The Aspergillus nidulans Zn(II)2Cys6transcription factor AN5673/RhaR mediates L-rhamnose utilization and the production of α-L-rhamnosidases

Abstract

BackgroundVarious plant-derived substrates contain L-rhamnose that can be assimilated by many fungi and its liberation is catalyzed by α-L-rhamnosidases. Initial data obtained in our laboratory focussing on two Aspergillus nidulans α-L-rhamnosidase genes (rhaA and rhaE) showed α-L-rhamnosidase production to be tightly controlled at the level of transcription by the carbon source available. Whilst induction is effected by L-rhamnose, unlike many other glycosyl hydrolase genes repression by glucose and other carbon sources occurs in a manner independent of CreA. To date regulatory genes affecting L-rhamnose utilization and the production of enzymes that yield L-rhamnose as a product have not been identified in A. nidulans. The purpose of the present study is to characterize the corresponding α-L-rhamnosidase transactivator.ResultsIn this study we have identified the rhaR gene in A. nidulans and Neurospora crassa (AN5673, NCU9033) encoding a putative Zn(II)2Cys6 DNA-binding protein. Genetic evidence indicates that its product acts in a positive manner to induce transcription of the A. nidulans L-rhamnose regulon. rhaR-deleted mutants showed reduced ability to induce expression of the α-L-rhamnosidase genes rhaA and rhaE and concomitant reduction in α-L-rhamnosidase production. The rhaR deletion phenotype also results in a significant reduction in growth on L-rhamnose that correlates with reduced expression of the L-rhamnonate dehydratase catabolic gene lraC (AN5672). Gel mobility shift assays revealed RhaR to be a DNA binding protein recognizing a partially symmetrical CGG-X11-CCG sequence within the rhaA promoter. Expression of rhaR alone is insufficient for induction since its mRNA accumulates even in the absence of L-rhamnose, therefore the presence of both functional RhaR and L-rhamnose are absolutely required. In N. crassa, deletion of rhaR also impairs growth on L-rhamnose.ConclusionsTo define key elements of the L-rhamnose regulatory circuit, we characterized a DNA-binding Zn(II)2Cys6 transcription factor (RhaR) that regulates L-rhamnose induction of α-L-rhamnosidases and the pathway for its catabolism in A. nidulans, thus extending the list of fungal regulators of genes encoding plant cell wall polysaccharide degrading enzymes. These findings can be expected to provide valuable information for modulating α-L-rhamnosidase production and L-rhamnose utilization in fungi and could eventually have implications in fungal pathogenesis and pectin biotechnology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0161-9) contains supplementary material, which is available to authorized users.

Gene expression patterns and dynamics of the colonization of common bean (Phaseolus vulgaris L.) by highly virulent and weakly virulent strains of Fusarium oxysporum

“…FNR1 has been described to mediate the adaptation to nitrogen-poor conditions in planta ( Divon et al, 2006 ), such as those potentially found inside the xylem vessels. The A. nidulans rhaR gene encodes a transcription factor that positively regulates the expression of both the enzymes that liberate L -rhamnose from complex plant substrates (alpha-L-rhamnosidases) as well as those involved in the assimilation of this sugar, and hence confers A. nidulans the ability to adapt its metabolism to the availability of L -rhamnose ( Tamayo-Ramos et al, 2012 ; Pardo and Orejas, 2014 ). Interestingly, RNA-seq data obtained from the Aspergillus Genome database (AspGD) suggest that rhaR could be preferentially expressed under nitrogen limiting conditions.…”

The Two-Component System RsrS-RsrR Regulates the Tetrathionate Intermediate Pathway for Thiosulfate Oxidation in Acidithiobacillus caldus

“…EMSA assays were performed as described in Pardo and Orejas (2014). Initially, a 15 μL reaction mixture containing 1.5 μL 10 × Binding Buffer, 1 μg of salmon sperm DNA, and 1 μg RsrR was mixed and used as the reaction solution.…”

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.…”