RNA-seq of ptrlbd39/22 double mutants

We analyzed laser capture microdissection-based transcriptomes of Populus trichocarpa TW-forming tissues representing various stages of TW development. We revealed that PtrLBD39 is the most significantly tension stress-induced transcription factor gene, which has a phylogenetically paired LBD homologous gene, PtrLBD22. These two genes were sharply induced by tension stress and were highly expressed in the TW-forming tissues. CRISPR-based knockout of PtrLBD39/22 led to a severely inhibited TW formation with low cellulose and high lignin, whereas overexpression of PtrLBD39 resulted in an opposite effect. Transcriptomic analyses of CRISPR-based double mutants of PtrLBD39/22 showed that these two genes regulate a set of TW-related genes. Moreover, chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PtrLBD39 direct targets. We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network (TRN) mediated by PtrLBD39. The network consists of three layers including transcription factors, secondary cell wall component genes and other potential regulators. Our work suggested that PtrLBD39 and PtrLBD22 specifically regulate TW formation by mediating a TW-specific TRN in Populus.