Supporting Information Methods S1
Construction of the cDNA libraries
cDNA library CACE/Sanger sequencing. ERM was grown as specified by Douds et al.
(2002). Approximately 35 very active split plate G. intraradices-Daucus carota cultures
were maintained with liquid M media (without sucrose) on the fungal side of the split
plate. ERM with some new spore tissues was harvested two times per plate to yield
several aliquots of ~7-13 mg. RNA was isolated from 10 mg of tissue using the Qiagen
RNeasy Plant mini kit and concentrated to >50 ng/uL using a Qiagen mini-elute clean
up kit. RNA quality was determined by electrophoresis on an Agilent Bioanalyzer. The
SMART library construction Kit (Clontech) was used for cDNA synthesis from 90 ng
of total RNA followed by directional cloning in SfiI-digested Triplex2 vector. Inserts
were size-selected with Chromaspin 1000 columns (Clontech). Phages were converted
to plasmid subclones (pTriplEx) by cre-lox excision in E. coli strain BM25.8. cDNAs
were sequenced from the 5' end with the 5'-Triplex sequencing primer (Tpx) and the 3'
end with the T7 primer (T7).
cDNA library CCHU/Sanger sequencing. Sterile spores collected from G. intraradices
in vitro Daucus carota cultures were germinated on water-agar medium for three days.
Total RNA was extracted using Trizol reagent, and precipitated in EtOH. RNA quality
was determined by electrophoresis on an Agilent Bioanalyzer. The library was made
from oligo(dT)-primed cDNA and cloned into vector pDNR-LIB (Clontech) using the
SMART library construction Kit (Clontech). To generate the first strand, polyA RNA
was primed with an oligo(dT) primer (5'-ATT CTA GAG GCC GAG GCG GCC GAC
ATG (T)30VN-3', where V = A, G, or C). The second strand was synthesized by PCR
using primer 5' AAG CAG TGG TAT CAA CGC AGA GTG GCC ATT ACG GCC
GGG-3' followed by digestion of the SfiI sites. cDNA was size selected from 2 to 8kb
using 1.1% agarose gel electrophoresis, then ligated into pDNR-L and used for Sanger
sequencing.
cDNA library EXTA/454 sequencing. Aliquots of about 500,000 sterile G. intraradices
spores were either germinated in 50 ml water containing 225 nM apigenin for 7 days at
25°C, 2% CO2 or in 20 ml water containing 10-8 M of GR24 (a synthetic strigolactone)
at 30°C, 2% CO2 for 6 days. Germinated spores were harvested in liquid nitrogen. Total
RNA was extracted from apigenin-induced spores using SV Total RNA Isolation Kit
(Promega) and from GR24-induced spores using RNeasy Plant Mini Kit (Qiagen).
PolyA-RNA was prepared from total RNA either by using Oligotex Kit (Qiagen) or
Invitrogen’s Dynabeads sytem. cDNA was synthesized from both samples using the
SMART library construction Kit (Clontech) and purified using CHROMA-Spin-200DEPC columns (Clontech) before pooling of the two samples (1:1) for 454
pyrosequencing.
cDNA library EXTB/454 sequencing. ERM was grown from G. intraradices-Daucus
carota cultures as specified by Douds et al. (2002). Approximately 35 very active split
plate cultures were maintained with liquid M medium (without sucrose) on the fungal
side of the split plate. ERM with some new spore tissues was harvested two times per
plate to yield several aliquots of ~7-13 mg tissue. RNA was isolated from 10 mg of
tissue using the Qiagen RNeasy Plant Mini kit and concentrated to >50 ng/uL using a
Qiagen mini-elute clean up kit. RNA quality was determined by electrophoresis using
an Agilent Bioanalyzer. 90 ng of RNA was converted to cDNA using a SMART PCR
cDNA synthesis kit (Clontech) and the "non-library" options. cDNAs were amplified
according to the manufacturer’s recommendations and used for 454 pyrosequencing.
cDNA library AKNA/454 sequencing/oligoarray. M. truncatula (cv. A17) plants (two
trifoliate leaf stage) were grown in pots with 10 plants per pot and inoculated with 1,000
G. intraradices spores per plant. The experiment included three pots (three biological
replicates) of mock-inoculated plants and three pots of G. intraradices-inoculated
plants. Plants were fertilized with 100 mL/pot of Hoagland solutions containing full
strength nitrogen and low Pi (20 µM), twice weekly. Roots were harvested at 27 days
post inoculation and frozen in liquid nitrogen. Total RNA was extracted from
mycorrhizal roots with Trizol reagent (Invitrogen Corporation, Carlsbad, CA) with
additional phenol (pH 6.6): chloroform (1:1, v/v) and chloroform purification steps.
RNA from mycorrhizal roots was treated with Turbo DNase I (Ambion Inc., Austin,
TX), and column purified with an RNeasy MinElute CleanUp kit (Qiagen). RNA
quality and integrity were checked prior to cDNA synthesis using the Bio-Rad Experion
analyzer and Experion RNA StdSens analysis kit. Total RNA preparations (three
biological replicates) were amplified using the SMART PCR cDNA Synthesis Kit
(Clontech) according to the manufacturer’s instructions. cDNAs were pooled after
purification using QIAquick PCR purification Kit (Qiagen) and used for 454
pyrosequencing. These three cDNA samples were used in the oligoarray transcript
profiling.
cDNA library AKNB/454 sequencing. Aliquots of about 100,000 sterile G. intraradices
spores from in vitro culture were activated in 6ml of M medium containing 1.5.10-6 M
apigenin for 24h at 30°C, 2% CO2. Spores were immediately frozen in liquid nitrogen.
Total RNA was extracted using a RNeasy Plant Mini Kit (Qiagen) with RLT lysis
buffer. RNA quality and integrity were checked prior to cDNA synthesis using the BioRad Experion analyzer and Experion RNA StdSens analysis kit. Total RNA
preparations (three biological replicates) were amplified using the SMART PCR cDNA
Synthesis Kit (Clontech) according to the manufacturer’s instructions. cDNAs were
pooled after purification using QIAquick PCR purification Kit (Qiagen) and used for
454 pyrosequencing.
cDNA library AKNC/454 sequencing. Aliquots of about 100,000 sterile G. intraradices
spores from in vitro culture were activated in 6 ml of M medium containing 10-8 M
GR24, a synthetic strigolactone, for 24h at 30°C, 2% CO2. Spores were immediately
frozen in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit
(Qiagen) with RLT lysis buffer. RNA quality and integrity were checked prior to cDNA
synthesis using the Bio-Rad Experion analyzer and Experion RNA StdSens analysis kit.
Total RNA preparations (three biological replicates) were amplified using the SMART
PCR cDNA Synthesis Kit (Clontech) according to the manufacturer’s instructions.
cDNAs were pooled after purification using QIAquick PCR purification Kit (Qiagen)
and used for 454 pyrosequencing. The same three replicates of activated spores were
used for oligoarray analysis.
cDNA library AKND/454 sequencing. For LMD, M. truncatula seedlings were
inoculated with G. intraradices using the Millipore sandwich method described by
Guether et al. (2009a). After about two months, mycorrhizal roots were dissected into 5
mm pieces and placed in RNase-free tubes containing freshly prepared Farmer's fixative
(absolute ethanol/glacial acetic acid 3:1 v/v). After a step under vacuum at room
temperature for 20 min, fixative solution was changed and root samples were incubated
overnight at 4°C. Roots were then dehydrated in a graded series of ethanol (70, 90, 100
and 100 % in sterilized water) followed by two steps in Neoclear (Merck), with each
step on ice for 30 min. Neoclear was gradually replaced with paraffin (Paraplast Plus;
Sigma). About 10 Paraplast Plus chips were added to 20 ml of fresh Neoclear and
samples were leaved for 2 h at room temperature and then for 3 h at 58 °C. Once the
chips dissolved at 58 °C, the mixture was replaced with molten Paraplast Plus at 58 °C,
and the medium was changed twice approximately at 4-5 h intervals before embedding
in pure paraffin. Root samples were embedded in paraffin in Petri dishes and stored at 4
°C. A Leica AS LMD (Leica Microsystem, Inc) was used to collect 10,000 arbusculecolonized cortical cells from paraffin root sections as described by Balestrini et al.
(2007). Total RNA were extracted from microdissected arbuscule-colonized cells using
the Pico Pure Kit (Arcturus Engineering). DNase treatment of total RNA was performed
using RNAse-free DNase Set (Qiagen). RNA quality and integrity were checked prior
to cDNA synthesis using a Bio-Rad Experion analyzer and Experion RNA StdSens
analysis kit. Total RNA preparations were amplified using the SMART PCR cDNA
Synthesis Kit (Clontech) according to the manufacturer’s instructions and used for 454
pyrosequencing.