These results point toward DELLA1 being a posi-

tive regulator of cortical radial expansion and, as described, a

negative regulator of cortical cell length. Unexpectedly, all pro-

motion effects of MIG1 OE on cell morphology were fully

reversed by GA, including longitudinal cell growth, which was

also abolished when MIG1 was ectopically expressed in the

D18DELLA1 background.

To understand the biological relevance of MIG1 in cortex re-

modeling, we did morphometric analyses of mycorrhizal roots.

Symbiosis formation induced a significant width increase in ar-

buscule-containing cells of the same magnitude as that induced

by MIG1 OE. Arbuscule formation on MIG1 OE plants did not

further enhance the effect on cell width, nor did it have any effect

on cell length (Figure S2K). Morphometric changes in response

to AM fungi are consistent with previous reports [43, 44] and sug-

gest that MIG1 symbiotic function might be to control primarily

the radial expansion of cortical cells invaded by the fungus.

But how does MIG1 control radial expansion in the cortex?

Our results show thatMIG1OE phenocopies the inhibitory effect

of D18DELLA1 and PAC, suggesting a negative role of GA

signaling in arbuscule-containing cells. This is consistent with

the observed negative effect of GA [45] and with the essential

requirement of DELLA proteins [7, 18, 46] for arbuscule develop-

ment. The suppression of the MIG1 effect by GA and the

epistasis relation of MIG1 and DELLA1 further support the hy-

pothesis that MIG1 mediates its effect in cell expansion by inter-

fering with the GA signaling pathway rather than by indepen-

dently regulating downstream GA-effector genes. Therefore,

we considered whether MIG1 could interact with other GRAS

TFs and, in particular, with the DELLA proteins. In this line, sym-

biotic GRAS proteins have been shown to be part of a network

of TF complexes [6, 7, 18, 19, 21]. Bimolecular fluorescence

complementation in Nicotiana benthamiana showed that,

indeed, MIG1 interacts with DELLA1 and with NSP1 in the nu-

cleus, but it never did with RAM1, NSP2, or DELLA3 and only

with RAD1 and DELLA2 when MIG1 was fused to the C terminus

of yellow fluorescent protein (YFP) (Figures 3H, S3A, and S3B).

DELLA1 also interacts with NSP1, which interestingly, is also

induced in response to fungal signals; thus, our findings further

strengthen NSP1 involvement in the mycorrhiza symbiosis [22,

23, 47, 48]. In addition, we found that, in contrast to NSP1,

NSP2, andRAM1 [21, 31], MIG1 did not form homodimers or het-

erodimers with MIG2 or MIG3. These results indicate that MIG1

might be part of novel legume GRAS TF complexes involving

DELLA1 and NSP1, distinct from the complexes observed

involving DELLA1, RAM1, RAD1, and NSP2 [6, 8, 19, 21, 49].

This suggests that MIG1 complexes might control different

downstream targets, and, in agreement, we did not observe in-

duction of several of the described target genes [6, 49] but rather

a suppressive effect that was significant for EXO70I when ectop-

ically expressing MIG1 (Figure S3D). This is interesting because

EXO70I has been shown to be a target of the DELLA1-RAM1

transcriptional cascade controlling arbuscule branching [6].

(D andH)Morphometrical analysis of roots. Cortical cell width and length from arb

analyzed with n (roots) R 15; n (cells) R 89. A Mann-Whitney U test was used

significance: *p < 0.05; **p < 0.01; ***p < 0.001.

See also Figure S4 and Table S2.

EXO70I is a dedicated component of the exocyst complex

that, in arbuscule-containing cells, allows the formation of

specialized subdomains in the PAM [14].

MIG1 Downregulation Impairs Arbuscule DevelopmentTo challenge the hypothesis that the effect of MIG1 on radial

cortical cell morphology might impact on arbuscule develop-

ment, and given that no insertion mutants are available for

MIG1, we downregulated its expression using RNAi. Two

expression systems were used to inactivate MIG1: one under

the control of the arbuscule-containing-cell-specific promoter

MtPT4 from M. truncatula and the second was driven by the

constitutiveA. thaliana UBIQUITIN3 promoter [50]. Both systems

worked similarly on MIG1 expression, which was reduced by

62% (PMtPT4) and 70% (PAtUBI3) in colonized roots (Figures 4A

and S4A). Downregulation of MIG1 resulted in an increase in

smaller and distorted arbuscules (Figures 4B and S4B). Although

the frequency of mycorrhization was not affected (Figures 4C

and S4C), intercellular septated hyphae, indicators of fungal

apoptosis, were often observed. Consistent with this, the per-

centage of mature arbuscules was reduced inMIG1RNAi plants,

whereas the abundance of distorted arbuscules was significantly

increased (Figures 4C and S4C). Given the high similarity be-

tween MIG1 and its duplications, MIG2 and MIG3, their expres-

sion was also analyzed. Both genes were inactivated using the

PAtUBI3 (�50%), but they were not significantly downregulated

with PMtPT4 (Figures 4A and S4A). Interestingly, the inactivation

of the threeMIGs did not result in a stronger phenotype; further-

more, in PMtPT4:MIG1-RNAi roots, MIG2 and MIG3 were not able

to rescue the arbuscule phenotype caused by MIG1 downregu-

lation (Figure 4C). This indicates that, despite their similarity,

there is no functional redundancy, and solely the reduction in

MIG1 expression is responsible for the observed arbuscule

developmental phenotype.

MIG1 RNAi did not alter the expression of several mycorrhizal

marker genes, with the exception of NSP2, which was signifi-

cantly induced (Figure S4E). This suggests that MIG1 is a nega-

tive regulator of NSP2 in arbuscule-containing cells. Although

the role of NSP2 in AM symbiosis is not fully understood, its

proper spatiotemporal distribution controlled by the micro-

RNA171h seems to be key [51]. NSP2 was shown to be respon-

sible for cortical cell divisions during nodulation [52]; therefore, it

might be interesting to see whetherMIG1 expression in arbuscu-

lated cells contributes to negatively regulate nodule organogen-

esis when roots are challenged with both microbes.

Most interestingly, MIG1 downregulation by RNAi reduced

cortical cell size in line with the opposite effects observed in

MIG1OE roots (Figures 4D and S4D). This suggests that themal-

formed arbuscule phenotype could be related to the impossi-

bility of radial expansion in cells harboring arbuscules. Indeed,

repression of MIG1 in the D18DELLA1 background, which

results in constitutive radially expanded cells, restores the

normal arbuscule phenotype (Figures 4E–4H), thus indicating

uscule-containing cells weremeasured. At least three biological replicateswere

for calculating the level of significance. Different letters or asterisks indicate



that radial cell expansion could be critical for proper arbuscule

development.

ConclusionsHere, we have identified a novel component of the transcriptional

reprogramming imposed by AM fungi on their host plants. It is

likely that induction of MIG1 by fungal signals serves to regulate

the expression of downstream components required for the cell

morphology changes occurring during arbuscule formation. We

envisage a model in which MIG1 acts as an integrator of fungal

signals into the GA signaling pathway by recruiting DELLA1 as

a transcriptional coactivator. In ourmodel, DELLA1 acts as ama-

jor repressor of longitudinal cortex cell growth, possibly at the

endodermis as shown in Arabidopsis [9], but it serves, in addi-

tion, as a positive regulator of cortical radial cell expansion. Dur-

ing symbiosis, MIG1 recruits DELLA1 to the promoter of genes

responsible for radial cell growth, resulting in a radial expansion

of arbuscule-containing cells. Because DELLA1 has already

been shown to be a key activator of genes required for arbuscule

branching which are somehow repressed in MIG1 OE roots, our

model implies a dual role for DELLA1 in arbuscule-containing

cells. Proper arbuscule development requires a coordinated

recruitment of DELLA1 to enable arbuscule branching through

RAM1 and MIG1-mediated radial cell expansion.

AM fungi are obligate biotrophic fungi that require nutrients

from their host to complete their life cycle. Our results here

show that AM fungal signals can finely adjust the plant develop-

mental program in order to promote their accommodation in the

cortex. Elucidating the mechanisms of how rhizospheric mi-

crobesmanipulate their hosts can further help us to better under-

stand the general mechanisms of plant development.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

four figures, and two tables and can be found with this article online at

http://dx.doi.org/10.1016/j.cub.2016.07.059.

AUTHOR CONTRIBUTIONS

N. Requena and C.H. designed the experiments and wrote the manuscript.

C.H., H.K., N. Rieger, S.W., and S.H. carried out the experiments.

ACKNOWLEDGMENTS

Financial support was provided by the German Science Foundation (DFG

Re1556/6-2; DFG Re1556/7-2). We are thankful to Dr. H. Slater and Dr. B.

Hause for their comments to the manuscript. We also thank Dr. F. Krajinski

for providing the plasmids for RNAi, Dr. M. Harrison for her gift of the double

DELLA mutant, and Dr. G. Jurges for her help with the cross-sections. We

also thank Dr. B. Ebner for his advice on the statistical analysis.

Received: February 2, 2016

Revised: June 30, 2016

Accepted: July 22, 2016

Published: September 15, 2016

REFERENCES

1. Berendsen, R.L., Pieterse, C.M., and Bakker, P.A. (2012). The rhizosphere

microbiome and plant health. Trends Plant Sci. 17, 478–486.

2. Tkacz, A., and Poole, P. (2015). Role of root microbiota in plant productiv-

ity. J. Exp. Bot. 66, 2167–2175.


3. Nihorimbere, V., Ongena, M., Smargiassi, M., and Thonart, P. (2011).

Beneficial effect of the rhizosphere microbial community for plant growth

and health. Biotechnol. Agron. Soc. 15, 327–337.

4. Gutjahr, C., and Paszkowski, U. (2013). Multiple control levels of root

system remodeling in arbuscular mycorrhizal symbiosis. Front. Plant Sci.

4, 204.

5. Evangelisti, E., Rey, T., and Schornack, S. (2014). Cross-interference

of plant development and plant-microbe interactions. Curr. Opin. Plant

Biol. 20, 118–126.

6. Park, H.J., Floss, D.S., Levesque-Tremblay, V., Bravo, A., and Harrison,

M.J. (2015). Hyphal branching during arbuscule development requires

reduced arbuscular mycorrhiza1. Plant Physiol. 169, 2774–2788.

7. Floss, D.S., Levy, J.G., Levesque-Tremblay, V., Pumplin, N., and Harrison,

M.J. (2013). DELLA proteins regulate arbuscule formation in arbuscular

mycorrhizal symbiosis. Proc. Natl. Acad. Sci. USA 110, E5025–E5034.

8. Pimprikar, P., Carbonnel, S., Paries, M., Katzer, K., Klingl, V., Bohmer,

M.J., Karl, L., Floss, D.S., Harrison, M.J., Parniske, M., and Gutjahr, C.

(2016). A CCaMK-CYCLOPS-DELLA complex activates transcription of

RAM1 to regulate arbuscule branching. Curr. Biol. 26, 987–998.

9. Ubeda-Tomas, S., Swarup, R., Coates, J., Swarup, K., Laplaze, L.,

Beemster, G.T., Hedden, P., Bhalerao, R., and Bennett, M.J. (2008).

Root growth in Arabidopsis requires gibberellin/DELLA signalling in the

endodermis. Nat. Cell Biol. 10, 625–628.

10. Bonfante, P., and Genre, A. (2008). Plants and arbuscular mycorrhizal

fungi: an evolutionary-developmental perspective. Trends Plant Sci. 13,

492–498.

11. Genre, A., Ivanov, S., Fendrych, M., Faccio, A., Zarsky, V., Bisseling, T.,

and Bonfante, P. (2012). Multiple exocytotic markers accumulate at the

sites of perifungal membrane biogenesis in arbuscular mycorrhizas.

Plant Cell Physiol. 53, 244–255.

12. Ivanov, S., Fedorova, E.E., Limpens, E., De Mita, S., Genre, A., Bonfante,

P., and Bisseling, T. (2012). Rhizobium-legume symbiosis shares an

exocytotic pathway required for arbuscule formation. Proc. Natl. Acad.

Sci. USA 109, 8316–8321.

13. Pumplin, N., Zhang, X., Noar, R.D., and Harrison, M.J. (2012). Polar local-

ization of a symbiosis-specific phosphate transporter is mediated by

a transient reorientation of secretion. Proc. Natl. Acad. Sci. USA 109,

E665–E672.

14. Zhang, X., Pumplin, N., Ivanov, S., and Harrison, M.J. (2015). EXO70I

Is Required for Development of a Sub-domain of the Periarbuscular

Membrane during Arbuscular Mycorrhizal Symbiosis. Curr. Biol. 25,

2189–2195.

15. Huisman, R., Hontelez, J., Mysore, K.S., Wen, J., Bisseling, T., and

Limpens, E. (2016). A symbiosis-dedicated SYNTAXIN OF PLANTS 13II

isoform controls the formation of a stable host-microbe interface in sym-

biosis. New Phytol. 211, 1338–1351.

16. Kosuta, S., Chabaud, M., Lougnon, G., Gough, C., Denarie, J., Barker,

D.G., and Becard, G. (2003). A diffusible factor from arbuscular mycor-

rhizal fungi induces symbiosis-specific MtENOD11 expression in roots

of Medicago truncatula. Plant Physiol. 131, 952–962.

17. Kuhn, H., Kuster, H., and Requena, N. (2010). Membrane steroid-binding

protein 1 induced by a diffusible fungal signal is critical for mycorrhization

in Medicago truncatula. New Phytol. 185, 716–733.

18. Yu, N., Luo, D., Zhang, X., Liu, J., Wang, W., Jin, Y., Dong, W., Liu, J., Liu,

H., Yang, W., et al. (2014). A DELLA protein complex controls the arbuscu-

lar mycorrhizal symbiosis in plants. Cell Res. 24, 130–133.

19. Gobbato, E., Marsh, J.F., Vernie, T., Wang, E., Maillet, F., Kim, J., Miller,

J.B., Sun, J., Bano, S.A., Ratet, P., et al. (2012). A GRAS-type transcription

factor with a specific function in mycorrhizal signaling. Curr. Biol. 22,

2236–2241.

20. Rich, M.K., Schorderet, M., Bapaume, L., Falquet, L., Morel, P.,

Vandenbussche, M., and Reinhardt, D. (2015). The petunia GRAS

transcription factor ATA/RAM1 regulates symbiotic gene expression and


http://refhub.elsevier.com/S0960-9822(16)30852-1/sref1




































































fungal morphogenesis in arbuscular mycorrhiza. Plant Physiol. 168,

788–797.

21. Xue, L., Cui, H., Buer, B., Vijayakumar, V., Delaux, P.M., Junkermann, S.,

and Bucher, M. (2015). Network of GRAS transcription factors involved in

the control of arbuscule development in Lotus japonicus. Plant Physiol.

167, 854–871.

22. Delaux, P.M., Becard, G., and Combier, J.P. (2013). NSP1 is a component

of the Myc signaling pathway. New Phytol. 199, 59–65.

23. Takeda, N., Tsuzuki, S., Suzaki, T., Parniske, M., and Kawaguchi, M.

(2013). CERBERUS and NSP1 of Lotus japonicus are common symbiosis

genes that modulate arbuscular mycorrhiza development. Plant Cell

Physiol. 54, 1711–1723.

24. Maillet, F., Poinsot, V., Andre, O., Puech-Pages, V., Haouy, A., Gueunier,

M., Cromer, L., Giraudet, D., Formey, D., Niebel, A., et al. (2011). Fungal

lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza.

Nature 469, 58–63.

25. Chen, A., Gu, M., Sun, S., Zhu, L., Hong, S., and Xu, G. (2011).

Identification of two conserved cis-acting elements, MYCS and P1BS,

involved in the regulation of mycorrhiza-activated phosphate transporters

in eudicot species. New Phytol. 189, 1157–1169.

26. Lota, F., Wegmuller, S., Buer, B., Sato, S., Br€autigam, A., Hanf, B., and

Bucher, M. (2013). The cis-acting CTTC-P1BS module is indicative for

gene function of LjVTI12, a Qb-SNARE protein gene that is required for ar-

buscule formation in Lotus japonicus. Plant J. 74, 280–293.

27. Liu, X., and Widmer, A. (2014). Genome-wide comparative analysis of

GRAS gene family in Populus, Arabidopsis and rice. Plant Mol. Biol. 32,

1129–1145.

28. Huang, W., Xian, Z., Kang, X., Tang, N., and Li, Z. (2015). Genome-wide

identification, phylogeny and expression analysis of GRAS gene family

in tomato. BMC Plant Biol. 15, 209.

29. Hogekamp, C., Arndt, D., Pereira, P.A., Becker, J.D., Hohnjec, N., and

Kuster, H. (2011). Laser microdissection unravels cell-type-specific tran-

scription in arbuscular mycorrhizal roots, including CAAT-box transcrip-

tion factor gene expression correlating with fungal contact and spread.

Plant Physiol. 157, 2023–2043.

30. Morohashi, K., Minami, M., Takase, H., Hotta, Y., and Hiratsuka, K. (2003).

Isolation and characterization of a novel GRAS gene that regulates

meiosis-associated gene expression. J. Biol. Chem. 278, 20865–20873.

31. Hirsch, S., Kim, J., Munoz, A., Heckmann, A.B., Downie, J.A., and

Oldroyd, G.E. (2009). GRAS proteins form a DNA binding complex to

induce gene expression during nodulation signaling in Medicago trunca-

tula. Plant Cell 21, 545–557.

32. Hirano, K., Kouketu, E., Katoh, H., Aya, K., Ueguchi-Tanaka, M., and

Matsuoka, M. (2012). The suppressive function of the rice DELLA protein

SLR1 is dependent on its transcriptional activation activity. Plant J. 71,

443–453.

33. Shive, J.B., and Sisler, H.D. (1976). Effects of ancymidol (a growth retar-

dant) and triarimol (a fungicide) on the growth, sterols, and gibberellins

of Phaseolus vulgaris (L.). Plant Physiol. 57, 640–644.

34. Tanimoto, E. (1987). Gibberellin-dependent root elongation in lactuca-sat-

iva: recovery from growth retardant-suppressed elongation with thick-

ening by low concentration of Ga3. Plant Cell Physiol. 28, 963–973.

35. Baluska, F., Parker, J.S., and Barlow, P.W. (1993). A role for gibberellic-

acid in orienting microtubules and regulating cell-growth polarity in the

maize root cortex. Planta 191, 149–157.

36. Wang, G.L., Que, F., Xu, Z.S., Wang, F., and Xiong, A.S. (2015). Exogenous

gibberellin altered morphology, anatomic and transcriptional regulatory

networks of hormones in carrot root and shoot. BMC Plant Biol. 15, 290.

37. Libbenga, K.R., and Harkes, P.A. (1973). Initial proliferation of cortical cells

in the formation of root nodules in Pisum sativum L. Planta 114, 17–28.

38. Hodges, C.F., and Taylor, D.P. (1966). Host-parasite interaction of a root-

knot nematode and creeping bentgrass, Agrostis palustris. Phytopathology

56, 88–91.

39. Xiao, T.T., Schilderink, S., Moling, S., Deinum, E.E., Kondorosi, E.,

Franssen, H., Kulikova, O., Niebel, A., and Bisseling, T. (2014). Fate map

of Medicago truncatula root nodules. Development 141, 3517–3528.

40. Paquette, A.J., and Benfey, P.N. (2005). Maturation of the ground tissue of

the root is regulated by gibberellin and SCARECROW and requires

SHORT-ROOT. Plant Physiol. 138, 636–640.

41. Fiorilli, V., Vallino,M., Biselli, C., Faccio, A., Bagnaresi, P., andBonfante, P.

(2015). Host and non-host roots in rice: cellular and molecular approaches

reveal differential responses to arbuscular mycorrhizal fungi. Front. Plant

Sci. 6, 636.

42. Ubeda-Tomas, S., Beemster, G.T., and Bennett, M.J. (2012). Hormonal

regulation of root growth: integrating local activities into global behaviour.

Trends Plant Sci. 17, 326–331.

43. Berta, G., Trotta, A., Fusconi, A., Hooker, J.E., Munro, M., Atkinson, D.,

Giovannetti, M., Morini, S., Fortuna, P., Tisserant, B., et al. (1995).

Arbuscular mycorrhizal induced changes to plant growth and root system

morphology in Prunus cerasifera. Tree Physiol. 15, 281–293.

44. Balestrini, R., Cosgrove, D.J., and Bonfante, P. (2005). Differential location

of alpha-expansin proteins during the accommodation of root cells to an

arbuscular mycorrhizal fungus. Planta 220, 889–899.

45. El Ghachtouli, N., Martin-Tanguy, J., Paynot, M., and Gianinazzi, S. (1996).

First report of the inhibition of arbuscular mycorrhizal infection of Pisum

sativum by specific and irreversible inhibition of polyamine biosynthesis

or by gibberellic acid treatment. FEBS Lett. 385, 189–192.

46. Foo, E., Ross, J.J., Jones, W.T., and Reid, J.B. (2013). Plant hormones in

arbuscular mycorrhizal symbioses: an emerging role for gibberellins. Ann.

Bot. (Lond.) 111, 769–779.

47. Camps, C., Jardinaud, M.F., Rengel, D., Carrere, S., Herve, C., Debelle, F.,

Gamas, P., Bensmihen, S., and Gough, C. (2015). Combined genetic and

transcriptomic analysis reveals three major signalling pathways activated

by Myc-LCOs in Medicago truncatula. New Phytol. 208, 224–240.

48. Hohnjec, N., Czaja-Hasse, L.F., Hogekamp, C., and Kuster, H. (2015). Pre-

announcement of symbiotic guests: transcriptional reprogramming by

mycorrhizal lipochitooligosaccharides shows a strict co-dependency on

the GRAS transcription factors NSP1 and RAM1. BMCGenomics 16, 994.

49. Floss, D.S., Levesque-Tremblay, V., Park, H.J., and Harrison, M.J. (2016).

DELLA proteins regulate expression of a subset of AM symbiosis-induced

genes in Medicago truncatula. Plant Signal. Behav. 11, e1162369.

50. Devers, E.A., Teply, J., Reinert, A., Gaude, N., and Krajinski, F. (2013).

An endogenous artificial microRNA system for unraveling the function of

root endosymbioses related genes in Medicago truncatula. BMC Plant

Biol. 13, 82.

51. Hofferek, V., Mendrinna, A., Gaude, N., Krajinski, F., and Devers, E.A.

(2014). MiR171h restricts root symbioses and shows like its target NSP2

a complex transcriptional regulation in Medicago truncatula. BMC Plant

Biol. 14, 199.

52. Oldroyd, G.E., and Long, S.R. (2003). Identification and characterization of

nodulation-signaling pathway 2, a gene of Medicago truncatula involved in

Nod actor signaling. Plant Physiol. 131, 1027–1032.




















































































































Current Biology, Volume 26

Supplemental Information

Symbiotic Fungi Control Plant Root

Cortex Development through the Novel

GRAS Transcription Factor MIG1

Carolin Heck, Hannah Kuhn, Sven Heidt, Stefanie Walter, Nina Rieger, and NataliaRequena

Figure S1. Expression and phylogenetic analysis of several GRAS proteins. Related to Figure 1 and 2. (A, D) Roots of

four 10 days old M. truncatula plants were incubated in spore extract (SE), SE fractions or mock solution for 6 h or 24 h.

Expressions of M. truncatula GRAS genes were analyzed via qPCR and normalized to the housekeeping gene MtTEF1a.

(A) Expression levels of GRAS genes in response to SE treatment relative to mock control are presented. (B) Identities

between MIG1, MIG2 and MIG3 sequences in the open reading frame on nucleotide and aminoacid level. (C) Schematic

presentation of MIG1, MIG2 and MIG3 gene organization on chromosome 2, drawn in scale. (D) Expression levels of

MIG1, MIG2 and MIG3 in response to treatment with SE fractions. SE was separated into supernatant (SN), pellet (P) and

lipid phase (LP). (n = 2. Data represent mean ± SD. Over a 2-fold expression was interpreted as upregulation in response to

SE (dashed line)). (E) Phylogenetic analysis of the MIG1-clade. The unrooted tree was generated based on an amino acid

alignment with GRAS proteins from different plant species. The sequences were recieved by a BLASTp search with the N-

terminus of MtMIG1 as query sequence. The alignment of the protein sequences was performed using Clustal W. The

phylogenetic tree was constructed with the neighbour-joining method using MEGA7, 1000 bootstrap (%-value labeled at

each node), p-distance and a pairwise gap deletion. MIG1, MIG2 and MIG3 are labeled with pink dots, sequences used in

secondary structure prediction are labeled with orange dots.

0

2

4

6

8

6 h SE 24 h SE

Exp

ressio

n r

el.

to m

ock

Medtr2g034280.1 RAM1

TF80 Medtr4g104020.1

Medtr1g086970.1 TF124

Medtr5g009080.1 Medtr7g109580.1

Medtr2g082090.1 Medtr5g097480.1

A

B

DNA: 86.26 %

protein: 82.17 %

DNA: 86.17 %

protein: 82.24 %

DNA: 84.10 %

protein: 79.18 %

MIG2 MIG3

MIG1

D

0

4

8

12

16

20

mock SE SN P LP mock SE SN P LP E

xp

ressio

n re

l. to

mo

ck MIG1 MIG2 MIG3

6 h 24 h

Medtr2g034260

MIG3 MIG1 MIG2

Medtr2g034250 Medtr2g034280 C

Figure S1

MIG

Secondary structure prediction

E

*

***

0

200

400

600

800

Ro

ot d

iam

ete

r [µ

m]

0%

20%

40%

60%

80%

Nu

mb

er

of ro

ots

[%

]

Root diameter [µm]

mock GA3 PAC C

A B

Figure S2. Morphometrical analyses of M. truncatula roots. Related to Figure 3. (A) Overexpression of MIG1 was

confirmed via qPCR in MIG1 OE roots. n ≥ 4. Student’s t-Test was used to calculate confidence level. (B, E) Analyses of

EV and MIG1 OE hairy roots grown on mock, GA3 or PAC-supplemented medium for two weeks. Three biological

replicates were analysed with n (roots) ≥ 90, n (cells) > 560. (C, G, J) Analyses of WT grown on mock, GA3 or PAC-

supplemented medium for two weeks. n (roots) > 20, n (cells) > 360. (D, H) Analyses of double mutant della1/della2 (DD)

compared to segregated WT grown on media for two weeks. n (roots) ≥ 36. (G, I) Analyses of composite plants

transformed with EV, MIG1 OE, D18DELLA1 and D18DELLA1+MIG1 OE grown on media for six weeks. n (roots) ≥ 58.

(B-E) Analyses of root diameter. Average and histograms organizing the root diameters in frequencies of distribution are

shown. (F-I) Analyses of root cortex layers. Average and histograms organizing the number of cortex layers in frequencies

of distribution are shown. (J) Morphometric analyses of cell width and length. Box-and-whisker plot show first, second and

third quartil, highest and lowest datum within 1.5 interquartile range (IQR). (K) Morphometrical analyses of control (EV)

and MIG1 OE roots after 5 weeks of growth inoculated with R. irregularis (myc) or without (nm = non myc). Cortical cell

width and length were measured in arbuscule-containing cells (myc) or corresponding cells (nm). Four biological replicates

were analysed with n (roots) ≥ 14; n (cells) ≥ 200. (Data represent mean ± SD. Mann-Whitney U test was used for

calculating the level of significance in morphometrical analyses, different letters or * indicate significance with p-value <

0.05; **p-value < 0.01; ***p-value < 0.001).

Figure S2

*

0

0.04

0.08

0.12

EV MIG1 OE

Exp

ressio

n r

el. to

M

tTE

F1

a

MIG1

**

***

0

2

4

6

8

Num

ber

of

cort

ex layers

0

20

40

60

80

100

2 3 4 5 6 7 8

Num

ber

of

roots

[%

]

Number of cortex layers

mock GA3 PAC

G

0%

10%

20%

30%

40%

50%

<1

00

1

00

-15

0

15

0-2

00

2

00

-25

0

25

0-3

00

3

00

-35

0

35

0-4

00

4

00

-45

0

45

0-5

00

5

00

-55

0

55

0-6

00

>

60

0

Nu

mb

er

of ro

ots

[%

] mock

0%

20%

40%

60%

80%

<1

00

1

00

-15

0

15

0-2

00

2

00

-25

0

25

0-3

00

3

00

-35

0

35

0-4

00

4

00

-45

0

45

0-5

00

5

00

-55

0

55

0-6

00

>

60

0

GA3

0%

10%

20%

30%

40%

<1

00

1

00

-15

0

15

0-2

00

2

00

-25

0

25

0-3

00

3

00

-35

0

35

0-4

00

4

00

-45

0

45

0-5

00

5

00

-55

0

55

0-6

00

>

60

0

PAC EV

MIG1 OE

Root diameter [µm]

***

0

100

200

300

400

500

WT DD

Ro

ot d

iam

ete

r [µ

m] D

0%

10%

20%

30%

40%

50%

Nu

mb

er

of ro

ots

[%

]

Root diameter [µm]

WT DD

H

0%

20%

40%

60%

80%

2 3 4 5 6 7 8

Num

ber

of

roots

[%

]

Number of cortex layers

EV

MIG1 OE

D18DELLA1

D18DELLA1 + MIG1 OE

I

*

*

0

30

60

90

120

Cell

length

[µ

m] **

0

10

20

30

40

50

Cell

wid

th [µm

]

J

0%

20%

40%

60%

Num

ber

of ro

ots

[%

]

Root diameter [µm]

EV MIG1 OE D18DELLA1 D18DELLA1 + MIG1 OE

E

0

20

40

60

80

4 5 6

mock

Nu

mb

er

or

roo

ts [%

]

0

20

40

60

80

2 3 4

GA3

0

10

20

30

40

50

5 6 7

PAC EV

MIG1 OE

Numer of cortex layers

F

0

20

40

60

2 3 4 5 6 7 8

Num

ber

of

roots

[%

]

Number of cortex cell layers

WT

DD ***

0

1

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3

4

5

6

7

WT DD

Num

ber

of

cort

ex layers

a b b

b

0

10

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nm myc

Cell

wid

th [µm

]

EV MIG1 OE

a a b

ab

0

25

50

75

100

nm myc

Cell

length

[µ

m]

K

NS

P1-Y

FP

C

MIG

1-Y

FP

N

DE

LLA

1-Y

FP

C

MIG

1-Y

FP

N

NS

P1-Y

FP

C

DE

LLA

1-Y

FP

N

DE

LLA

1-Y

FP

C

+ N

SP

1-Y

FP

N

Figure S3

Figure S3. Interaction study of MIG1 with other GRAS proteins via BiFC in N. benthamiana and AM marker

expression in mycorrhized MIG1 OE roots. Related to Figure 3. (A-C) The C-terminal or N-terminal part of Split-YFP

was fused N-terminally to the GRAS proteins. Pictures were taken 2 d after co-infiltration with A. tumefaciens strain

GV3101 into N. benthamiana leaves. (A) MIG1, DELLA1 and NSP1 interact with each other in the nucleus. (B) Summary

of all combinations tested in the BiFC interaction study, including controls. Green shows positive interaction in both

directions. Yellow shows positive interaction in only one direction (+), the second direction could not confirm this

interaction (-). Red means no interaction detected. (C) As negative control EV was co-infiltrated. No specific YFP-signal

could be detected. (D) Expression of marker genes in mycorrhized (5 wpi) EV and MIG1 OE composite plants. Genes

connected to arbuscule branching show less induction in MIG1 OE roots compared to EV roots (Data represent single

values of 4 biological replicates. Student’s t-Test was used to calculate confidence level, * indicate significance with p-

value < 0.05.) .

EV

-YF

PC

DE

LLA

1-Y

FP

N

DE

LLA

1-Y

FP

C

EV

-YF

PN

EV

-YF

PC

NS

P1-Y

FP

N

NS

P1-Y

FP

C

EV

-YF

PN

MIG

1-Y

FP

C

EV

-YF

PN

EV

-YF

PC

MIG

1-Y

FP

N

C

A

BiFC

MIG

1

MIG

2

MIG

3

RA

D1

1g

086970

DE

LL

A1

DE

LL

A2

DE

LL

A3

RA

M1

NS

P1

NS

P2

MIG1 - -

MIG2

MIG3

RAD1 +

1g086970

DELLA1

DELLA2 +

DELLA3

RAM1

NSP1

NSP2

YFPC

YF

PN

B

D

0

0.003

0.006

0.009

Expre

ssio

n r

el. to M

tTE

F1a

RAM1

0

0.02

0.04

0.06

RAD1

0

0.2

0.4

0.6

SCP1

0

0.01

0.02

0.03

Vapyrin

*

0

0.01

0.02

0.03

0.04

EXO70I

EV

MIG1 OE

ac a

b

c

0

0.0003

0.0006

0.0009

Expre

ssio

n r

el. to M

tTE

F1a MIG1

A

a a

b

c

0

0.002

0.004

0.006

MIG3

a a

b

a

0

0.002

0.004

0.006

MIG2

EV B PAtUBI3

:MIG1-RNAi

*

0

20

40

60

80

100

EV MIG1-RNAi

%

F ma da C

nm myc

Figure S4. Downregulation of MIG1 with PAtUBI3

:MIG1-RNAi results in a distorted arbuscule phenotype. Related to

Figure 4. Composite plants were grown with (myc) or without (nm = non myc) R. irregularis and harvested 5 wpi. (A-D)

Analyses of plants transformed with MIG1-RNAi or EV construct driven by the ubiquitous AtUBI3 promoter. (A)

Downregulation of MIG1 was confirmed via qPCR. In addition expressions of the duplications MIG2 and MIG3 were

analysed. PAtUBI3:MIG1-RNAi leads to a significant downregulation of all three MIGs. (B) Visualization of representative

fungal structures in EV and MIG1-RNAi lines stained with WGA-FITC. Scale bar represents 100 µm. (C) Quantification of

mycorrhizal colonization. The frequency of colonization (F%) is comparable in MIG1-RNAi and EV roots, whereas

abundance of mature arbuscules (ma%) is lower and abundance of distorted arbuscules (da%) significantly higher in MIG1-

RNAi plants. Results of this figure mirror the downregulation of MIG1 driven by the MtPT4 promoter shown in Figure 4.

The total number of analysed root fragments were 224 in PAtUBI3:EV and 284 in PAtUBI3:MIG1-RNAi. (D) Morphometrical

analysis of roots transformed with control (EV) or MIG1-RNAi (PAtUBI3:MIG1-RNAi). Cortical cell width and length from

arbuscule-containing cells were measured. Three biological replicates were analysed with n (roots) ≥ 18; n (cells) ≥ 212.

(E) Expression of mycorrhizal marker genes were analyzed in mycorrhized MIG1-RNAi roots and compared to the

corresponding EV. Only the expression of NSP2 was significantly changed. Expression of NSP1, RAD1, RAM1, DELLA1,

DELLA2, SCP1, AMT2;4, AMT2;5, PT4, BCP1a, BCP1b, VAPYRIN, LEC5, EXO70I and STR were tested and showed no

significant differences. (Data represent mean ± SD. n ≥ 3 (n denotes the number of biological replicates). (Student’s t-Test

was used to calculate confidence level, different letters or * indicate significance with p-value < 0.05; **p-value < 0.01,

***p-value < 0.001.)

Figure S4

**

*

0

0.004

0.008

0.012

PAtUBI3 PMtPT4

Expre

ssio

n r

el. to

MtT

EF

1a

NSP2

EV

MIG1-RNAi

E

**

0

20

40

60

80

EV

MIG

1-

RN

Ai

Cell

length

[µm

]

***

0

10

20

30

EV

MIG

1-

RN

Ai

Cell

wid

th [µm

]

D

Table S2. Oligonucleotides and corresponding gene numbers used in this study.

All gene and accession numbers refer to the NCBI gene/protein database.

Primer name Sequence Gene/Accession number

Cloning BiFC constructs

MtDELLA1 ORF F ATG AAG AGA GAA CAC CAA GAA AGT MTR_3g065980

MtDELLA1 ORF R TCA CTT GGA CTC ATT TTG TGG AAG

MtDELLA2 ORF F ATG AAG AGA GAG CAT AAG CTT G ACJ83347.1 AFK49468.1 MtDELLA2 ORF R TCA GTG CGA AAC CAC CAC TGA G

MtDELLA3 ORF F ATG GAA ATA GTT TCA GAT TCT TCT TC Mt3.5 contig_55897

MtDELLA3 ORF R TCA ACA ATC AAA ACG CAG TGT TTC

MtRAD1 ORF F ATG TCA CCT GCA CTT TAT GCT A MTR_4g104020

MtRAD1 ORF R TCA GCA TTT CCA GCA AGA AAC TG

MtMIG1 ORF F ATG ATG GAA AAC TTG TGG GAA TTT G MTR_2g034280

MtMIG1 ORF R TCA AAT GAA TTT CCA AAC AGA AAC AGA G

MtMIG2 ORF F ATG GAA AAC TTG TGC AAT TTT GGT G MTR_2g034260


MtMIG3 ORF F ATG GAA AAC TTA TAC AAT TTG GGT G MTR_2g034250

MtMIG3 ORF R TCA GAT GAA TTT CCA AAC AGA AAC

MtNSP1 ORF F ATG ACT ATG GAA CCA AAT CCA AC MTR_8g020840

MtNSP1 ORF R CTA CTC TGG TTG TTT ATC CAG T

MtNSP2 ORF F ATG GAT TTG ATG GAC ATG GAT G MTR_3g072710

MtNSP2 ORF R CTA TAA ATC AGA ATC TGA AGA AGA AC

MtRAM1 ORF F ATG ATC AAT TCA CTT TGT GGA AGC TC MTR_7g027190

MtRAM1 ORF R TCA GCA TCG CCA TGC AGA AG

Cloning RNAi, promoter, overexpression constructs

MtMIG1-RNAi_BamHI F att gga tcc GAA GTG CAT AAA GAA AAT ACT AG

MTR_2g034280

MtMIG1-RNAi_XmaI R acc cgg gCA TCA AAG TGA ACC ATG TCA

MtMIG1-RNAi_MluI F aat acg cgt GAA GTG CAT AAA GAA AAT ACT AG

MtMIG1-RNAi_BspEI R taa tcc gga CAT CAA AGT GAA CCA TGT CA

MtMIG1 ORF F cac cAT GAT GGA AAA CTT GTG GGA ATT TGG


PMtMIG1 CACC 2kb F cac cCT GAT CAT ATC TTG TAG CTC ATC

PMtMIG1 CACC 230bp F cac cTC TAC TGG ACG GAT TTA CG

PMtMIG1 R AGA TTC AAA CAA ATT TTT CTT TAG TAT TAT G

P35S-MIG1OE XhoI F atc tcg agG GTC AAC ATG GTG GAG C

P35S-MIG1OE XhoI R cgc tcg agT CAA ATG AAT TTC CAA ACA GAA ACA

PPT4-MIG1RNAi XhoI F atc tcg agG ACT CGA TCC ACA ACA AAG

PPT4-MIG1RNAi XhoI R atc tcg agG GAC AAT CAG TAA ATT GAA CG

MtDELLA1 ORF F cac cAT GAA GAG AGA ACA CCA AGA AAG T

MTR_3g065980 MtDELLA1 ORF R TCA CTT GGA CTC ATT TTG TGG AAG

5'P-MtD18DELLA1 F P-GAT GTA GCT CAA AAG CTT GAA C

5'P-MtD18DELLA1 R P-TCC ACC ACC GTT GGT TTC

qPCR oligonucleotides

MtTEF1a-qPCR F TAC TCT TGG AGT GAA GCA GAT G MTR_6g021800

MtTEF1a-qPCR R GTC AAG AGC CTC AAG GAG AG

Mt2g082090-qPCR F GAC AAG GGA CAT GCT TGA TA MTR_2g082090

Mt2g082090-qPCR R CCT GTT GAA GAA AGG TGT TG

MtTF80-qPCR F CTG CTT GGA ATC TAC TGT TAC MTR_3g022830

MtTF80-qPCR R GTG GTC TGT CAC TCC AAC

Mt5g009080-qPCR F TAA TGG TTC GAC TCT GAT GG MTR_5g009080

Mt5g009080-qPCR R CAA ACA GCA TCT TCT CAA CC

Mt5g097480-qPCR F GTT CAA CTG CAT AAC CTT GC MTR_5g097480

Mt5g097480-qPCR R CCC TAT GAT TCT GAG TGC TG

Mt7g109580-qPCR F ATT GTT GCG TCA GAA GGT AG MTR_7g109580

Mt7g109580-qPCR R AAC ACT TCC CAT TCT TTT CCA C

MtTF124-qPCR F TCA GCT TCT TTA CCA AAC CAC MTR_8g442410

MtTF124-qPCR R TGC TTC CTC TAC ATG CAA ATT C

MtDELLA2-qPCR F AGC CTG AGA CAA GAG ACC ACJ83347.1 AFK49468.1 MtDELLA2-qPCR R ACC ACC AGC AGC AAG CTA

MtRAD1-qPCR F GAG GAG GAG AAT GAG TAG G MTR_4g104020

MtRAD1-qPCR R CAC AAG CTA CTA ATA TAG GGT C

Mt1g086970-qPCR F GGT TAG GAA TGT GAA GAT TGA TG MTR_1g086970

Mt1g086970-qPCR R CCC TAA TAT GAT CAA ACA ACC C

MtMIG1-qPCR F GGA GTT GAA AGA AAG AGT ACA G MTR_2g034280

MtMIG1-qPCR R GCA CCT TGT CCT TTA AAG TTA TA

MtMIG2_3-qPCR F CAT TGT CTT CTT GTT GGA TGG A MTR_2g034260

MtMIG2-qPCR R ACA CCA ACT AAC CAC ACA T

MtMIG2_3-qPCR F CAT TGT CTT CTT GTT GGA TGG A MTR_2g034250

MtMIG3-qPCR R GTA ATT ATA CTA ATA AGA ACC ATG GA

MtMIG-ORF-qPCR F TTT GGC TGA AGG AGT TGA AAG MTR_2g034280

MtMIG-ORF-qPCR R TCC ATC CAA CAA GAA GAC AAT

MtPT4-qPCR F GTG CGT TCG GGA TAC AAT ACT MTR_1g028600

MtPT4-qPCR R GAG CCC TGT CAT TTG GTG TT

MtRAM1-qPCR F ACC GTT AAC CGT CTC CAC MTR_7g027190

MtRAM1-qPCR R CTC TAC CTT AGC CCT TGG

MtSCP1-qPCR F GTT CAC CGT CGA ATT AAT GG MTR_3g079590

MtSCP1-qPCR R AAC ACT CTC ACA ACA ATA GAC TA

MtVapyrin-qPCR F AGA GAT AAC ATA TCA TCC TCC AC MTR_6g027840

MtVapyrin-qPCR R TCT TGG TAG TGA ACC AAT CAC

MtEXO70I-qPCR F AGC AGT GGC AGA AGA ATC AG MTR_1g017910

MtEXO70I-qPCR R CCA AGC AAC CCT TGC AAC

MtNSP2-qPCR R1 GTT TAG CTA GTA TAG ATT AGC AC MTR_3g072710

MtNSP2-qPCR F1 GTG AAG AGG AGA GAA GGT C

RiTEF-qPCR F TGT TGC TTT CGT CCC AAT ATC DQ282611.1

RiTEF-qPCR R GGT TTA TCG GTA GGT CGA G

Cloning constructs for transactivation assay in yeast

AtRGA_NdeI F gcg cat atg ATG AAG AGA GAT CAT CAC CAA At2g01570

AtRGA_BamHI R atg gat ccT CAG TAC GCC GCC GTC

MtMIG1_NdeI F ggc ata tgA TGA TGG AAA ACT TGT GGG AAT

MTR_2g034280 MtMIG1_BamHI R1 cag gat ccT CAA ATG AAT TTC CAA ACA GAA AC

MtMIG1_BamHI R2 gga tcc TGA TGG TTG GTA ATA TTG ATT GTC

MtDELLA2_NdeI F gcg cat atg ATG AAG GAG ATC ATC ACC ACJ83347.1 AFK49468.1

MtDELLA2_BamHI R1 gga tcc GTG TGC AAT GTG CAA ATA TGC

MtDELLA2_BamHI R2 gga tcc GGT GTC GTT TGA AAG ATG TTG

Supplemental Experimental Procedures

Biological material and transformation of plant roots

Medicago truncatula Jemalong A17, M truncatula R108 double mutant della1/della2 and the

segragated WT [S1] were grown on Fahraeus medium [S2] in a growth chamber at 25 °C with a 16 h day/ 8 h

night photoperiod.

M. truncatula Jemalong A17 roots were transformed with Agrobacterium rhizogenes ARquaI [S3] after

Boisson-Dernier et al. [S2]. Transformed roots were selected visually based on the constitutive expression of the

inserted DsRED1 cassette. Composite plants were grown in 50 ml tubes with a mixture of sand:gravel (4:1) and

watered with half strength Long Ashton nutrient solution [S4] twice a week (5 ml). Plants were grown for 5

weeks at 25 °C with a 16 h day/ 8 h night photoperiod. Excised hairy roots were cultivated on modified Minimal

(M)-medium supplemented with 1 % saccharose at 27 °C in the dark [S5].

Nicotiana benthamiana was used for transient expression of proteins and grown in soil at 22 °C, with a

16 h day/ 8 h night photoperiod.

Rhizophagus irregularis DAOM 197198 [S6] was cultivated in monaxenic culture as describe before

[S5].

Generation of constructs used in this study

For promoter reporter assays, a 1937 bp or a 230 bp fragment upstream of the start codon ATG of

MIG1 were cloned into the binary Gateway vector pPGFPGUS-RedRoot [S5]. For overexpression analyses, the

ORF of MIG1 was amplified from cDNA (1737 bp) and cloned into the destination vector 2xP35S-pKGW-

RedRoot. This binary Gateway vector was constructed by cloning the DsRED1 cassette (AatII and KpnI) [S5,

S7] and the 2x35S promoter (AatII) into the vector pKGW [S8]. Silencing of MIG1 was achieved using the

vectors pRED-RNAi either with the promoters of AtUBI3 or MtPT4 [S9]. A 315 bp RNAi construct covering the

region from -62 to +253 with respect to the start codon ATG (+1) was amplified from cDNA and cloned in sense

(BamHI and XmaI) and antisense (MluI and BspEI) direction, with spacer sequence forming a hairpin structure.

Sequence of MIG1-RNAi contsruct:

gaagtgcataaagaaaatactagttgaaactcataatactaaagaaaaatttgtttgaatctatgatggaaaacttgtgggaatttggtgagttcagttttgatacaacga

aaaatgttaaattttctgtagcagaagatctaggagactgtaatggaattgagtctctttgctctaattttggtttctttgaagatgatccatcacaagaagaagagcttcttc

tttccactaatcaacaaaagtatcaccaccaaccatatctagattatgaagcttttgataacttcaacattgacatggttcactttgatg

The non-degradable DELLA protein ∆18DELLA1 was constructed after Floss et al. [S1]. The DELLA1 ORF

was amplified from cDNA (1785 bp) and cloned into the vector pENTR™/D-TOPO® (Invitrogen). The amino

acid sequence MDELLAALGYKVRSSDMA including the DELLA motif was deleted by invers amplifying

DELLA1-pENTR™/D-TOPO® with 5’ phosphorylated primers leaving out the 54 nucleotides. The linear PCR

amplicon was religated with the T4-Ligase. The construct was integrated into the binary Gateway vector

pPCGFP-RR [S8], modified according to Kuhn et al. [S5], resulting in a P35S-∆18DELLA1 construct. For

simultaneous transformation of ∆18DELLA1 and overexpression of MIG1, the sequence 2xP35S-MIG1 was

amplified from MIG1-2xP35S-pKGW-RedRoot and cloned into ∆18DELLA1-pPCGFP-RR (XhoI). For

simultaneous transformation of ∆18DELLA1 and downregulation of MIG1, the sequence PMtPT4-MIG1RNAi

including the terminator was amplified from MIG1RNAi-pRED-RNAi(PMtPT4) and cloned into ∆18DELLA1-

pPCGFP-RR (XhoI).

For the transactivation assay in yeast the full length ORF of MtDELLA2, AtRGA and MtMIG1 as well as the N-

terminal part of MtMIG1 (324 bp) and MtDELLA2 (243 bp) were amplified from cDNA and cloned into the bait

vector pGBKT7 (NdeI and BamHI; Clontech). For BiFC experiments N-terminal fusions of the different ORFs

to the N- or C-terminal YFP-halves were constructed using the pCR8/GW/TOPO and the binary Gateway

vectors P35S-pSPYNE-GW and P35S-pSPYCE-GW [S10]. All primers used in this manuscript are listed in

Supplementary Table S2.

Mycorrhizal colonization and treatment of plant roots

Wildtype M. truncatula plants were inoculated with R. irregularis as described before [S5]. Inoculation

of composite plants, grown as described above, was carried out using R. irregularis inoculum from colonized

carrot roots in monoaxenic culture (1 plate inoculum for 400 ml substrate). Plants were then watered with half

strength low P (20 µM) Long Ashton nutrient solution [S4] twice a week.

For hormonal treatment M. truncatula seedlings or hairy roots were grown on medium supplemented

with Gibberellic Acid GA3 (Sigma-Aldrich), Paclobutrazol (PAC; Sigma-Aldrich) or mock control (EtOH) for

two weeks. Working concentrations of GA3 and PAC were 1 µM in 0,001 % EtOH each.

Spore extract (SE) treatment was carried out using 10 days old M. truncatula plants. Spores of R.

irregularis were morsed in liquid nitrogen and dH2O was added to a concentration of 200 mg/ml SE. The roots

of 4 plants were incubated for 15 min, 30 min, 6 h or 24 h in 2 ml spore extract diluted in liquid M-medium

(1:8). Fractionation of the SE was done by centrifugation. The treatments with the upper lipid phase (LP),

aqueous supernatant (SN) and pellet (P) were carried out with the same procedure as for the SE treatment.

Transactivation assay in yeast

For the transactivation test in Saccharomyces cerevisiae sequences of GRAS proteins were inserted into

the bait vector (pGBKT7) and transformed into the yeast strain AH109 (Clontech Laboratories, Inc). Colonies

were tested for self-activation on SD-WHA medium (single dropout medium w/o tryptophan, histidine and

adenine). For the spotting tests, 10 µl of a 1:10, 1:100 or 1:1000 dilution from overnight cultures were spotted

onto selection plates. Transformation and growth protocols were carried out according to the Yeast Protocols

Handbook (Clontech Laboratories, Inc).

Histochemical GUS analysis

Promoter reporter studies were carried out using 5 wpi (weeks post inoculation) mycorrhizal or control

composite plants. Roots were incubated in fixation solution (50 mM NaH2PO4, 1 mM Na2EDTA, 0,37 %

formaldehyde) for 30 min. Incubation in staining solution (50 mM NaH2PO4, 1 mM Na2EDTA, 1 mM X-GlcA

(Apollo Scientific; stock solution 0,1 M in DMF)) was carried out ON at 37 °C. The staining was fixed in 50 %

EtOH before counterstaining for fungal structures with WGA-fluorescein according to Rech et al. [S11].

Gene expression analysis

RNA extraction, cDNA synthesis and quantitative real time PCR (qPCR) were carried out as described

in Kuhn et al. [S5] with minor changes: for qPCR 1 µl cDNA of a 1:5 dilution was used per well as template.

The PCR protocol was selected as follows: 5 min 95 °C, 15 s 95 °C, 20 s 56 °C, 30 s 72 °C (40 cycles). Plant

transcript levels were normalized to the translation elongation factor 1-alpha of M. truncatula (MtTEF1a) [S12].

Transcript levels of genes were determined in at least three biological replicates with three technical replicates

per reaction. All primers are listed in Supplementary Table S2.

Bimolecular fluorescence complementation (BiFC)

For BiFC assays, leaves of 4-5 weeks old N. benthamiana plants were infiltrated with A. tumefaciens

GV3101 transformed with P35S-pSPYNE-GW and/or P35S-pSPYCE-GW [S10] expressing the genes of interest

fused to the YFP-halves or empty vector (EV) control. The p19 protein of tomato bushy stunt virus was used to

suppress gene silencing [S13]. Cells were kept 2-3 h in infiltration solution (10 mM MES-KOH (pH 5.6), 10

mM MgCl2, 150 µM Acetosyringon (in DMSO)) with an OD600 of 0.8 at RT before co-infiltration with a ratio of

1:1:1. Images were taken 2 days after infiltration.

Phenotypical analysis and quantification of mycorrhization

For phenotypical analysis and quantification of mycorrhizal roots, fungal structures were stained with

WGA-fluorescein as described in Rech et al. [S11]. Quantification of mycorrhizal structures were carried out

according to Trouvelot et al. [S14] using the program MYCOCALC (http://www2.dijon.inra.fr/mychintec

/Mycocalc-prg/download.html). F % represents the frequency of mycorrhiza in the root system. The intensity of

mature (ma %) and distorted arbuscules (da %) in the root fragments were calculated equally to arbuscule

abundance (a %) [S14].

For morphometrical analysis of cell length and width, as well as root diameter images were taken

randomly from actively growing secondary roots. The analyzed roots were always of the same age and compared

to the corresponding control roots (mock-treated and/or transformed with corresponding empty vector). Sites of

the root meristem, emergence and onsets of lateral roots were avoided. Measurements were done with Fiji

software (http://fiji.sc/Fiji).

For cross sections secondary hairy roots were fixed in 4 % paraformaldehyde and dehydrated in an

EtOH series. The root fragments were embedded in Paraplast Plus and sectioned (10 µm) using a mechanical

microtome. The cross sections were stained with 0.1 % (w/v) Toluidine Blue O (Carl Roth) and mounted in

Entellan new (Merck).

Confocal microscopy

All microscopical analyses were done using a Leica TCS SP5 (DM5000) confocal microscope with

conventional PMT detectors and the color camera Leica DFC295.

WGA-fluorescein stained roots and eYFP in BiFC analyses were both excited with an argon laser at

488 nm and 514 nm, respectively. Emission was detected from 505–525 nm (Fluorescein) or from 530–605 nm

(eYFP).

Images were processed using the Fiji software.

In silico, phylogenetic analysis and secondary structure prediction

In silico expression data was obtained from the M. truncatula gene expression atlas

(http://mtgea.noble.org/v3/) [S15].

Amino acid alignments were conducted using Clustal W within the MEGA7 software

(http://www.megasoftware.net/) [S16] with default settings and the protein weigh matrix PAM. The respective

trees were built with MEGA7 using the neighbour-joining algorithm, a bootstrap value of 1000, p-distance and

pairwise gap deletion. GRAS protein sequences from A. thaliana, M. truncatula, S. lycopersicum [S17] and O.

sativa were retrieved from the databases https://www.arabidopsis.org/, http://jcvi.org/medicago/,

https://solgenomics.net/ and http://rice.plantbiology.msu.edu/index.shtml, respectively.

Secondary structure predictions on the N-terminus of 18 proteins from the MIG1-clade (Figure S2)

were performed using the following 11 web servers:

ProteinPredict (https://www.predictprotein.org/), Jpred (http://www.compbio.dundee.ac.uk/jpred/), PSIPRED (ht

tp://bioinf.cs.ucl.ac.uk/psipred/), PSSpred (http://zhanglab.ccmb.med.umich.edu/PSSpred/), SABLE (http://sable

.cchmc.org/), SCRATCH (http://scratch.proteomics.ics.uci.edu/), Frag1D (http://frag1d.bioshu.se/pred/), Genesil

ico (http://iimcb.genesilico.pl/), APSSP2 (http://www.imtech.res.in/raghava/apssp2/), 1D (http://biomine.ece.ual

berta.ca/1D/1D.html), YASPIN (http://www.ibi.vu.nl/programs/yaspinwww/). A helix structure was accepted if

for a given amino acid at least 6 prediction tools considered it as helical (> 50 %). The prediction for DNA-

binding was performed by PredictProtein (https://www.predictprotein.org/).

Statistical analyses

Unless otherwise stated, all data shown represent the mean of biological replicates, error bars represent

SD (standard deviation). Two tailed Student’s T-test was used for pairwise comparisons of qPCR data and

quantified data of mycorrhizal colonization. Mann Whitney U test was used for comparison of morphometrical

analyses of roots and cells. The niveau of significance is indicated by asterisks (*p < 0.05; **p < 0.01; ***p <

0.001) or letters (different letters mean significant differences with p-value < 0.05). The statistical test and value

of n are indicated in the corresponding Figure legends.

Supplemental References

S1. Floss, D.S., Levy, J.G., Levesque-Tremblay, V., Pumplin, N., and Harrison, M.J. (2013). DELLA

proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci U S A

110, E5025-5034.

S2. Boisson-Dernier, A., Chabaud, M., Garcia, F., Bécard, G., Rosenberg, C., and Barker, D.G. (2001).

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing

and endomycorrhizal symbiotic associations. Mol Plant Microbe Interact 14, 695-700.

S3. Quandt, H.J., Pühler, A., and Broer, I. (1993). Transgenic root nodules of Vicia hirsuta: A fast and

efficient system for the study of gene expression in indeterminate-type nodules. Mol Plant Microbe

Interact 6, 699-706.

S4. Hewitt, E.J. (1966). Sand and water culture methods used in the study of plant nutrition, (London:

Commonwealth Bureau).

S5. Kuhn, H., Küster, H., and Requena, N. (2010). Membrane steroid-binding protein 1 induced by a

diffusible fungal signal is critical for mycorrhization in Medicago truncatula. New Phytol 185, 716-

733.

S6. Schenck, N., and Smith, G. (1982). Additional new and unreported species of mycorrhizal fungi

(Endogonaceae) from Florida. Mycologia 74, 77-92.

S7. Limpens, E., Mirabella, R., Fedorova, E., Franken, C., Franssen, H., Bisseling, T., and Geurts, R.

(2005). Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by

DMI2. Proc Natl Acad Sci U S A 102, 10375-10380.

S8. Karimi, M., Inze, D., and Depicker, A. (2002). GATEWAY vectors for Agrobacterium-mediated plant

transformation. Trends Plant Sci 7, 193-195.

S9. Devers, E.A., Teply, J., Reinert, A., Gaude, N., and Krajinski, F. (2013). An endogenous artificial

microRNA system for unraveling the function of root endosymbioses related genes in Medicago

truncatula. BMC Plant Biol 13, 82.

S10. Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C.,

Schumacher, K., Oecking, C., et al. (2004). Visualization of protein interactions in living plant cells

using bimolecular fluorescence complementation. Plant J 40, 428-438.

S11. Rech, S.S., Heidt, S., and Requena, N. (2013). A tandem Kunitz protease inhibitor (KPI106)-serine

carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago

truncatula. Plant J 75, 711-725.

S12. Wulf, A., Manthey, K., Doll, J., Perlick, A.M., Linke, B., Bekel, T., Meyer, F., Franken, P., Küster, H.,

and Krajinski, F. (2003). Transcriptional changes in response to arbuscular mycorrhiza development in

the model plant Medicago truncatula. Mol Plant Microbe Interact 16, 306-314.

S13. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient expression system

in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J

33, 949-956.

S14. Trouvelot, A., Kough, J., and Gianinazzi-Pearson, V. (1986). Estimation of VA mycorrhizal infection

levels. Research for methods having a functional significance. In Physiological and Genetic Aspects of

Mycorrhizae, G.-P. V. and S. Gianinazzi, eds. (Paris: INRA Press), pp. 217-221.

S15. Benedito, V.A., Torres-Jerez, I., Murray, J.D., Andriankaja, A., Allen, S., Kakar, K., Wandrey, M.,

Verdier, J., Zuber, H., Ott, T., et al. (2008). A gene expression atlas of the model legume Medicago

truncatula. Plant J 55, 504-513.

S16. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: Molecular

evolutionary genetics analysis version 6.0. Mol Biol Evol 30, 2725-2729.

S17. Huang, W., Xian, Z., Kang, X., Tang, N., and Li, Z. (2015). Genome-wide identification, phylogeny

and expression analysis of GRAS gene family in tomato. BMC Plant Biol 15, 209.

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