The Transcriptional and Translational Response of Neurospora crassa to Salt and Light

Abstract

Many different response-pathways enable microorganisms to respond to changes in the environment. Two environmental conditions that elicit responses in the model filamentous fungus Neurospora crassa are light and osmotic stress. The light response is controlled by transcription factors (TFs) —including the blue-light photo receptor WC-1. WC-1 forms a heterodimeric complex with WC-2; this complex, referred to as the white collar complex (WCC) binds to the promoters of multiple genes and regulates their transcription. Among the WCC targets are other TFs. To determine the downstream impact of removing WC-1, WC-2, and light-responsive TFs, we grew deletion-strains of 11 known light-responsive TFs (including ∆wc-1 and ∆wc-2) whose promoters are bound by WCC. Strains were grown for 24hr then either exposed to 1hr of light or kept in the dark. RNA-seq analyses were then performed. We discovered that these 11 TFs play important roles in the transcript-level light response and established that two previously uncharacterized genes, NCU00275 and NCU09615, have major roles in multiple light-regulated pathways. In N. crassa, osmotic stress activates an osmosensing mitogen activated kinase (OS MAPK) pathway. This pathway activates numerous kinases, including RCK-2, which phosphorylates translation elongation factor 2 (EF2) in response to salt stress. Phosphorylation of EF2 reduces translation. We examined the effects of both salt stress and deletion of rck-2 by RNA-seq and ribosome profiling (Ribo-seq). We found that salt stress has large transcript-level and translation-level impacts on wild type N. crassa that differ from what is observed in S. cerevisiae. In the N. crassa ∆rck-2 mutant, multiple pathways that are normally repressed following salt stress lose repression at both a transcriptional and translational levels. However, deletion of rck-2 did not impact transcript levels, but did impact translation in the absence of salt. Genes involved in respiration and the electron transport chain have increased translation efficiency in ∆rck2 as compared to wild type.