Appendix S1. Experimental procedures for the identification of Tnt1 insertion mutants, the
heterologous expression assay and the Affymetrix GeneChip hybridizations.
Affymetrix GeneChip hybridization
A total of 2.5µg of genomic DNA per sample was randomly biotin-labelled with the BioPrime DNA
Labelling System (Invitrogen) using the manufacturers protocol with the following modification. The
reaction time was completed in four hours and the Klenow enzyme was increased to 2µL per 500ng
gDNA. Purification of the biotin-labelled products was performed utilizing the PureLink PCR
Purification Kit (Invitrogen) following the manufacturers protocol. The purified biotin-labelled
products were sized (~120bp) using the DNA 12000 chip run on the 2100 Bioanalyzer (Agilent
Technologies) and quantification of the purified products was performed using the ND-1000
(Nanodrop Technologies). 15µg of biotin labelled DNA was hybridized to the Affymetrix Medicago
GeneChip following the manufacturer’s standard protocol for RNA analysis. Following hybridization,
the chips were stained, washed, and scanned using the standard Affymetrix RNA analysis protocol.
Microarray hybridizations using RNA as a starting material were carried out as previously described
(Benedito et al. 2008). The plants used for the FNB4 RNA based microarray (Figure 4a) was grown in
turface media for one week then inoculated with S. meliloti rhizobia and harvested after 24 h. The
microarray data in Figure 4 is part of a larger work that will be described in detail elsewhere. Briefly
mutants (dmi1-2, dmi3-1, nfp-1, nsp1-1, nsp2-2, ern1-1, nin-1) were grown on solid BNM media
(Ehrhardt et al. 1992) with 0.1 µM aminoethoxyvinylglycine (AVG) with and then transferred to 0.1
nM NF in buffer or just buffer for 6 hours at which point RNA was harvested after removal of the root
tip and the top part of the root.
Reverse screening of Tnt1 mutant DNA pools
Approximately 9,000 tobacco retrotransposon Tnt1 insertion lines were available at The Samuel
Roberts Noble Foundation (Tadege et al. 2008). Individual genomic DNA isolated from each
insertion line was pooled into 4 levels of genomic DNA pools, which consist of 18 super-pools, 90
pools, 900 mini-pools and 9,000 individual lines. Two forward primers, Tnt1-F (5’-
ACAGTGCTACCTCCTCTGGATG-3’) and Tnt1-F1 (5’TCCTTGTTGGATTGGTAGCCAACTTTGTTG-3’), and two reverse primers, Tnt1-R (5’CAGTGAACGAGCAGAACCTGTG-3’) and Tnt1-R1 (5’TGTAGCACCGAGATACGGTAATTAACAAGA-3’), were designed from both ends of Tnt1. Two
pairs of gene-specific nested primers designed from VPY coding DNA sequence VIPER-F1 (5'tcttcaccaaaccccttcac-3') and VIPER-R3 5'-caaatcacacttgcaactcca-3'); VIPER-F2 (5'ttccagcaacaaaaatcttcc-3'), and VIPER-R2 (5'-tcaaatattccaaatcacacttgc-3') were designed. Four primer
combinations of Tnt1-F, Tnt1-R, VIPER-F1 and VIPER-R3 are used for first round PCR with 18
super-pool DNA as templates. The 2nd-round PCR are carried out using the nested primer
combinations with 1:50 dilutions of the 1st-round PCR reactions as templates. At this point the
identity of the PCR products were confirmed by sequencing and the PCR screening continues in this
fashion using the specific primer pairs in pools, mini-pools and finally on DNA from the individual
lines.
References:
Benedito, V.A., Torres-Jerez, I., Murray, J.D., Andriankaja, A., Allen, S., Kakar, K., Wandrey, M.,
Verdier, J., Zuber, H., Ott, T., Moreau, S., Niebel, A., Frickey, T., Weiller, G., He, J., Dai, X.,
Zhao, P.X., Tang, Y. and Udvardi, M.K. (2008) A gene expression atlas of the model legume
Medicago truncatula. Plant J, 55, 504-513.
Ehrhardt, D.W., Atkinson, E.M. and Long, S.R. (1992) Depolarization of alfalfa root hair membrane
potential by Rhizobium meliloti Nod factors. Science, 256, 998-1000.
Tadege, M., Wen, J., He, J., Tu, H., Kwak, Y., Eschstruth, A., Cayrel, A., Endre, G., Zhao, P.X.,
Chabaud, M., Ratet, P. and Mysore, K.S. (2008) Large-scale insertional mutagenesis using
the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J, 54, 335-347.