Direct Target Network of the Neurospora crassa Plant Cell Wall Deconstruction Regulators CLR-1, CLR-2, and XLR-1

Abstract

Fungal deconstruction of the plant cell requires a complex orchestration of a wide array of intracellular and extracellular enzymes. In Neurospora crassa, CLR-1, CLR-2, and XLR-1 have been identified as key transcription factors regulating plant cell wall degradation in response to soluble sugars. The XLR-1 regulon was defined using a constitutively active mutant allele, resulting in hemicellulase gene expression and secretion under noninducing conditions. To define genes directly regulated by CLR-1, CLR-2, and XLR-1, we performed chromatin immunoprecipitation and next-generation sequencing (ChIPseq) on epitope-tagged constructs of these three transcription factors. When N. crassa is exposed to plant cell wall material, CLR-1, CLR-2, and XLR-1 individually bind to the promoters of the most strongly induced genes in their respective regulons. These include promoters of genes encoding cellulases for CLR-1 and CLR-2 (CLR-1/CLR-2) and promoters of genes encoding hemicellulases for XLR-1. CLR-1 bound to its regulon under noninducing conditions; however, this binding alone did not translate into gene expression and enzyme secretion. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest paralog in Saccharomyces cerevisiae, Gal4p. Coimmunoprecipitation studies showed that CLR-1 and CLR-2 act in a homocomplex but not as a CLR-1/CLR-2 heterocomplex.

Regulation of the lignocellulolytic response in filamentous fungi

“…Together, CLR-1 and CLR-2 transcriptionally activate genes that make up the full cellulolytic response. However, unlike clr-2, constitutive expression of clr-1 does not cause expression of target genes, although CLR-1 binds cis-regulatory elements even under noninducing conditions Craig et al, 2015). These data indicate that CLR-1 also requires activation/derepression.…”

“…These three species have a conserved 5 0 -GGCTWWW-3 0 consensus sequence in the 5 0 cis regulatory elements of the target genes van Peij et al, 1998a;Zeilinger et al, 1998). The promiscuity of regulons amongst homologous transcription factors is further reflected as N. crassa CLR-2 has a consensus sequence nearly identical with its closest yeast paralogue, Gal4, but without a clear function in the N. crassa galactose utilization pathway (Bram and Kornberg, 1985;Craig et al, 2015;Giniger et al, 1985). The evolution of CLR-2/ ClrB and XYR1/XlnR/XLR-1 as the major transcription factors of CAZymes is poorly understood and may represent a functional shift in environmental niches from an ancestral lifestyle.…”

Network of nutrient-sensing pathways and a conserved kinase cascade integrate osmolarity and carbon sensing in Neurospora crassa

“…S1A) (4). Expression of clr-2 is directly regulated by CLR-1, and constitutive expression of clr-2 is sufficient to activate the cellulolytic response (15,16). However, transcription of clr-1 does not result in the activation of cellulase gene expression in the absence of an inducer (4).…”

Fungal sensing of host environment

“…In Candida glabrata, a high-affinity glucose sensor sucrose non-fermenting 3 was recently identified, which helps the fungi to survive in a glucose-limited environment (Ng et al, 2015). Sensing of carbohydrates enables the plant destructing fungus Neurospora crassa to only induce the gene coding for transporters and enzymes when nutrients are available (Craig JP, 2015). Depending on the carbon source used for growth, fungi also reshape their cell wall in order to improve osmotic resistance and elasticity (Ene IV, 2015).…”