Netprimer software

Routine C. autoethanogenum genomic DNA extraction for PCR diagnostics was done with PureLink Genomic DNA extraction kit (Invitrogen™, Thermo Fischer Scientific), while plasmid DNA was extracted from E. coli using FavorGen FavorPrep™ Plasmid DNA Extraction Mini Kit (Favorgen Biotech Corp). All purified genomic and plasmid DNA fragments were quantified using the NanoDrop™ 1000 Spectrophotometer (Thermo Fischer Scientific). PCR for amplification of DNA fragments for sequencing and cloning, routine screening, and analytical procedures was performed using the Phusion™ High-Fidelity DNA Polymerase (Thermo Fischer Scientific). All primers used in this study (see Supplementary Table S1) were designed using the NetPrimer software (PREMIER Biosoft International) and synthesised by Integrated DNA Technologies. PCR products and DNA fragments were purified from agarose gels with FavorPrep Gel/PCR Purification kit (Favorgen Biotech Corp).

Long-range primers were designed from DNA sequence ID ENSFCAT00000026034 and ENSFCAT00000009625 for Hspb1 and Tp53 through the application of Primer3 and NetPrimer software (PREMIER Biosoft International, Palo Alto, CA) (19 ,20 ). Primer express software (Applied Biosystem, USA) was used to design the primer-probe sequences of the GAPDH gene as an endogenous control for normalization (21 (link)). Hspb1and Tp53 primer-probes expression assays were purchased with FAM flourophore while GAPDH probe was labeled with VIC reporter dye on 5´ end and TAMRA as a quencher on the 3´ (Table 2).

Total RNA was extracted with the RNeasy Mini Kit (Qiagen, http://www.qiagen.com/) from 100 mg frozen material of five primary roots per biological replicate, followed by RNase-free DNAse I treatment (Fermentas, http://www.thermoscientificbio.com/fermentas/). For cDNA synthesis 1 µg of total RNA was amplified using the Revert Aid H Minus First Strand cDNA Synthesis Kit (Fermentas). To clone the open reading frames of ZmIAA2 (GRMZM2G159285), ZmIAA11 (GRMZM2G059544), ZmIAA15 (GRMZM2G128421), ZmIAA20 (GRMZM5G864847) and ZmIAA33 (GRMZM2G359924) oligonucleotide primers were designed by PrimerPlus3 software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and checked with NetPrimer Software (PREMIER Biosoft, http://www.premierbiosoft.com/). PCR amplicons of the Aux/IAA open reading frames generated by specific oligonucleotide primers were introduced into the vector pGEM-t-easy (Promega, http://www.promega.de/). Subsequently, the conserved degron-Sequence VGWPPV in domain II was mutated in all Aux/IAA genes via the GENEART site-directed mutagenesis system (Life technologies, http://www.lifetechnologies.com/). Either the first proline residue was substituted by serine (VGWSPV) or the second proline was replaced by leucine (VGWPLV). The oligonucleotides were designed according to the manufacturer's suggestions (Table S1).

Mesocarp tissue samples taken from fruit equator area were frozen in LN and kept in falcon tubes at −80°C for later analyses. Total RNA was extracted from each sample using the Spectrum™ Total RNA kit (Sigma Aldrich, Louis, MO, USA). First-strand cDNA was synthesized from the RNA, using the MAXIMA^TM cDNA kit (Thermo Scientific, Waltham MA USA), and stored at −20°C for later analyses. The qPCR reactions were performed using a StepOne® Real-Time PCR System (Applied Biosystems) and Fast SYBR^TM Green Master Mix (Applied Biosystems, cat no. 4385616) and gene-specific primers. Primer sequences were designed using the primer-designing tool Primer-BLAST (NCBI), and the primers' compatibilities were evaluated by NetPrimer software (PREMIER Biosoft International). The expression data were analyzed by Ct value normalized to the control gene UBI3 and quantified by the Delta-Delta cycle threshold method (Livak and Schmittgen, 2001 (link)). All experiments were carried out with a non-template control, and repeated for three biological experiments. The list of primers used in the qPCR analysis is shown in Supplementary Table 1.