A RsrC-RsrA-RsrB transcriptional circuit positively regulates polysaccharide-degrading enzyme biosynthesis and development in Penicillium oxalicum

Abstract

Filamentous fungi produce polysaccharide-degrading enzymes, which is controlled by poorly understood transcriptional circuits. Here we show that a circuit comprising RsrC-RsrA-RsrB (Rsr: production of raw-starch-degrading enzyme regulator) that positively regulates production of raw starch-degrading enzymes in Penicillium oxalicum. Transcription factor (TF) RsrA is essential for biosynthesis of raw starch-degrading enzymes. RsrB and RsrC containing Zn2Cys6- and C2H2-zinc finger domains, act downstream and upstream of RsrA, respectively. RsrA activates rsrB transcription, and three nucleotides (G^-286, G^-287 and G^-292) of rsrB promoter region are required for RsrA, in terms of TF, for binding. RsrB_165-271 binds to DNA sequence 5'-TCGATCAGGCACGCC-3' in the promoter region of the gene encoding key raw-starch-degrading enzyme PoxGA15A. RsrC specifically binds rsrA promoter, but not amylase genes, to positively regulate the expression of rsrA and the production of raw starch-degrading enzymes. These findings expand complex regulatory network of fungal raw starch-degrading enzyme biosynthesis.

Fig. 1. Analysis of RsrB (POX_g08691) functions and protein sequences.

a Production of raw starch-degrading enzymes (RSDEs) and soluble starch-degrading enzymes (SSDEs) (b) by P. oxalicum mutant ΔrsrB, parental strain Δku70 and complementation strain CrsrB in the presence of soluble corn starch (SCS). Lowercase letters represent p < 0.05. Different letters indicate significant differences, evaluated by one-way ANOVA. c Conserved domains in RsrB. Grey and green areas indicate the Gal4-like Zn2Cys6 zinc finger domain (Gal4) and Fungal_TF_MHR domain, respectively. d Phylogenetic analysis of RsrB and its homologues. The cladogram was constructed by MEGA version X using the neighbour-joining method and a Poisson model. Values displayed on branches were calculated using 1000 bootstrap replicates. e Yeast self-activation assay. Yeast cells carrying different lengths of RsrB peptides were cultured on SDO (SD/-Trp) and SDO/X/A (SD-Trp/+ X-α-Gal /+ aureobasidin A) for 4 days. f Effects of rsrB overexpression on RSDE and SSDE (g) production of P. oxalicum. All tested P. oxalicum strains were cultured in medium containing soluble corn starch for 2–4 days after transfer from glucose. Each mutant included three independent transformants. Results are mean ± SD. All experiments were performed at least three times. Uppercase and lowercase letters represent p < 0.01 and p < 0.05, respectively. Different letters indicate significant differences, evaluated by one-way ANOVA.

Fig. 2. Effects of RsrB on gene expression in P. oxalicum in the presence of SCS.

a Volcano plot indicating differentially expressed genes (DEGs). DEGs were screened and detected with thresholds of 0 <False Discovery Rate (FDR) ≤ 0.05. b KEGG annotation of DEGs modulated by RsrB. c DEGs encoding amylases. d DEGs encoding putative sugar transporters. e DEGs encoding putative transcription factors. f Real-time reverse transcription quantitative PCR (RT-qPCR) analysis of genes encoding major amylases, as well as sporulation-regulatory gene brlA (g). All P. oxalicum strains were cultured for 4–24 h in the presence of SCS. Gene expression in ΔrsrB was normalised to the level of Δku70. Results are mean ± SD. **p < 0.01 and *p < 0.05 indicate significant differences between ΔrsrB and Δku70, calculated by Student’s t test. PoxGA15A, raw starch-degrading glucoamylase; POX_b02418, glucoamylase; Amy13A, α-amylase.

Fig. 3. In vitro electrophoretic mobility shift assay (EMSA) indicating RsrB binding to target genes, and identification of the key DNA sequence bound.

a–c Each reaction contained Trx-His-S-tagged rRsrB_165–271 (0–3 μg) and 6-carboxyfluorescein-tagged probe (~50 ng). Trx-His-S peptide and bovine serum albumen (BSA) served as controls, along with the promoter region of the β-tubulin gene. Competitive probes were DNA fragments without 6-carboxyfluorescein. PoxGA15A, raw starch-degrading glucoamylase gene; POX_b02418, glucoamylase gene; amy13A, α-amylase gene; brlA, conidiation regulatory gene. d Schematic diagram indicating truncated promoter region of PoxGA15A probes for in vitro EMSA. e, f In vitro EMSA between rRsrB_165–271 and truncated probes of PoxGA15A. Each reaction contained Trx-His-S-tagged rRsrB_165–271 (3 μg) and 6-carboxyfluorescein-tagged probe (~50 ng). g MEME-predicted conserved DNA sequence bound by rRsrB_165–271. DNA sequences were from promoter regions of PoxGA15A, POX_b02418 and amy13A. h EMSA indicating the binding of mutated PoxGA15A probes by rRsrB_165–271 (3 μg). Mutated PoxGA15A probe has ‘A’ instead of T and C at the 1st and 15th positions, as shown in panel D.

Fig. 4. Analysis of the regulatory relationship between RsrA and RsrB.

a Real-time reverse transcription quantitative PCR (RT-qPCR) indicating expression of rsrB in mutant ΔrsrA on soluble corn starch (SCS). P. oxalicum strains were cultured for 4–48 h after transfer. mRNA levels in mutant ΔrsrA were normalised to the levels in parental strain Δku70 at the corresponding timepoints. **p < 0.01 and *p < 0.05 by Student’s t test represent significant differences between deletion mutant ΔrsrA and Δku70. Results are means ± SD. b In vitro EMSA between RsrA and rsrB probe. Recombinant rRsrA_830–883 (0–3 μg) and rsrB probe (50 ng) were loaded. Trx-His-S peptide and bovine serum albumen (BSA) served as controls, along with the promoter region of the β-tubulin gene. Competitive probes were DNA fragments without 6-carboxyfluorescein. c MEME-predicted conserved DNA sequences bound by rRsrA_830–883. DNA sequences were from the promoter regions of PoxGA15A, POX_b02418, amy13A and rsrB. d EMSA indicating the binding of mutated rsrB probes by rRsrA_830–883 (3 μg). Mutated probes have ‘A’ or ‘T’ instead of ‘G at the 1st, 6th and 7th positions, as shown in (c). e EMSA between mutated rRsrA_830–883 (3 μg) and rsrB probes. In mutated rRsrA_830–883, R866 is exchanged for A or K. ‘–’ indicates no protein added. (f, g) Production of RSDEs and SSDEs by P. oxalicum parental strain Δku70, deletion mutants ΔrsrB and ΔrsrA, and double mutant ΔrsrBΔrsrA. These strains were cultured in medium containing SCS for 2–4 days after transfer from glucose. Each mutant included three independent transformants. Results are mean ± SD. All experiments were performed at least three times. Lowercase letters represent p < 0.05. Different letters indicate significant differences, evaluated by one-way ANOVA. RSDE raw starch-degrading enzyme, SSDE soluble starch-degrading enzyme.

Fig. 5. Effects of RsrC (POX_a01508) on binding to the promoter region of rsrA, sequence and phylogenetic analyses, and measurement of transcriptional activation ability.

a Y1H assay. Yeast cells carrying DNA fragments upstream of rsrA were cultured on SD/-Leu and SD/-Leu/AbA²⁰⁰ for 3 days. b Conserved domains in RsrC. Purple areas indicate Cys2His2 (C2H2)-type zinc finger domains. c Phylogenetic analysis of RsrC and its homologues. The cladogram was constructed by MEGA version X with the neighbour-joining method and a Poisson model. Values displayed on branches were calculated using 1000 bootstrap replicates. Yeast self-activation assay. Yeast cells carrying different lengths of RsrC peptides (d) were cultured on SDO (SD/-Trp) and SDO/X/A (SD-Trp/+ X-α-Gal /+ aureobasidin A) for 3 days (e).

Fig. 6. Effects of RsrC on amylase production and phenotypes of P. oxalicum.

a Production of RSDEs and SSDEs (b) by P. oxalicum mutant ΔrsrC, parental strain Δku70, complementation strain CrsrC and overexpression strain OrsrC in the presence of SCS. Lowercase letters represent p < 0.05. Different letters indicate significant differences, evaluated by one-way ANOVA. c Colony observation of P. oxalicum mutant ΔrsrC, parental strain Δku70, complementation strain CrsrC and overexpression strain OrsrC on PDA, glucose and SCS plates for 4–5 days.

Fig. 7. Effects of RsrC on gene expression in P. oxalicum in the presence of SCS.

a Volcano plot showing DEGs. DEGs were selected with threshold p ≤ 0.05. b KEGG annotations of DEGs modulated by RsrC. c DEGs encoding carbohydrate-active enzymes (CAZymes). d DEGs encoding amylolytic genes. e DEGs encoding putative transcription factors. f DEGs encoding factors related to gene transcription. g RT-qPCR analysis of genes encoding major amylases, as well as key regulatory gene rsrA (h). All P. oxalicum strains were cultured for 12–48 h in the presence of SCS. Gene expression in ΔrsrC was normalised to the level of Δku70. Results are mean ± SD. **p < 0.01 and *p < 0.05 indicate significant differences between ΔrsrC and Δku70, calculated by Student’s t test. PoxGA15A, raw starch-degrading glucoamylase; POX_b02418, glucoamylase.

Fig. 8. Analysis of regulatory relationship between RsrC and RsrA.

a, b Production of RSDEs and SSDEs by P. oxalicum parental strain Δku70, deletion mutants ΔrsrC and ΔrsrA, overexpression strain OrsrA, mutant OrsrAΔrsrC, and double mutant ΔrsrAΔrsrC. These strains were cultured in medium containing soluble corn starch for 2–4 days after transfer from glucose. Each mutant included three independent transformants. Results are mean ± SD. All experiments were performed at least three times. Lowercase letters represent p < 0.05. Different letters indicate significant differences, evaluated by one-way ANOVA. RSDE, raw starch-degrading enzyme; SSDE, soluble starch-degrading enzyme. c Schematic diagram indicating the truncated promoter region of rsrA for Y1H assay. d Y1H assay between RsrC and the truncated region of upstream DNA sequence of rsrA. Yeast cells carrying DNA fragments upstream of rsrA are cultured on SD/-Leu and SD/-Leu/AbA²⁰⁰ for 3 days.

Fig. 9. Co-regulation between RsrA, RsrB and RsrC in P. oxalicum.

a Number of DEGs between RsrA, RsrB and RsrC regulons. Thresholds were set at False Discovery Rate ≤0.05 or p < 0.05. b KEGG annotations of genes co-regulated by RsrA, RsrB and RsrC. c Genes encoding CAZymes, TFs, sugar transporters and amylolytic enzymes regulated by RsrA, RsrB and RsrC.

Fig. 10. Proposed model of RsrC-RsrA-RsrB regulation in P. oxalicum in the presence of soluble corn starch.

a Model of RsrC-RsrA-RsrB regulation. PoxGA15A, raw starch-degrading glucoamylase. b Regulatory network at 24 h of induction. Dashed lines indicate indirect regulation or non-confirmation.