Methods
RNA isolation
For RNA isolation, total RNA was extracted by using an RNeasy Plant Mini Kit
(QIAGEN Cat#74903, Germany) following the manufacturer’s instructions. Samples of 2
g total RNA isolated from roots and leaves were reverse-transcribed in a 25-l reaction
using
MMLV-RT
(Promega
Cat#M1701,
Madison,
WI,
USA)
following
the
manufacturer’s instructions. For PCR amplification, 1 l of RT reaction was used. The
PCR reactions were carried out in 25 l with 0.5 M of each primer.
Isolation of cDNA encoding the MsDMI3 gene, phylogenetic analysis and plasmid
construction
Full-length cDNA of the MsDMI3 gene was amplified by PCR (5´ATGGGATATGGAACAAGAAAACTCTC-3´
and
5´-
TTATGGACGAATAGAAGAGAGAACTAC-3´). This fragment was cloned into a
pGEM-t easy vector. The sequencing reactions were performed by Macrogen (Korea).
Phylogenetic analysis of DMI3 protein was conducted by using MEGA 4.0
(www.megasoftware.net). This analysis was constructed using the neighbor-joining (NJ)
method with genetic distances computed using Poisson correction model. MsDMI3 with a
truncated C-t autoinhibitory domain, named MsDMI3/1-340, was amplified by PCR (5´CCTCTAGAATGGGATATGGAACAAGAAAACTCTC-3´
and
5´-
CCGAGCTCTTAAACACTAGCAATTGCAGCTGC-3´, where XbaI and SacI restriction
sites are underlined) and this amplification fragment was digested with XbaI and SacI, and
cloned into pBI121 (AF485783). The resulting plasmid, named 35S-MsDMI3/1-340 (Fig.
S3), was sequenced by primer walking.
Plant transformation
Medicago sativa (Alfalfa Forage Genetics 969) seeds were surface-sterilized with
concentrated sulfuric acid (H2SO4) for 10 min and rinsed with sterile water. The seeds were
germinated for 72 h on semi-solid agar medium (7 g/l) in growth chamber at 25°C. For
production of spontaneous nodules in alfalfa, non-transformed and non-infected plants were
grown in 25 ml MS medium without nitrogen in glass flasks under axenic condition. For
construction of DMI3 induced-nodules in alfalfa, the vector 35S-MsDMI3/1-340 was
transformed into alfalfa through the mediation of Agrobacterium rhizogenes MSU440. To
obtain rhizobium-infected alfalfa plants, seeds were inoculated with a Sinorhizobium
meliloti B399 suspension (≈109 CFU/ml) from a stationary-phase culture. Agrobacterim
rhizogenes-transformed and non-transformed alfalfa plants infected with rhizobial strains
were respectively transferred to 25 ml MS medium supplemented or not with 300 µg ml -1
cefotaxime in glass flasks under axenic conditions. Plants were incubated for 21 days at
23ºC under a 10/14 h (day/night) photoperiod and 100 µE m-2 s-1 of light intensity provided
by fluorescent lamps. All nodules were checked by PCR analysis. Four sets of primer pairs,
virCfor (5´-ATCATTTGTAGCGACT-3´) and virCrev (5´-AGCTCAAACCTGCTTC-3´),
nifHfor
(5´-GGCTACAAAGGCATCAAATGCGTGGA-3´)
TACTGGATCACCGTCATCTTCCTGAG-3´),
and
AATfor
nifHrev
(5´(5´-
CAATTTCGCATCTCATTAAGATCG-3´)
and
ACCACATCCCAAATAAATAAGATTCTAAC-3´),
GATGCCTCTGCCGACAGTGGTCCC-3´)
and
AATrev
and
35SMsDMI3/1-340for
35SMsDMI3/1-340rev
(5´(5´(5´-
TTAAACACTAGCAATTGCAGCTGC-3´) were evaluated for virC from Agrobacterium,
nifH from Sinorhizobium, aspartate aminotransferase from alfalfa and 35SMsDMI3/1-340
vector detection by PCR.
Gel electrophoresis of PCR products from transformed (MsDMI3-induced nodules)
and non-transformed (spontaneous and nitrogen-fixing nodules) alfalfa as well as from
Agrobacterium and Sinorhizobium strains was carried out to show the presence or absence
of 35S-MsDMI3/1-340, the virC gene and the nifH gene (Fig. S4). The virC gene was
present in Agrobacterium Ti and Ri plasmids, whereas the nifH gene was found in
Sinorhizobium. Therefore, the amplification of the virC and nifH genes indicates the
presence of Agrobacterium and Sinorhizobium strains, respectively. 35S-MsDMI3/1-340
PCR products were present in transgenic root lines but not in non-transformed root
cultures, whereas a PCR product corresponding to a fragment of the virC gene was absent
in all roots. In addition, the nifH gene was detected only in alfalfa roots inoculated with
Sinorhizobium. Thus, spontaneous and DMI3-induced nodules were not contaminated with
Agrobacterium and/or Sinorhizobium strains and nodules in transgenic alfalfa roots were
actually transformed with 35S-MsDMI3/1-340.
Moreover, when surface-sterilized spontaneous and DMI3-induced nodules were
crushed and their contents plated in LB medium, no viable bacteria were recovered.
Additionally, the contents of nonsterilized nodules were unable to induce nodulation when
used to inoculate axenic alfalfa roots. These results suggest that spontaneous and DMI3induced nodules were not associated by bacteria. To confirm the absence of rhizobium in
spontaneous nodules and nodules induced by MsDMI3/1-340 overexpression, spontaneous
and DMI3-induced nodules were excised from roots, surface-sterilized with mercuric
chloride, crushed individually in Hoagland solution and plated in LB medium. In five
independent experiments, ten nodules were crushed individually without prior surface
sterilization in 250 μl of Hoagland solution and the suspensions were used to inoculate 7day-old alfalfa plants, in groups of five, grown in growth pouches. In both cases,
Sinorhizobium meliloti B399-induced nodules were used as positive control.
ATP, ADP, leghaemoglobin and oxygen content determinations
ATP and ADP were measured with a cycling assay according to Gibon and coworkers procedure with slight modifications. For ATP quantification, aliquots of extracts (5
ml) or standards (0±50 pmol) were distributed directly into a microplate, and 95 ml 100
mM buffer containing 2 units glycerol-3-phosphate oxidase, 130 units catalase, 0.4 unit
glycerol-3-phosphate oxidase, 0.05 unit TPI, 0.12 mmol NADH and 1 mmol 3phosphoglycerate or 0.5 mmol ATP, absorbance followed for 20 min, 0.05 unit of 3phosphoglycerate kinase and 0.05 unit of glyceraldehyde- 3P dehydrogenase added (both in
1 ml 100 mM Tricine buffer), and absorbance monitored for another 21 min. In order to
determine the concentration of ADP, 60 ml Tricine buffer containing 0.1 unit pyruvate
phosphate dikinase, 0.05 unit pyrophosphatase, 1 mmol inorganic phosphate and 2 mmol
pyruvate was added to 20 ml aliquots of extracts or ADP standard (0±50 pmol) in 2 ml
microtubes, incubated 60 min at room temperature, heated (5 min, 90°C), cooled,
centrifuged, the supernatants transferred to a microplate, 20 ml of 100 mM buffer
containing 0.05 units glycerokinase, 2 units glycerol-3-phosphate oxidase, 0.4 unit
glycerol-3-phosphate dehydrogenase, 130 units catalase, 0.12 mmol NADH, 0.25 mmol
glycerol and 50 pmol ADP added, absorbance read for 20 min, 1 unit myokinase (in 1 ml
100 mM Tricine buffer) added, and absorbance monitored for a further 20 min.
Concentrations of NADPH and NADP+ were determined using the Dalton method. Nodules
were crusher with liquid N2. The liquid N2 was allowed to boil off and 40 mg of the frozen
powder was added to 1 ml of either 5% trichloroacetic acid. The extract was placed in a
boiling water bath for 3 min, cooled to 4°C and then centrifuged at 12000 g for 2 min. 20 μl
of the supernatant was used in an enzymatic cycling assay based on alcohol dehydrogenase.
The leghemoglobin (Lb) levels in nodule soluble extracts were quantified by the
spectrophotometry method. Quantifications of leghaemoglobin in envelope extracts were
prepare from reduced (Na2S204) + CO minus reduced difference spectra, using standards
of purified mixed leghaemoglobin. Free-oxygen was quantified using a needle-type fiberoptic oxygen microsensor with a tip diameter of 50 μM. Oxygen levels of the central zone
of the nodules are expressed as a percentage of the concentration in air.
Nucleotide sequence accession number
The nucleotide sequences of MsDMI3 gene obtained here have been deposited in
the EMBL Nucleotide Sequence Database Accession No.: GQ890699.