Fig 1.
Construction of RNA interference cassettes.
RNA interference cassettes were constructed based on the pSilent-1 plasmid [25]. The inserted fragments were obtained by PCR following restriction enzyme digestion. Primers are indicated in black boxes. IS, inserted fragment; IT, intron 2 of cutinase (CUT) gene from Magnaporthe oryzae; PtrpC, promotor of trpC from A. nidulans; TtrpC, trpC terminator of A. nidulans; Hygr, hygromycin resistance; Ampr, ampicillin resistance. Restriction enzyme sites are also indicated.
Fig 2.
Gene expression of gna-1 and pkaR in RNAi mutants.
(A) Transcript levels of the gna-1 were detected by qRT-PCR in the wild-type, pG and pGP transformants using the primers qGNA(s) and qGNA(as). (B) Transcript levels of pkaR were measured by qRT-PCR in the wild-type and pGP transformants using the primers qPKAR(s) and qPKAR(as). Transcripts of gna-1 and pkaR were normalized against actin amplified with primers qActin(s) and qActin (as) (S1 Appendix). There is significantly difference between the mutant and wild-type as indicated by an asterisk (p-value <0.05 with T-test analysis) or by two asterisks (p-value <0.01 with T-test analysis). Experiments were performed in triplicate.
Fig 3.
Knock-down of gna-1 leads to deficiency in melanin, perithecia, and ChA production in C. globosum.
(A) Colony morphology of silenced mutants of gna-1, or gna-1/pkaR double knock-down. All mutants were inoculated in PDA medium supplemented with 100 mg/L hygromycin B and incubated at 28°C for 9 days. (B) Light microscopy involving mycelium and perithecia formation in C. globosum NK102 (WT) and RNAi mutant pG14 and pGP6. Scale bar represents 20 μm.
Fig 4.
Gene expression of pks-1 in RNAi mutants.
Transcript levels of the pks-1 were detected by qRT-PCR in the wild-type, pG and pGP transformants using the primers qPKS(s) and qPKS(as). There is significantly difference between the mutant and wild-type as indicated by two asterisks (p-value <0.01 with T-test analysis), ns: no significant difference. Experiments were performed in triplicate.
Fig 5.
Diminished biosynthesis of ChA.
(A) HPLC analysis on the production of ChA in the wild-type and the RNAi mutants. Arrow indicates the peak of ChA. (B) The cAMP assay for the silenced mutants. Relative expression of gene CgcheA from the wild-type (WT) and the transformant pG14 in the presence of 8-Br-cAMP(C) and H-89(D) at indicated concentrations.
Table 1.
Yields of chaetoglobosin A in different strains.
Fig 6.
Quantification of the gene candidates likely involved in ChA biosynthesis by qRT-PCR.
(A) Relative expression levels by qRT-PCR analysis of six key genes, chosen from the RNA-seq profiling and found in the chaetoglobosin biosynthetic gene cluster, in the transformants compared with the wild-type (WT) strain. The six genes are C6 zinc finger protein (CHGG_01237), transposase (CHGG_01238), PKS−NRPS hybrid gene cluster, CgcheA (CHGG_01239), P450 oxygenase (CHGG_012421), FAD-dependent oxidoreductase (CHGG_012422) and P450 oxygenase (CHGG_01243). All transcripts were normalized against actin cDNA amplified with primers qActin(s) and qActin(as) (S1 Appendix). (B) Decreased expression levels of CglaeA in pG14 and restoration in pGP6. There is significantly difference between the mutant and wild-type as indicated by two asterisks (p-value <0.001 with T-test analysis), ns: no significant difference. Experiments were performed in triplicate.
Table 2.
Expression variation of genes putatively related to ChA biosynthesis detected by RNA-seq profiling.
Fig 7.
Suggested schematic on the regulatory network responsible for ChA and melanin biosynthesis of C. globosum.