Fast Green

Total RNAs of leaves were isolated by TRIzol Reagent (Invitrogen, USA). The first-strand synthesis of cDNAs was carried out by SuperScript® III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instruction. Semi-quantitative RT-PCR was performed for 25 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C. qPCR was performed in StepOne™ Real-Time PCR System (Applied Biosystems, USA) using the Fast SYBR Green Master Mix reagent (Applied Biosystems) by the manufacturer’s instructions, and the thermal cycle used was as follows: 95°C for 20 s; and 40 cycles of 95°C for 3 s, and 60°C for 30 s. OsRAc1 (GenBank accession: X16280), a rice constitutively expressed gene of Actin, was used as a standardization control, using the primer pair 5’-GGAACTGGTATGGTCAAGGC-3’ and 5’-AGTCTCATGGATACCCGCAG-3’ for semi--quantitative RT-PCR, 5’-TGGCATCTCTCAGCACATTCC-3’ and 5’-TGCACAATGGATGGGTCAGA-3’ for qPCR. Gene-specific primers of candidate genes for semi-quantitative PCR and qPCR are listed in Table 2 and Table 3, respectively. Independent biological repetitions of each experiment were performed three times.Table 2

Gene-specific primers for RT-PCR in this study

aSpot id	bAccession	Forward primer (5’-3’)	Reverse primer (5’-3’)	c Tm (°C)
1	LOC_Os12g37540	AACAAGGTAGGGATAGTTACTT	CCTTGTATGTGGGTTTTTTAGAA	55
2	AK067692	AATGAAATCTTGCTTGCTGC	CTAAATCTTCTTGGGACATA	60
3	AK067692	GCCTACTTCTTCACATTCAC	ATTTCATTACCTTCACGAGC	55
4	AK067732	ACCCGCTTTATTCTGCTG	ATCCTTTTGACACAGTATGG	60
5	AK104875	AGATGCTGTGCTTGATGCCT	CAATGACGAAGCGACCAGAT	55
6	AK105059	CAAACAGGGTGAAGAGCCAG	CTCGCATTTAGCCAGGGACA	62
7	AK065872	ACGCCGACAAGAATCCCAAC	CAATACGACCAGCCCCAACA	60
8	AK064960	TTCATCACCACCGACTACAT	AACCCTCAACAATACCAAAC	55
9	AK060847	GAAGCTGAAGAAGCAGGTGACATC	CGAAGACGAGCTCACACTGGAAG	55
10	AK104332	ATGGGTGAATTCTGTGGTGAG	CCCTTCTTGATGATGTCTGCC	58
11	AK102889	CTCGTTGCGGTAGTGCTGCT	AATGAAATCTTGCTTGCTGC	55
12	AK099598	TGGCAGCGAAGACAAACAAC	CTGGAAGAGCACCGACGAAA	62
13	AK063934	GCTTGAGATTTGATGTTGAG	GTCCTCTGCGTATTTTTCTG	62
14	AK070067	CGACTTCTCCACCCTACTAT	ATGATGTTGGTGATGACGCC	55

^a Spot identity of protein in 2-D gel image; ^bmRMA accession of the corresponding protein in GenBank (http://www.ncbi.nlm.nih.gov/), except for spot 1 protein whose gene accession was only deposited in TIGR database (http://rice.plantbiology.msu.edu/index.shtml); ^cAnneal temperature of primer pair used in RT-PCR.

Table 3

Gene-specific primers for qPCR in this study

Gene name	Forward primer (5’-3’)	Reverse primer (5’-3’)
APX7	ATACGCAGAGGACCAAGAAGCAT	CTACGAGCAAGATAAATAGCAGA
Chia2a	CCAACATCATCAACGGCGGCAT	TTGGGATACTACATCACTACAT

To evaluate the performance of the primer pairs amplifying target DNA from a heterogeneous pool of DNA in environmental samples, all primer pairs were tested in a qPCR set-up. A 2-fold dilution series (1∶1 to 1∶64) was made from twelve DNA samples (ranging from 5 ng µl⁻¹ to 78 pg µl⁻¹, including one no-template control (NTC) for each sample). Amplification was performed in optical 96-well plates using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Green chemistry. PCR conditions were as follows: initial denaturation at 95°C for two minutes, followed by 40 cycles of 95°C (30 s), 55°C (30 s) and 72°C (60 s) and a final extension phase at 72°C for 10 minutes followed by the generation of a dissociation curve to verify amplification specificity. These qPCR conditions were chosen to mimic the PCR conditions used during the PCR step prior to emPCR and amplicon pyrosequencing. Reactions contained 2.5 µL template DNA, 5 µL 2× Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), 0.3 µl forward and reverse primers (3.3 µM each) and 1.9 µL nuclease-free H₂O in a total volume of 10 µL. PCR efficiencies (E) were calculated as E = (10^−1/slope−1)×100.
To assess a potential PCR-bias at the phylum level, DNA was extracted from 15 pure cultures provided by the Mycothèque de l'Université Catholique de Louvain (BCCM/MUCL) including 5 basidiomycetes (Lentinula edodes (MUCL 44827), Agrocybe praecox (MUCL 46727), Coniophora marmorata (MUCL 39471), Suillus luteus (UH-Slu-LM8-n1) and Antrodia vaillantii (MUCL 54533)), 5 ascomycetes (Cladosporium cladosporioides (MUCL 53652), Cryptosporiopsis radicicola (MUCL 53485), Monilinia laxa (MUCL 30841), Arthroderma otae (MUCL 39756) and Galactomyces geotrichum (MUCL 52377)), 2 glomeromycetes (Rhizophagus clareus (MUCL 46238) and Rhizophagus sp. (MUCL 41833)) and 3 zygomycetes (Mortierella verticillata (MUCL 9658), Absidia corymbifera (MUCL 38907) and Mucor hiemalis (MUCL 15439), also see Table S4). DNA was extracted from cultures using the DNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen, Venlo, Netherlands). DNA concentrations extracted from pure cultures used for qPCR ranged from 5 ng µl⁻¹ to 20 ng µl⁻¹. PCR bias at the phylum level was tested according to the qPCR protocol described above.

Six DEGs were validated through a real-time qPCR analysis (Table S5). Three DEGs were randomly chosen, in addition to the most downregulated high-affinity nitrate transporter (NTR2:6) and one NADH-nitrate reductase, which are related to nitrate uptake, and a silicon efflux transporter (LSI3) related to the deposition of silicon in spore valves. Two genotyped strains of C. socialis, namely APC12 and MCA6 were used for this purpose: the former strain is the one used for the transcriptome experiment, while MCA6 is a freshly established strain isolated at station LTER-MC in the Gulf of Naples and for which the D1–D3 region of the nuclear-encoded large subunit ribosomal DNA (partial 28S rDNA) has been sequenced as in [70 ] to confirm its identity.
Triplicate cultures of both strains were maintained in control and low N media, with the same nutrient concentrations used for the RNA-seq experiment. Cells were harvested on day 2 in the control, when the percentage of spores was zero, and on day 3 in the treatments, when the percentage of spores was ~ 33 and ~ 38% for APC12 and MCA6, respectively, corresponding to the ones recorded at T3 of the transcriptome experiment. RNA extraction and purification were performed as illustrated above. Total RNA was reverse-transcribed using the QuantiTect® Reverse Transcription Kit (Qiagen, Venlo, Limburgo, Nederlands).
RTqPCR amplification was performed with cDNA diluted 1:10, in a 10 µl reaction containing each primer at a final concentration of 1 µM and Fast SYBR Green Master mix with ROX (Applied Biosystems) using a ViiA™ 7 Real-Time PCR System (Applied Biosystems by Life Technologies, Carlsbad, CA, USA) and the following cycling parameters: 95 °C for 20 s, 40 cycles at 95 °C for 1 s, 60 °C for 20 s, 95 °C for 15 s, 60 °C 1 min, and a gradient from 60 °C to 95 °C for 15 min. Raw results were processed using the ViiA™ 7 Software and exported into Microsoft Excel for further analyses. The reference gene used was the tubulin gamma chain (TUB G) designed using sequence information from the transcriptome and the software Primer3Plus v.2.4.2 ([71 (link)]). The sequences for the forward and reverse primers are 5’- TGCAGAGTTTGGTCGATGAG -3’and 5’-GGAAGCCAAAGAGTCTGCTG-3’, respectively, yielding a PCR product of 197 bp (Table S5). Primers for all other tested DEGs were designed using the same approach. log2(FC)s were obtained with the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS) ([72 (link)]). A pairwise fixed reallocation randomisation test has been used to identify statistically significant results (P ≤ 0.05).