Computational protocol: Hop/Sti1 – A Two-Faced Cochaperone Involved in Pattern Recognition Receptor Maturation and Viral Infection

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Protocol publication

[…] RNA was isolated from wild-type Nicotiana tabacum plants and from NtHop/Sti1-silenced plants, both PVY-infected and uninfected, as described before (). The obtained RNA was then used to generate cDNA, using the Bio-Rad iScript cDNA synthesis kit (Bio-Rad) for analysis of PR-gene expression or the RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific) for analysis of bZIP60 splicing status, PVY genome detection and analysis of Hop/Sti1 expression.For PVY RNA detection, cDNA was amplified using Taq polymerase and oligonucleotide primers specific for the viral capsid protein (CPfw: 5′-CGGAGTTTGGGTTATGATGG-3′; CPrev: 5′-ACGCTTCTGCAACATCTGAG-3′). The band intensities in the successional agarose gel were determined using the ImageJ software. Quantitative real-time PCR analyses were performed using specific oligonucleotide primers for Nicotiana tabacum PR-1 and PR-2 (PR1-for: 5′-TTGCCGTGCCCAAAATTCTC-3′; PR1-rev: 5′-ACAATCTGCAGCCAATTGGG-3′; PR2-for: 5′-TGCTCCTGCCATGCAAAATG-3′; PR2-rev: 5′-ATACTATCTTTGGGCGGGTTGG-3′), Hop/Sti1 (Hop-fw: 5′-AGACGAAGAACGCGAGAAAG-3′; Hop-rev: 5′-TATATGCCCTCGGGTCTTTG-3′) or spliced bZIP60 (bZIP60F: 5′-GCAGAAAATCAAGCTTTGCGCT-3′; bZIP60SR: 5′-AGGGAACCCAACAGCAGACT-3′) as reported earlier (). For normalization of target gene expression, the expression of either actin or ubiquitin was determined (Act-for: 5′-GCTGGATTTGCTGGTGATG-3′; Act-rev: 5′-TCCTTCTGTCCCATTCCGAC-3′; UBQ10fw: 5′-AGATCCAGGACAAGGAAGGTATTC-3′; UBQ10rev: 5′-CGCAGGACCAAGTGAAGAGTAG-3′). As a reference dye, SYBR green was used (Brilliant II SYBR® MM or Brilliant III SYBR® MM, Agilent, Santa Clara, CA, United States). The reactions were performed in triplicates in Agilent MX3000P or AriaMx real-time thermocyclers. Data evaluation was performed either by the Stratagene MxPro software (Agilent) or (when the AriaMx thermocycler was used) according to . [...] Bonferroni-corrected student’s t-tests were carried out in Microsoft Excel 2010 (Microsoft, Redmond, WA, United States), while analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference test was performed using IBM SPSS Statistics 24 (IBM, Armonk, NY, United States). […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Nicotiana tabacum, Potato virus Y
Diseases Virus Diseases