ABSTRACT: Purpose: To explore the pathway cross-talk of endoplasmic reticulum (ER) stress response and cellulases synthesis in Neurospora crassa. Methods: To identify ER stress response targets(ESRTs), conidial suspensions of WT were obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium (1x vogel’s salts , 1% w/v crystalline cellulose (Avicel PH-101; Sigma-Aldrich), 0.2% w/v NH4NO3 ,pH 7.0 )for 36 hours (25ºC, 200 rpm, constant light) followed by the addition of dithiothreitol (DTT, final concentration of 0.1mM for mild stress level while 5mM for acute stress level) or tunicamycin (TM, final concentration of 5ug/mL for mild stress level while 40ug/mL for acute stress level) and growth for 1 more hour.To identify which ESRTs regulated by IRE-1/Hac-1 mediated UPR pathway,conidial suspensions of the Ire-1 and Hac-1 KO mutants as well as their parental strains (FGSC#2489 and FGSC#9720, respectively) were obtained and 1 x 10^6 cells/ml incubated in minimal medium containing 2% sucrose for 16 hours, then young hyphae was harvested and transferred into cellulases induction medium as mentioned above culture for 4 hours followed by the addition of DTT (final concentration of 5mM)(or H2O as mock)and growth for 1 more hour.For Hac-1 KO mutant and FGSC#9720,cultures were plus with final concentration of 100ug/ml L-histidine.To explore the function of transcription factors RES-1, RES-2 and RRG-2 in response to ER stress and cellulases synthesis, each conidial suspension of the three strains was obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium for 36 hours followed by the addition of dithiothreitol (DTT, final concentration of 5mM ) (or H2O as mock)and growth for 1 more hour.Cells were harvested by vacuum filtration with Whatman filter paper, frozen immediately in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. An additional clean-up including the on-column DNase I treatment was performed by using the RNeasy mini kit (Qiagen).The thirteen 49-nt single-end RNA-seq libraries were generated commercially at Beijing Genomics Institute (BGI, Shenzhen, China) while eight 90-nt paired-end RNA-seq libraries were generated at Genewiz Co. Ltd (Suzhou) by using the HiSeq™ platform.Sequencing data was handled essentially with Bowtie2, Tophat2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Results: Transcriptional profiling of ER stress during cellulase synthesis in N.crassa uncovered a set of 766 genes was identified as the ER stress response target genes (ESRTs). Of these 766 genes, canonical UPR components IRE-1 and HAC-1 regulated 223 and 186 genes, respectively. In screening 527 available ESRT mutants, we found 249 that exhibited DTT sensibility, including 100 targets with unknown function. Disruption of ire-1 or hac-1 renders N. crassa cells seriously deficient in cellulase secretion. Further investigation of the 39 transcription factors (TFs) that showed differential expression under ER stress uncovered three novel TFs (RES-1, RES-2, RRG-2) that markedly affected cellulase secretion. Conclusions: Here, we determined the transcriptional scope of the ER stress response during lignocellulase synthesis in the model cellulolytic fungus N. crassa. Through genome-wide mutant screening and analysis, dozens of novel genes were discovered to be involved in the process. The findings of this work will be useful for strain improvement to facilitate lignocellulase and biomass-based chemical production.
