Supporting Information Methods S1
Supporting information about production of transgenic plants overexpressing NFP,
microarray experiments and chitin oligomers preparation.
Production of transgenic plants overexpressing NFP
The coding sequence of NFP was cloned in frame with a C-terminal 3xFLAG epitope
tag and in between the constitutive CaMV 35S promoter and terminator, in the pBin+ vector.
The plasmid was introduced into Agrobacterium tumefaciens strain AGL1, which was used to
produce transgenic plants by the leaf disc method (1). Transgenic plants were selected on
kanamycin and tested for expression of NFP by immunoblot analysis of root extracts (2),
using anti-FLAG antibodies (Sigma). Seeds from the F3 generation of a homozygous line
(M11/D6/8), expressing the NFP-3xFLAG protein, were used for the experiments described
in Fig. 2.
AFFYMETRIX Array hybridization
Three independent biological replicates were performed for WT and nfp-2
lines, non-inoculated or inoculated 1 dpi. For each biological repetition RNA samples were
extracted from roots of ten 15 d-old plants inoculated or not. Following extraction RNA
samples were checked for their integrity on The Agilent 2100 bioanalyzer according to the
Agilent Technologies (Waldbroon, Germany). One μg of total RNA was used to synthesize
biotin-labeled cRNAs with the One-cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA).
Superscript II reverse transcriptase and T7-oligo (dT) primers were used to synthesize the
single strand of cDNA at 42°C during 1 h followed by the synthesis of the double stranded
cDNA by using DNA ligase, DNA polymerase I and RNaseH during 2 h at 16°C. Clean up of
the double-stranded cDNA was performed with Sample Cleanup Module (Affymetrix)
followed by in vitro transcription (IVT) in presence of biotin-labeled UTP using GeneChip®
IVT labelling Kit (Affymetrix, Santa Clara, CA, USA). Quantity of the labelled-cRNA with
RiboGreen® RNA Quantification Reagent (Turner Biosystems, Sunnyvale, CA, USA) was
determined after cleanup by the Sample Cleanup Module (Affymetrix). Fragmentation of 15
μg of labelled-cRNA was carried out for 35 min at 94°C, followed by hybridization during 16
h at 45°C to Affymetrix GeneChip® Medicago Genome Array representing c. 61,200 probe
sets: 32,167 M. truncatula EST/mRNA-based probe sets; 18,733 M. truncatula IMGAG and
phase 2/3 BAC prediction-based probe sets; 1,896 M. sativa EST/mRNAbased probe sets; and
8,305 S. meliloti gene prediction-based probe sets. After hybridization, the arrays were
washed with 2 different buffers (stringent: 6X SSPE, 0.01% Tween-20 and non-stringent: 100
mM MES, 0.1M [Na+], 0.01% Tween-20) and stained with a complex solution including
Streptavidin R-Phycoerythrin conjugate (Invitrogen/molecular probes, Carlsbad, CA, USA)
and anti-Streptavidin biotinylated antibody (Vectors laboratories, Burlingame, CA, USA).
The washing and staining steps were performed in a GeneChip® Fluidics Station 450
(Affymetrix). The Affymetrix GeneChip® Medicago Genome Arrays were finally scanned
with the GeneChip® Scanner 3000 7G piloted by the Command Console Launcher Tool. The
raw CEL files were imported in R software for data analysis.The data were normalized with
the gcrma algorithm, available in the Bioconductor package (3). To determine differentially
expressed genes, we performed a usual two group t-test that assumes equal variance between
groups. The variance of the gene expression per group is a homoscedastic variance, where
genes displaying extreme variance (too small or too large) were excluded. The raw p-values
were adjusted by the Bonferroni method, which controls the Family Wise Error Rate (FWER)
(4). Genes with a P-value lower than a Bonferroni-corrected P-value threshold of 9.82318E07 is declared differentially expressed.
Chitin oligomers preparation
Preparation of a chitin oligomers elicitor was adapted from(5): crab shell chitin (Sigma
C3387) was finely ground, deproteinized by incubation in 1 M NaOH at 20°C for 1 h, washed
in distilled water, and lyophilized. Partial hydrolysis was performed by incubation in 12 N
HCl at 40°C for 100 min. After neutralization with KOH, the hydrolysate was dialyzed
against distilled water at 1 kDa mol weight cut-off, lyophilized, and then analyzed on a
BioGel P4 column (24x250 mm) in 100 mM NH4HCO2, pH 6.8. The column was calibrated
with 1.1, 5 and 50 kDa dextran standards. Fractions corresponding to apparent molecular
weights between 2,000 and 3,000 Da, and between 1,000 and 2,000 Da, were separately
collected, resulting in two pools, which were lyophilized and solubilized in UHQ water.
MALDI-TOF mass spectrometry analysis of the two samples, using 2,5-dihydroxybenzoic
acid as the matrix in positive ion mode, detected the presence of chitoheptamer only in the
pool of higher molecular weight, which was retained for plant elicitation.
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