L. japonicus nfr5 , lhk1-1 nin-2 - PNAS€¦ · Materials and Methods Soil and plant material....

Materials and Methods

Soil and plant material.

Seeds of L. japonicus WT, ecotype Gifu B-129 and the corresponding symbiosis-deficient

mutants nfr5-2, nfr5-3, lhk1-1 and nin-2 were grown in soil batches collected from an

agricultural field located at the Max Planck Institute for Plant Breeding Research in Cologne,

Germany (50.958N, 6.865E) in the following seasons: CAS8-spring 2013, CAS9-fall 2013,

CAS10-spring 2014. The field was not cultivated in past years, no fertiliser or pesticide

treatment was administered at the harvesting site. For each genotype and soil batch, 3

biological replicas were obtained, and the samples were harvested from 10 weeks -old plants

grown in the greenhouse (day/night cycle 16/8h, light intensity 6000 LUX, temperature: 20

°C, relative humidity: 60%). Optional supplementation of the soil with potassium nitrate was

done by addition of KNO3 to a final concentration of 10 mM to the tap water used for plant

watering. For nitrogen supplementation experiment, plants were grown in CAS11 soil,

collected in fall 2015 from a location described before.

Sample and 16S library preparations.

Fragments of the root systems (4 cm-long starting 1 cm below hypocotyl) were washed and

ultrasound treated to separate the rhizosphere, root and nodule compartments. First wash

containing the root-adhering soil layer defined the rhizosphere compartment. Nodules and

visible primordia, were separated from washed root fragments of nodulating genotypes (WT

and lhk1-1) with a scalpel. Root samples were exposed to 10 cycles of 30” ultrasound

treatment, and the nodules to 3 cycles in order to avoid tissue out-burst. Homogenized

samples were transferred to lysis buffer and DNA extraction was performed following the

manufacturer’s protocol (MP Bioproducts). DNA concentrations were measured

fluorometrically (Quant-iT™ PicoGreen dsDNA assay kit, Life Technologies, Darmstadt,

Germany), and finally adjusted to 3,5 ng/μl. Primers targeting the variable V5-V7 regions of

bacterial 16S rRNA genes (799F and 1193R) (1, 2) were used for amplification. For each

sample, triplicate amplifications were performed using three independent PCR mixtures (9

replicates per sample in total, along with no template controls). Amplification products were

purified, combined and subjected to 454 sequencing.

Quantitative RT-PCR.

WT and mutant root samples were used for mRNA isolation using oligodT DYNA beads

following the protocol of the manufacturer (Invitrogen). The mRNA was subsequently used

as template for cDNA synthesis using an oligodT primer. The same cDNA pool was used for

amplification of all tested transcripts in each sample. Quantitative PCR was performed on

the LightCycler (Roche Molecular Biochemicals) using FastStart DNA master SYBR greenI kit

(Roche). The relative quantification software (Roche) was used to determine the efficiency-

corrected relative transcript concentration, normalized to a calibrator sample. ATP, UBC,

PP2A were used as housekeeping genes. For each genotype, normalized relative ratios of the

target genes and the three independent housekeeping genes have been calculated using the

Relative Quantification Software (Quant) from Roche. The geometric mean of the relative

expression ratios for the three biological and three technical replicates and the

corresponding 95% intervals of confidence have been calculated. Primers used for transcript

amplification are presented in Dataset S3.

Computational analyses.

The 16S rRNA gene sequences were processed using a combination of custom scripts as well

as tools from the QIIME (3) and USEARCH (4) pipelines (QIIIME-ready mapping files are

provided in the Dataset S4 and S5). First, reads were truncated to an even length (290 bp)

using the truncate_fasta_qual_files.py QIIME script. Libraries were demultiplexed

(split_libraries.py) and only reads with a quality score Q > 25 were retained for subsequent

analysis. After dereplication and removal of singletons we conducted de novo clustering of

sequences into OTUs using the UPARSE algorithm (5) at 97% identity. Next, chimeric

sequences were filtered using the UCHIME algorithm (6) implemented in the USEARCH

pipeline and the ‘gold’ database (http://drive5.com/uchime/gold.fa) as a reference. An

additional filtering step was performed by first aligning all OTU representative sequences to

the greengenes database (7) using PyNAST (8; align_seqs.py script in QIIME) and

subsequently discarding sequences not aligned to the database at least at 75% identity. OTU

representative sequences were classified taxonomically using the RDP classifier (9) and the

uclust algorithm (4) trained on the greengenes database. The resulting OTU table was used

in all subsequent statistical analyses of differentially abundant taxa as well analyses of alpha -

and beta-diversity.

Indices of α-diversity (Shannon, chao1 and number of observed OTUs) were calculated after

subsampling to an even depth of 1000 reads (QIIME script alpha_diversity.py). Measures of

β-diversity (Bray-Curtis and weighted and unweighted UniFrac; 10) were calculated on a

normalized OTU table (CSS method with QIIME script function beta_diversity.py). Principle

Coordinate Analysis (PCoA) was done by classical multidimensional scaling of β -diversity

distance matrices using the cmdscale function in R. Canonical Analysis of Principle

Coordinates (11; CAP) was computed using the capscale function implemented in the vegan

R library (12), by constraining for the variable of interest and conditioning for the remaining

http://drive5.com/uchime/gold.fa

factors. Statistical significance was determined using a permutation-based ANOVA test

implemented in anova.cca function using 5,000 permutations. Statistical analyses of

differentially abundant OTUs were performed using the edgeR library (13). Briefly, we first

obtained normalization factors using the function calcNormFactors and subsequently

estimated common and tag-wise dispersions for a Negative Binomial Generalized Linear

Model (GLM) using the estimateGLMCommonDisp and estimateGLMTagwiseDisp functions,

respectively. In order to test for differential OTU abundances, we then fit a negative

binomial generalized log-linear model to the read counts for OTU using the glmFit function.

P values were corrected for multiple tests using the approach of (14) with α=0.05.

Metabolite analysis

Root nitrate contents were determined by ion chromatography method as previously

described (15). Approximately 20 mg plant tissue was homogenized and extracted in 1 mL of

sterile water for 1 hour at 4°C. The extracts were heated for 15 min at 95°C and centrifuged

for 15 min at 13,000 rpm. Ten µL of the extracts were analysed on Dionex IonPac AS22 RFIC

4x250 mm analytical column (Thermo Scientific) using a carbonate buffer (4.5 mM Na2CO3,

1.4 mM NaHCO3) as eluent at 1 mL/min in an isocratic mode and a Dionex ICS-1100 ion

chromatography system. Proteins were extracted in 10 mM Tris/HCl buffer, pH 8 and

determined with a Bio-Rad Protein Assay Kit using bovine serum albumin as standard.

References

1. Bulgarelli D, et al. (2012) Revealing structure and assembly cues for Arabidopsis root-

inhabiting bacterial microbiota. Nature 488(7409):91-95.

2. Chelius MK, Triplett EW (2001) The Diversity of Archaea and bacteria in association

with the roots of Zea Mays. Microb. Ecol. 41:252-263

3. Caporaso JG, et al. (2010) PyNAST: a flexible tool for aligning sequences to a template

alignment. Bioinformatics 26(2):266-267.

4. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST.

Bioinformatics 26(19):2460-2461.

5. Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon

reads. Nature methods 10(10):996-998.

6. Edgar RC, Haas BJ, Clemente JC, Quince C, & Knight R (2011) UCHIME improves

sensitivity and speed of chimera detection. Bioinformatics 27(16):2194-2200.

7. DeSantis TZ, et al. (2006) Greengenes, a chimera-checked 16S rRNA gene database

and workbench compatible with ARB. Applied and environmental microbiology 72(7):5069-

5072.

8. Caporaso JG, et al. (2010) QIIME allows analysis of high-throughput community

sequencing data. Nature methods 7(5):335-336.

9. Wang Q, Garrity GM, Tiedje JM, & Cole JR (2007) Naive Bayesian classifier for rapid

assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microb

73(16):5261-5267.

10. Lozupone C, Lladser ME, Knights D, Stombaugh J, & Knight R (2011) UniFrac: an

effective distance metric for microbial community comparison. The ISME journal 5(2):169-

172.

11. Anderson MJ & Willis TJ (2003) Canonical analysis of principal coordinates: A useful

method of constrained ordination for ecology. Ecology 84(2):511-525.

12. Oksanen J, et al. (2015) R Package ‘vegan’ 2.3-3. Community Ecology Package.

13. Robinson MD, McCarthy DJ, & Smyth GK (2010) edgeR: a Bioconductor package for

differential expression analysis of digital gene expression data. Bioinformatics 26(1): 139-

140.

14. Benjamini Y & Hochberg Y (1995) Controlling the false discovery rate - a practical and

powerful approach to multiple testing. J Roy Stat Soc B Met 57(1):289-300.

15. Koprivova A, Harper AL, Trick M, Bancroft I, Kopriva S (2014) Dissection of control of

anion homeostasis by associative transcriptomics in Brassica napus. Plant Physiol,

166(1):442-450.

Fig. S1. Harvesting procedures applied for compartment separation of nodulating (a)and non-nodulating (b) genotypes of Lotus japonicus.

a

b

DNA

extra

ctio

nRhizosphere of Roots

Roots 16S

rRN

A C

omm

unity

Pro

flin

g

Isolation andWashing

Sonication

Isolation andWashing

Compartmentseparation DN

A ex

tract

ion

Rhizosphere of Rootsand Nodules

Pooled Nodules

Roots without Nodules

16S

rRN

A C

omm

unity

Pro

flin

g

Sonication

Fig. S2. Analisys of α-diversity (within sample diversity) based on the Shannon indexin the soil (n=27), rhizosphere (n=38), root (n=62) and nodule (n=18) compartments.Each dot corresponds to an individual sample, colored by compartment and each hostgenotype is represented by a different shape. Each sample was rarefied to an depth of1,000 reads before the analysis.

2

4

6

8

Soil Rhizosphere Root Nodule

Sh

an

no

n i

nd

ex

rhizosphereroot

soil

compartment

nodule

genotype

lhk1-1

soil

nfr5-3

nin-2

gifu (wild-type)

nfr5-2

−0.2

0.0

0.2

−0.4 −0.2 0.0 0.2PCoA 1 (26.62%)

PC

oA

2 (

11

.27

%)

genotype

nfr5-2

gifu (wild-type)

rhizosphereroot

soil

compartment

−0.2

0.0

0.2

0.4

−0.4 −0.2 0.0 0.2PCoA 1 (31.38%)

PC

oA

2 (

13

.67

%)

genotype

nfr5-2

gifu (wild-type)

rhizosphereroot

soil

compartment

−0.2

0.0

0.2

−0.50 −0.25 0.00 0.25PCoA 1 (31.64%)

PC

oA

2 (

9.7

99

%)

genotype

hit1-1

soil

nfr5-3

nin-2

gifu (wild-type)

rhizosphereroot

soil

compartment

CAS10

CAS9

CAS8

Fig. S3. PCoA plots of Bray-Curtis distances for samples from each soil batch(a, CAS8, n=63; b, CAS9, n=31 and c, CAS10, n=72). Each point corresponds to a differentsample, colored by compartment. Host genotype is represented by different shapes.

a

b

c

Fig. S4. Constrained PCoA plot of Bray-Curtis distances constrained by genotype after in silicodepletion of nodule-enriched OTUs (9.72% of variance explained, P<0.001; n=164). Eachpoint corresponds to a different sample, colored by compartment and each host genotypeis represented by a different shape. The percentage of variation indicated in each axiscorresponds to the fraction of the total variance explained by the projection.

−0.2

−0.1

0.0

0.1

0.2

−0.2 −0.1 0.0 0.1CPCoA 1 (83.52%)

CP

Co

A 2

(1

6.4

8%

)

genotype

lhk1-1

soil

nfr5-3

nin2

gifu (wild-type)

nfr5-2

rhizosphereroot

soil

compartment

9.72 % of variance (P<0.001)

Wild-type

Root Rhizosphere

Soil

nfr5−2

Root Rhizosphere

Soil

Fig. S5. Ternary plots depicting compartment relative abundance of all OTUs (>5 ‰) for wild-type samples (a, gifu) and mutant samples (b, nfr5-2) in CAS8 soil. Each point corresponds to an OTU and its position represents its relative abundance with respectto each compartment. Colored circles represent OTUs enriched in one compartmentcompared to the others (green in root, orange in rhizosphere and brown in root samples).

a b

Soil

Rhizosphere

Root

Enriched OTUs

Soil

Rhizosphere

Root

Enriched OTUs

Wild-type

Root Rhizosphere

Soil

nfr5−2

Root Rhizosphere

Soil

Fig. S6. Ternary plots depicting compartment relative abundance of all OTUs (>5 ‰) for wildtype samples (a, gifu) and mutant samples (b, nfr5-2) in CAS9 soil. Each point corresponds to an OTU and its position represents its relative abundance with respectto each compartment. Colored circles represent OTUs enriched in one compartmentcompared to the others (green in root, orange in rhizosphere and brown in root samples).

a b

Soil

Rhizosphere

Root

Enriched OTUs

Soil

Rhizosphere

Root

Enriched OTUs

nfr5−3Wild-type

Root Rhizosphere

Soil

Root Rhizosphere

Soil

Fig. S7. Ternary plots depicting compartment relative abundance of all OTUs (>5 ‰) for wildtype samples (a, gifu) and mutant samples (b, nfr5-3) in CAS10 soil. Each point corresponds to an OTU and its position represents its relative abundance with respectto each compartment. Colored circles represent OTUs enriched in one compartmentcompared to the others (green in root, orange in rhizosphere and brown in root samples).

a b

Soil

Rhizosphere

Root

Enriched OTUs

Soil

Rhizosphere

Root

Enriched OTUs

Root Rhizosphere

Soil

Root Rhizosphere

Soil

lhk1-1Wild-type

Fig. S8. Ternary plots depicting compartment relative abundance of all OTUs (> 5 ‰) for wildtype samples (a, gifu) and mutant samples (b, lhk1-1) in CAS10 soil. Each point corresponds to an OTU and its position represents its relative abundance with respectto each compartment. Colored circles represent OTUs enriched in one compartmentcompared to the others (green in root, orange in rhizosphere and brown in root samples).

a b

Soil

Rhizosphere

Root

Enriched OTUs

Soil

Rhizosphere

Root

Enriched OTUs

nin-2Wild-type

Root Rhizosphere

Soil

Root Rhizosphere

Soil

Fig. S9. Ternary plots depicting compartment relative abundance of all OTUs (> 5 ‰) for wildtype samples (a, gifu) and mutant samples (b, nin-2) in CAS10 soil. Each point corresponds to an OTU and its position represents its relative abundance with respectto each compartment. Colored circles represent OTUs enriched in one compartmentcompared to the others (green in root, orange in rhizosphere and brown in root samples).

a b

Soil

Rhizosphere

Root

Enriched OTUs

Soil

Rhizosphere

Root

Enriched OTUs

1 2 3

a b c a b c a b c

1 2 3

a b c a b c a b c

1 2 3

a b c a b c a b c

1 2 3

a b c a b c a b c

Wild-type nfr5-3 nin-2 lhk1-1O

TU

s en

rich

ed in

gifu

ro

ots

OT

Us

enric

hed

in m

utan

ts r

oots

[0,0.01)

[0.01,0.05)

[0.05,0.1)

[0.1,0.5)

[0.5,1)

[1,2)

[2,4)

[6,8)

[8,100)

R.A (%)

Fig. S10. (a) Heatmaps showing relative abundances of all OTUs found to be significantlyenriched in the root samples of wild-type plants grown in CAS10 soil with respect to themutants (top panel) or significantly enriched in the root samples of the mutants comparedto wild-type (bottom panel). (b) Heatmaps showing relative abundances of all OTUs foundto be significantly enriched in the rhizosphere samples of wild-type plants grown in CAS10soil with respect to the symbiosis- impaired mutants (top panel) or significantly enriched inthe rhizosphere samples of the mutants compared to wild-type (bottom panel). Labels at thebottom of each column correspond to biological (numbers) and technical replicates (letters).Enrichment tests were performed using the negative binomial generalized log-linear modeland P-values were FDR adjusted.

1 2 3

a b c a b c a b c

1 2

a b c a b c

1 2 3

a b c a b c a b c

1 2 3

a b a a

Wild-type nfr5-3 nin-2 lhk1-1

OT

Us

enric

hed

in g

ifu r

hiz

osph

ere

mut

ants

[0,0.01)

[0.01,0.05)

[0.05,0.1)

[0.1,0.5)

[0.5,1)

[1,2)

[2,4)

[6,8)

[8,100)

R.A (%)

a

b

0

20

40

60

−log10(P)

wild

-typ

e ro

ots

a

0

20

40

60

−log10(P)

mut

ant r

oots

b

0

20

40

60

−log10(P)

wild

-typ

e rh

izos

phe

re

c

0

20

40

60

−log10(P)

mut

ant r

hizo

sphe

re

d

0.001 0.04 0.08 0.12 0.16

relative abundance (%)significantly enriched n.s.

Gp1

ThermotogalesNeisseriales

ChlamydialesGemmatimonadales

Bacillales

XanthomonadalesPseudomonadales

MyxococcalesBurkholderiales

Sphingomonadales

Rhizobiales

Nitrospirales

Actinobacteridae

LegionellalesSphingobacterialesFlavobacteriales

Gp10Thiotrichales

OTUs enriched in wild-type roots with respect to mutant roots

FlavobacterialesXanthomonadalesBurkholderialesActinobacteridae Sphingobacteriales

FlavobacterialesCytophagales

OTUs enriched in mutant roots with respect to wilt-type roots

ThermotogalesMyxococcales

RhodocyclalesThermoleophilalesRubrobacteridae

Gp1Thermotogales

Gemmatimonadales XanthomonadalesPseudomonadales

BurkholderialesRhizobialesNitrospirales

Actinobacteridae FlavobacterialesGp22

Methylophilales / Neisseriales

OTUs enriched in wild-type rhizosphere with respect to mutant rhizosphere

SphingomonadalesSphingobacteriales

BurkholderialesActinobacteridae

OTUs enriched in mutant rhizospherewith respect to wilt-type rhizosphere

Fig. S11. Manhattan plots showing OTUs enriched in wild-type root compared to mutant rootsamples (a), depleted in the wild-type with respect to the mutants (b), enriched in the wild-typerhizosphere compared with respect to the mutants (c) or depleted in gifu rhizosphere withrespect to the mutant rhizosphere (d). Significantly enriched OTUs are depicted as full circles.The dashed line corresponds to the FDR corrected P-value threshold of significance (α=0.05).The color of each dot represents the different taxonomic affiliation of the OTUs (order level)and their sizes to their relative abundances in their respective samples (a, wild-type rootsamples; b, mutant root samples). Grey boxes are used to denote the different taxonomicgroups (order level).

Fig. S12. Relative abundances aggregated to the phylum taxonomic level showing acomparison between wild-type and the mutant root (a; n=74) and rhizosphere (b; n=63)samples.

Root

Rhizosphere

a

b

Mutant

Wild-type

Mutant

Wild-type

●●

● ●● ●● ●●

●

● ●

●●

Other

Firmicutes

Bacteroidetes

Actinobacteria

Proteobacteria

0% 20% 40% 60% 80%relative abundance

●●●

●●

Other

Firmicutes

Bacteroidetes

Actinobacteria

Proteobacteria

0% 20% 40% 60% 80%relative abundance

Fig. S13. Soil-grown wild-type and symbiotic mutants differ in the expression of earlysymbiotic genes, but show comparable immune- and symbiosis-related metabolic responses.Relative transcript levels of (a) immune-response genes (LjPR1b, LjErf1, LjMyc2 LjNpr1,Ljwrky70, LjJar1, LjIcs1, LjCoi1), (b) symbiosis-related metabolic responses (LjInv1, LjNod26,LjNod70, LjSut4) and (c) early symbiotic genes (LjNin, LjPeroxidase, LjThaumatin, LjPR1a).

a

b

cLj2g3v3373100.1_LjNin Lj0g3v0207139.1_LjThaumatin LjSGA_029184_LjPR1a

Lj6g3v2170750.1_LjPR1b

chr6.CM0314.280.nc_LjPeroxidase

Lj5g3v1167370.1_LjERF1

0

1

2

3

4

5

6

WT lhk1-1 nin2 nfr5-2

Lj0g3v0343449.1_LjMYC2

0

1

2

3

4

5


Lj6g3v1692500.1_LjNPR1

0

1

2

3

4


Lj1g3v1134120.1_LjWRKY70

0

0,5

1

1,5

2


Lj1g3v5033420.1_LjJAR1

0

0,1

0,2

0,3


Lj3g3v1970100_LjICS1

0

0,5

1

1,5


Lj0g3v0011599.1_LjCOI1

0

0,2

0,4

0,6

0,8

1

1,2


Lj5g3v1853130.1_LjINV1

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6


Lj5g3v0878400.1_LjNod26

0

0,005

0,01

0,015

0,02

0,025

0,03


Lj0g3v0003829.1_LjNod70

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6


Lj1g3v2096190.1_LjSut4

0

0,05

0,1

0,15

0,2

0,25

0,3


1

2

3


0

0,05

0,1

0,15

0,2

0,25


0

1

2

3

4

5


1

2

3

4

5

6

7

8

9

10


5

10

15

20

25

30

35

40

45

50


Fig. S14. Roots of soil-grown wild-type and symbiotic mutants have the same proteinconcentration but differ in nitrate pool. Concentration of (a) soluble proteins and (b) nitratein roots across Lotus genotypes. Bars correspond to the standard deviation between samples.

nmol

/ m

g F

W

μg

/ mg

FW

wild-ty

pe

(n=6

) lhk1-

1

(n=1

0)nfr5

-2

(n=8

)nfr5

-3

(n=1

0) nin2

(n=1

0)

wild-ty

pe

(n=3

) lhk1-

1

(n=5

)nfr5

-2

(n=4

)nfr5

-3

(n=5

) nin2

(n=5

)

Fig. S15. (a) Images depicting L. japonicus wild-type (a) and root nodule symbiosis-deficientmutant plants lhk1-1 (b) nfr5-2 (c) and nfr5-3 (d) and nin2 (e) grown in natural soil. The lowerpanels display an identical set of genotypes, wild-type (f), hit1-1 (g), nfr5-2 (h), nfr5-3 (i) andnin2 (j), grown in natural soil supplemented with 10 mM potassium nitrate. Scale barscorrespond to 1cm. (k) differences in nitrate concentrations in the roots of wild-type and Lotusmutants under both conditions. Differences in fresh weight of wild-type Lotus and mutantplants grown in natural soil (l) and in natural soil supplemented with potassium nitrate (m).

k l m

0

5

10

15

20

25

30

35

+ 0 mM KNO3 + 10 mM KNO3

nm

ol/m

g f

resh

mass

gifu nfr5-3nfr5-2 nin2 lhk1-10.00

0.25

0.50

0.75

1.00

1.25

fre

sh

we

igh

t

wild-type

mutant

+ 0 mM KNO3

q

q

q

0.00

0.25

0.50

0.75

1.00

1.25

fre

sh

we

igh

t

+ 10mM KNO3

wild-type

mutant

gifu nfr5-3nfr5-2 nin2lhk1-1 gifu nfr5-3nfr5-2 nin2lhk1-1

Fig. S16. (a) Constrained PCoA plot of Bray-Curtis distances between samples from KNO3treated CAS11 soil constrained by genotype (21.2% of variance, P>0.001; n=61).(b) Contrained PCoA plot of Bray-Curtis distances between CAS11 untreated samplesconstrained by genotype (21.8% of variance explained, P<0.001; n=58). Each pointcorresponds to a different sample, colored by compartment and each host genotype isrepresented by a different shape. The percentage of variation indicated in each axiscorresponds to the fraction of the total variance explained by the projection.

a

b KNO3-

PCoA constrained by genotype

21.8 % of variance (P<0.001)

KNO3+

PCoA constrained by genotype

21.2 % of variance (P<0.001)

−0.3

−0.2

−0.1

0.0

0.1

0.2

−0.1 0.0 0.1 0.2 0.3CPCoA 1 (86.09%)

CP

Co

A2

(13

.91

%)

−0.3

−0.2

−0.1

0.0

0.1

0.2

−0.1 0.0 0.1 0.2 0.3CPCoA 1 (88.52%)

CP

Co

A2

(11

.48

%)

genotype

soil

nfr5-3

gifu (wild-type)

nfr5-2

genotype

soil

nfr5-3

gifu (wild-type)

nfr5-2

rhizosphereroot

soil

compartment

rhizosphereroot

soil

compartment

L. japonicus nfr5 , lhk1-1 nin-2 - PNAS€¦ · Materials and Methods Soil and plant material....

Documents

Transcript of L. japonicus nfr5 , lhk1-1 nin-2 - PNAS€¦ · Materials and Methods Soil and plant material....