Fig 1.
Comparison of conidial stress resistance.
Conidia of A. fumigatus strain Af293 were harvested after 7 d of growth on PDA at 25, 37, and 45°C. The survival rates upon heat stress (60°C, 15 min) (A), oxidative stress (200 mM hydrogen peroxide, 15 min) (B), and UV stress (573 J m−2) (C) are shown. The survival rate (%) was calculated by dividing the number of colony-forming units (CFU) following the stress treatment by the number of CFU obtained in the absence of treatment. (D) Upon heat stress (60°C, 15 min) the survival rates of the conidia harvested after 3, 7, and 14 d of growth at 25 and 37°C were shown. Each sample was tested in 3 or 4 technical replicates. Error bars represent the standard deviation. Experiments were repeated twice, and representative experiments are shown. Statistical analysis (Student’s t-test) was performed, and significant differences were indicated as an asterisk (**:p<0.01; *:p<0.05).
Fig 2.
Polyols and trehalose contents of conidia.
(A) Polyols and trehalose were extracted from conidia and analyzed by high-performance liquid chromatography (HPLC). Error bars represent the standard deviation based on six independent replicates. (B) Expression analysis of trehalose metabolism related genes in the conidia from the 25 and 37°C cultures. RNA was extracted from Af293 conidia that were produced at the indicated temperature. The expression of tpsA-D, orlA, and treA,B was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Each sample was tested in three biological replicates. Error bars represent the standard deviation. Statistical analysis (Student’s t-test) was performed, and significant differences were indicated as an asterisk (**:p<0.01; *:p<0.05).
Fig 3.
Comparison of conidial pigmentation.
(A) Conidia of A. fumigatus strain Af293 were harvested from 25, 37, and 45°C cultures. The conidial suspensions were diluted to 1x108 conidia mL−1 and photographed. (B) Conidia were harvested after cultivation in the presence or absence of tricyclazole (TCZ). The conidial suspensions were diluted to 5x107 conidia mL−1 and photographed. (C) The conidia were harvested from 25 and 37°C cultures at days 3, 5, 7, and 10. The conidial suspensions were diluted to 1x108 conidia mL−1 and photographed. (D) Expression analysis of the DHN-melanin biosynthesis pathway in the conidia from the 25 and 37°C cultures. RNA was purified from Af293 conidia that were produced at the indicated temperature. The relative expression ratio of arp1, arp2, and pksP to actin gene was determined by qRT-PCR. Each sample was tested in three biological replicates. Error bars represent the standard deviation. Statistical analysis (Student’s t-test) was performed, and significant differences were indicated as an asterisk (*:p<0.05).
Table 1.
FPKM data for the genes that were enriched at different conidiation temperatures.
Fig 4.
The trypacidin (tpc) cluster is induced in conidia at a low temperature.
(A) Using data from the RNA sequencing analysis, expression values (FPKM) for the genes in the tpc cluster were depicted. The arrows shown below the graph indicate the direction and relative length of the genes. (B) The MIDDAS-M analysis revealed that the expression of clustered genes is coordinately altered in the conidia from 25 and 45°C cultures, compared with those from a 37°C culture.
Table 2.
FPKM data for secondary metabolism genes.
Fig 5.
Chromatograms from the HPLC analysis of the WT and ΔtpcC strains.
Samples were extracted from conidia that were harvested from 25 and 37°C cultures. The crude extract was injected into an HPLC Wakosil-II system with a 3C18 HG column, and it was monitored by a photodiode array detector. The chromatograms detected by the absorbance at 330 nm are shown. Arrows (a and b) indicate the peaks specific to WT conidia from the 25°C culture. Arrow a: trypacidin. Arrow b: monomethylsulochrin.
Fig 6.
Quantification of trypacidin production in clinical isolates.
(A) Samples were extracted from the conidia of clinical isolates as well as Af293 strains that were harvested from 25°C and 37°C cultures, and they were analyzed by HPLC. The amount of trypacidin was quantified using a standard curve. Each sample was tested in 3 to 5 replicates. Error bars represent the standard deviation. Significant differences (by Student’s t-test) between conidia from 25°C and 37°C cultures were indicated with asterisk (*: p<0.05). (B) Gene expression analysis of tpcC in the clinical isolates. The eight clinical isolates were grown on PDA for 7 d at 25 and 37°C. Then, conidia were harvested, and their RNA was extracted. The expression ratio of tpcC relative to actin was determined by qRT-PCR analysis. Error bars represent standard deviations based on three biological replicates.
Fig 7.
Effect of temperature on pigmentation in the clinical isolates.
(A) Comparison of conidial pigmentation. Conidia of the clinical isolates were harvested from 25 and 37°C cultures. Conidial suspensions were diluted to 108 conidia mL−1 and photographed. (B) Expression analysis of pksP. RNA was extracted from conidia that were produced on PDA at 25 and 37°C. The expression ratio of pksP was determined by qRT-PCR. Each sample was tested in three biological replicates. Error bars represent standard deviations. (C) Trehalose was extracted from the conidia of clinical isolates and quantified by glucose assay after trehalase digestion as described previously [5]. Error bars represent the standard deviation based on three independent replicates. Significant differences (by Student’s t-test) between conidia from 25°C and 37°C cultures were indicated with asterisk (**: p<0.01).