A novel Zn₂Cys₆ transcription factor BcGaaR regulates D‐galacturonic acid utilization in Botrytis cinerea

Abstract

D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2 Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.

An Evolutionarily Conserved Transcriptional Activator-Repressor Module Controls Expression of Genes for D-Galacturonic Acid Utilization inAspergillus niger

“…Phenotypic characterization of mutants lacking the GA-regulator in both B. cinerea and A. niger has shown that GaaR is required for growth on GA (Alazi et al 2016;Zhang et al 2016). Expression analysis in both fungi confirmed that GaaR is required for the induced expression of GA-responsive genes.…”

“…), and Dothideomycetes [Zymoseptoria (Mycosphaerella), Aureobasidium, and Cochliobolus spp.] (Zhang et al 2016). Synteny analysis of 17 species belonging to four classes of Pezizomycetes (Eurotiomycetes, Leotiomycetes, Sordariomycetes, and Dothideomycetes) revealed a strong conservation of the genomic clustering of gaaR and gaaX orthologs ( Figure 5 and Table S5).…”

“…Computational analysis has been used to identify the galacturonic acid (GA) responsive element (GARE) of GA-induced genes (Martens-Uzunova and Schaap 2008). The predicted sequence (CCNCCAA) was shown to be required for the induction of GA-responsive genes in A. niger (Niu et al 2015) and Botrytis cinerea (Zhang et al 2016). Furthermore, using the yeast one-hybrid method, it was shown in B. cinerea that the GaaR transcription factor interacts specifically with the GARE (Zhang et al 2016).…”

Regulation of the lignocellulolytic response in filamentous fungi

“…Several conserved zinc binuclear cluster transcription factors required for the regulation of genes encoding enzymes important for the transport and utilization of the component saccharides have been identified. In Botrytis cinerea and A. niger, the utilization of galacturonic acid, the most abundant pectin component, is regulated by GaaR (Alazi et al, 2016;Zhang et al, 2016), while in A. niger and A. nidulans, the utilization of rhamnose and arabinose, two minority pectin components, is regulated by RhaR and AraR, respectively (Battaglia et al, 2011a(Battaglia et al, , 2011bGruben et al, 2014;Pardo and Orejas, 2014) (Fig 1 and Table 1). The identification of additional transcription factors may be necessary to fully elucidate the regulation of pectin utilization.…”

The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

“…Previous studies, mostly in the highly pectinolytic fungi Aspergillus niger  ( A. niger ) and Botrytis cinerea ( B. cinerea ), have shown that individual sugars liberated from pectin by secreted enzymes, such as d -GalA, l -Ara, l -Rha and d -Gal, or their respective metabolic derivatives have an inducing potential for the corresponding gene products [25–27]. Recently, transcription factors (TFs) that are conserved across fungi and that mediate the response to some of these monosaccharides have been identified: TRC1 and RhaR for l -Rha in Pichia stipitis ( P. stipitis ) and A. niger , respectively [28, 29], GaaR for d -GalA in B. cinerea [30] and Ara1 for l -Ara in Magnaporthe oryzae ( M. oryzae ) [31]. While identified in different fungi, these TFs have in common that they are responsible for regulating a subset of genes involved in the degradation of a particular pectin domain or pectic polysaccharide: HG for GaaR, the RG-I backbone for RhaR, and the arabinan side chains of RG-I (or hemicellulosic arabinan) for Ara1.…”