A Homeodomain-Containing Transcriptional Factor Po Htf1 Regulated the Development and Cellulase Expression in Penicillium oxalicum

Abstract

Homeodomain-containing transcription factors (Htfs) play important roles in animals, fungi, and plants during some developmental processes. Here, a homeodomain-containing transcription factor PoHtf1 was functionally characterized in the cellulase-producing fungi Penicillium oxalicum 114-2. PoHtf1 was shown to participate in colony growth and conidiation through regulating the expression of its downstream transcription factor BrlA, the key regulator of conidiation in P. oxalicum 114-2. Additionally, PoHtf1 inhibited the expression of the major cellulase genes by coordinated regulation of cellulolytic regulators CreA, AmyR, ClrB, and XlnR. Furthermore, transcriptome analysis showed that PoHtf1 participated in the secondary metabolism including the pathway synthesizing conidial yellow pigment. These data show that PoHtf1 mediates the complex transcriptional-regulatory network cascade between developmental processes and cellulolytic gene expression in P. oxalicum 114-2. Our results should assist the development of strategies for the metabolic engineering of mutants for applications in the enzymatic hydrolysis for biochemical production.

Keywords: Penicillium oxalicum; cellulase; conidiation; development; homeodomain-containing transcription factors.

FIGURE 1

Analysis of the complete and homeodomain sequences of Htfs homologs. (A) Phylogenetic analysis of PoHtf1 homologs. The complete sequences of Htfs from Penicillium sp., Aspergillus sp., Trichoderma sp., Neurospora crassa, Fusarium graminearum, Pyricularia oryzae, and Podospora anserina were downloaded from NCBI. The phylogenetic tree was generated by MEGA 5.0 software using the neighbor-joining (NJ) method. (B) Sequence alignment of the Htfs homolog homeodomains. The alignment was performed by ClustalW Multiple Alignment function in the Bioedit tool. The consensus residues are labeled by asterisk (*).

FIGURE 2

Deletion and verification of PoHtf1. (A) PCR analysis of Pohtf1 deletion and complement. The confirmation of Pohtf1 deletion and complement was performed using three pairs of primers: primers inside the selected marker genes hph (hph-YZ-F/R) (lane 1–5) and ptrA (ptrA-YZ-F/R) (lane 6–10) and primers amplifying the target gene Pohtf1 (7199-hphF/7199-hphR) (lane 11–15). Primer sequences are listed in Supplementary Table 1. Lane 1, 6, 11 are the results of the WT strain P. oxalicum 114-2. Lane 2, 7, 12 are the results of Pohtf1-deletion strain. Lane 3–4, 8–9, and 13–14 are the results of two Pohtf1-complement strains. (B) Southern blot analysis of ΔPohtf1. After deletion of Pohtf1, a 4.8 kb-length fragment was hybridized in the ΔPohtf1 genome by Southern blot, while the fragment length in P. oxalicum 114-2 was 3.7 kb. (C) Southern blot strategy. A 2 kb-length fragment downstream of the target gene was amplified as the hybridization probe. P. oxalicum 114-2 genomic DNA was digested by a single restriction endonuclease, HindIII, and a 3.7 kb-length fragment containing the complete probe sequence was generated. The ΔPohtf1 genomic DNA was double digested with HindIII and EcoRI to generate a 4.8 kb-length fragment. (D) qRT-PCR analysis of Pohtf1 expression levels in the mutant strains. Pohtf1 expression levels in P. oxalicum 114-2, ΔPohtf1, and CPohtf1 were tested using primers RT-7199-F/R inside Pohtf1 (Supplementary Table 2).

FIGURE 3

Expression profile of 80 annotated cellulose hydrolyase genes in P. oxalicum 114-2 and ΔPohtf1. The expression levels (RPKM) of the 80 genes are labeled by green-yellow-red color scales in excel. The minimum value is 0, the median is 100, and the maximum value is 36,801.

FIGURE 4

Colony phenotypes of strains P. oxalicum 114-2, ΔPohtf1 and CPohtf1. Phenotype analysis was performed on Vogel’s medium plates containing 2% glucose or 1% cellulose, PDA plates and 10% wheat bran medium plates. The plates were incubated for 3 days at 30°C.

FIGURE 5

The effects of PoHtf1 on conidiation. (A) The conidiation curve of P. oxalicum 114-2, ΔPohtf1 and CPohtf1 on glucose plates. The plates were incubated at 30°C, and the spore numbers in a fixed area were counted at 24, 30, 36, 48, 60, and 72 h. (B) Expression of the major asexual development transcriptional regulators. All strains were incubated in the cellulase production medium for 4 h. The expression levels of brlA, stuA and fluC were measured by qRT-PCR.

FIGURE 6

Analysis of the cellulase activities of P. oxalicum 114-2, ΔPohtf1 and CPohtf1. (A) Filter Paper activity (FPA), (B) Endoglucanase, (C) Cellobiohydrolase and (D) β-glucosidase activities of strains P. oxalicum 114-2, ΔPohtf1 and CPohtf1 were assessed. All the strains were sampled and measured every 24 h from day 3 to day 6.

FIGURE 7

qRT-PCR analysis of the major cellulases and transcriptional regulators in P. oxalicum 114-2, ΔPohtf1 and CPohtf1. (A) Expression levels of the four cellulase genes, cbh1, eg1, bgl1, and bgl2; (B) Expression levels of the four transcriptional regulator genes, creA, clrB, xlnR, and amyR. All strains were incubated in the cellulase production medium for 4 h. The gene expression levels were measured by qRT-PCR.