Y2H-seq library screening of Mj-ISE6 using next-generation sequencing
This project contains data and code associated with the yeast two-hybrid sequencing (Y2H-seq) screening of the Meloidogyne javanica effector Mj-ISE6. The Y2H-seq workflow was performed following the protocol of (Erffelinck et al., 2018), with minor modifications.Experimental overview: Bait constructs were transformed into the Saccharomyces cerevisiae PJ69-4A strain. Competent bait yeast was then super-transformed with 50 ug of cDNA library plasmid DNA. Transformants were plated on: SD-LW (selection for successful transformation; determination of transformation efficiency), and SD-LWH + 3-AT (selection for bait-prey protein interactions). A transformation efficiency of >=1 x 10^6 independent clones was consistently achieved.Plant treatments and cDNA library construction: Four-week-old 'MoneyMaker' plants were treated with: 500 uM salicylic acid (SA), 50 uM jasmonate (JA), Agrobacterium tumefaciens strain K599 at OD600 = 0.1. Roots were harvested at the following timepoints: SA: 2 h, 1 day, 2 days. JA: 2 h, 1 dayAgrobacterium: 2 days, 6 days, 8 days RNA was extracted using the Qiagen RNA Maxiprep Kit, followed by mRNA isolation using the Poly(A)Purist MAG Kit (Thermo Fisher). cDNA library construction was performed with the CloneMiner II cDNA Library Construction Kit (Thermo Fisher).The resulting cDNA library contained 1.39 x 10^8 primary clones, with an average insert size of 1.3 kb.Y2H-seq processing and sequencing: Yeast colonies growing on a selective interaction medium were pooled. Plasmid DNA was extracted, and the prey inserts representing all potential interactors for the Mj-ISE6 bait were amplified for sequencing. A total of 1 ug of plasmid DNA was submitted to VIB Nucleomics for Illumina HiSeq 2000 sequencing (125-bp paired-end reads).CitationErffelinck M-L, Ribeiro B, Perassolo M, Pauwels L, Pollier J, Storme V, Goossens A. 2018. A user-friendly platform for yeast two-hybrid library screening using next-generation sequencing. PLoS ONE 13: e0201270.