Computational protocol: PiHOG1, a stress regulator MAP kinase from the root endophyte fungus Piriformospora indica, confers salinity stress tolerance in rice plants

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[…] P. indica HOG1 gene sequence was retrieved from P. indica genome (http://www.ncbi.nlm.nih.gov) by using tBLASTn and S. cerevisiae HOG1 protein amino acid sequence from the Saccharomycete Genome Database (SGD, http://www.yeastgenome.org/) as a query. Primers were designed using retrieved putative PiHOG1 sequence as template. PiHOG1 was PCR amplified using following program: 95 °C 2 min, 35 cycles (95 °C 1 min −56 °C 30 sec −72 °C 1 min 30 sec), 72 °C 5 min. PiHOG1 was cloned into pGEMT-easy vector (Promega, Finland). Cloning of the desired gene was confirmed by EcoRI restriction enzyme digestion and sequencing. Primers used for cloning and for the identification PiHOG1 genomic region and PiHOG1 cDNA are listed in Table S3. [...] Functional sites and their patterns in PiHOG1 protein were determined using the InterProScan data-bank (http://www.ebi.ac.uk_InterProScan/). For identification purposes, BLASTn and BLASTx algorithm (http://www.ncbi.nlm.nih.gov) were used. CLUSTALW was done by using CLUSTALW2 software (www.ebi.ac.uk_clustalw).Phylogenetic tree of P. indica PiHOG1 was constructed by the neighbour-joining (N-J) method using the MEGA7 software (http://www.megasoftware.net/). Two types of phylogenetic analyses were constructed, one with homologs proteins of HOG1 from kingdom Fungi only () and second with stress-activated MAP kinases (SAMAPKs), HOG1, Ser-Thr Kinases (STKs), Stress-induced MAP kinases (SIMPKs), P38 and Sty1 proteins from closely related as well as different groups like fungi, mammals, insects and plants (). [...] To find out the expression of genes of P. indica and rice plants during colonization, RNA was isolated from the non-colonized and colonized P. indica and rice plants. For this purpose, P. indica mycelia were grown in KF media for 7 days, filtered in minimal media and further grown for 3 days. Salinity treatment was given to acclimatized fungus by adding 0.5 M NaCl in MN medium (0.4 mM NaCl, 2.0 mM KH2PO4, 0.3 mM (NH4)2HPO4, 0.6 mM CaCl2, 0.6 mM MgSO4, 3.6 mM FeCl3, 0.2 mM Thiaminehydrochloride, 0.1% (w/v) Trypticase peptone, 1% (w/v) Glucose, 5% (w/v) Malt extract, 2 mM KCl, 1 mM H3BO3, 0.22 mM MnSO4.H2O, 0.08 mM ZnSO4, 0.021 mM CuSO4, pH 5.8). After 1 hr of salinity stress treatment, fungus was immediately filtered and stored in liquid nitrogen. In case of colonization, plant roots were submerged in MN media supplemented with 0.5 M NaCl for 1 hr and the samples were frozen immediately and the total RNA was isolated.HOG pathway related (PiHOG1, PiPBS2 and PiSSK2), regulated (PiGPD, PiSTL1, PiPFK26) and other osmoresponsive genes (PiHSP78, PiGRE2, PiENA1, PiPMR1 and PiPMC1) were explored by BLASTp search in the P. indica genome browser using the S. cerevisiae genes from the Saccharomyces Genome Database (SGD, http://www.yeastgenome.org/) as query. P. indica salinity tolerance genes and rice salinity tolerance genes were also selected for this study. The following cycles were used in the ABI 7500 Fast system (96 wells plates): pre-incubation at 95 °C for 5 min, denaturation 94 °C for 10 sec (4.8 C/s), annealing at 60 °C for 10 sec (2.5 °C/s), extension at 72 °C for 10 sec (4.8 °C/s), 40 cycles of amplification and final extension at 72 °C for 3 min. The Ct values were automatically calculated, the transcript levels were normalized against PiTef expression in case of P. indica and against OsGAPDH in case of rice and the fold change was calculated based on the non-treated control. Two-step Real time-PCR protocol was used in different conditions. Real time-PCR reactions were performed in an ABI 7500 Fast sequence detection system (Applied Biosystems, Life Technologies, USA). The Fold Change values were calculated using the expression, where ∆∆CT represents ∆CT condition of interest gene- ∆CT control gene. The fold expression was calculated according to the 2−∆∆CT method mentioned elsewhere. The primers used in this study are shown in . […]

Pipeline specifications

Software tools InterProScan, BLASTN, BLASTX, Clustal W, MEGA, BLASTP
Applications Phylogenetics, Genome data visualization
Organisms Saccharomyces cerevisiae, Oryza sativa, Ariomma indicum
Chemicals Sodium Chloride