An investigation into the possible regulation of the expression of genes by yapA in Talaromyces marneffei using the qRT- PCR method

An investigation into the possible regulation of the expression of genes by yapA in Talaromyces... Abstract The pathogenic dimorphic fungus Talaromyces marneffei is known to cause a fatal systemic mycosis in immunocompromised patients, especially in HIV patients in Southeast Asia. The basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. A prior study described that yapA was involved in the oxidative and nitrosative stress response in T. marneffei. Interestingly, an essential role of Saccharomyces cerevisiae Yap1p in the oxidative stress response is the activation of the transcription of its target genes. To identify the target genes of yapA in T. marneffei, the qRT-PCR method were used in this study. Investigation into the expression of genes which are probably regulated by yapA revealed that yapA controlled the expression of cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress conditions in all forms of conidium, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase with a similar relative expression level in all of the wild-type, mutant and complemented strains. These genes are involved in response against oxidative stress and nitrosative stress in this fungus. The data showed that they could be regulated by the yapA gene during stress conditions. Moreover, the yapA gene is also known to control red pigment production by inhibiting the regulation of the five polyketide synthase (pks) genes, pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) in the mould phase. In addition, it also regulates transcription in the laccase gene cluster including lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) in all stages of the T. marneffei lifecycle (conidia, mould, and yeast phase). This study suggests the importance of the role of the yapA gene in the stress response and virulence of T. marneffei. Talaromyces marneffei, yapA, bZIP transcription factor, stress response, qRT-PCR Introduction Talaromyces marneffei (Penicillium marneffei) is a pathogenic temperature-dependent dimorphic fungus that causes a systemic mycosis in immunocompromised patients, especially in patients diagnosed with human immunodeficiency virus (HIV) in Southeast Asia. In addition, the infection has also been reported as occurring in non–HIV-infected individuals with underlying immune defects, including patients with hyper immunoglobulin E (IgE) syndrome,1 x-link hyper IgM disease,2 SLE,3 and patients with impaired interferon γ (INF-γ) and TH17 response.4 The endemic areas of T. marneffei infection have been shown to be in Southeast Asia, particularly Thailand, Northeastern India, Hong Kong, Southern China, Vietnam, and Taiwan.5 However, there have been cases of T. marneffei infection in Ghana and Togo in patients who had never been in Asia.6,7Talaromyces marneffei is likely to be a primary pulmonary pathogen that disseminates to other internal organs via the hematogenous route. Once the conidia are inhaled into the lungs of the host, the conidia are phagocytosed and destroyed by the alveolar macrophages. However, in immunodeficient patients, the inhaled conidia could convert into yeast cells within the macrophages and spread to other organs.5,8 Resistance to stress conditions that are induced in the phagocytes is the key aspect of these fungi for intracellular survival. The basic leucine-zipper (bZip) transcription factor gene in Saccharomyces cerevisiae, namely, yap1 (yeast activating protein-1), is one of the most important determinants of the yeast's response to oxidative stress and is responsible for the transcriptional activation of various genes involved in ROS detoxification.9 An essential role of Yap1p in the oxidative stress response is the activation of transcription of its target genes. The Yap1p targets are those that are involved in the antioxidant defense mechanism including thioredoxin system genes and glutathione system genes. The yap1 target gene, thioredoxin encoding gene (trx2), is involved in the oxidative stress response. Also, the glutamylcysteine synthetase (gsh1) gene is one of several genes that are transcriptionally regulated by Yap1p.10 In addition, other glutathione genes, glutathione reductase (glr1), glutathione peroxidase (gpx2) and glutathione synthetase (gsh2), express dependently upon by the Yap1 transcriptional activator protein.11 Global genomic analysis of Δyap1 S. cerevisiae on exposure to H2O2 revealed that Yap1p could regulate the activities of the thioredoxin antioxidant system, glutathione system and phosphate pathways.12 This correlates with the inability of Δyap1 S. cerevisiae to adapt in stress conditions instigated by H2O2 and malondialdehyde.13 In the filamentous fungus Aspergillus fumigatus the Afyap1 gene regulates several defense genes against oxidative stress including the catalase gene, thioredoxin reductase, and other anti-oxidant genes.14 To date, the basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. Our previous study reported that the function of the T. marneffei yapA gene is related to the stress response and fungal growth, and it has been suggested that it may well be a potential virulence factor in T. marneffei.15 The objective of this study was to identify the genes that are transcriptionally regulated by yapA in T. marneffei under certain stress conditions, red pigment and laccase production by the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) method. Methods Fungal strains and culture conditions The genetically modified T. marneffei G809 strain (ΔligD::pyrG+niaD pyrG) modified from Talaromyces marneffei FRR2161 (CBS 334.59, ATCC18224),16 was kindly provided by Dr. Alex Andrianopoulos (Department of Genetics, The University of Melbourne, Parkville, Victoria, Australia). The strain G809, ΔyapA mutant (ΔligD pyrG− ΔyapA::niaD pyrG+) and complemented strain (ΔligD pyrG− ΔyapA::yapA niaD pyrG+) were cultured on ANM for 10 days. The genotypes of T. marneffei are described in Table 1. The conidia of wild-type, mutant and complemented strains were collected by cotton swab-scraping and resuspended in sterile normal saline containing tween 40. The suspension was filtered through sterile glass wool to remove the mycelial fragments. Table 1. Strains and genotypes of the T. marneffei in this study. Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] View Large Table 1. Strains and genotypes of the T. marneffei in this study. Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] View Large Culture conditions To study the expression of the genes possibly regulated by yapA, the expression patterns were determined from the asexual conidia, mycelia, and yeast phase. To investigate the asexual conidia, the conidia of wild-type, mutant, and complemented strains were collected from the cultures grown for 12 days at 25°C on ANM media. For mycelium phase, 108 conidia of wild-type, mutant, and complemented strains were cultured in 50 ml SDB at 25°C for 3 days in a shaking incubator with continuous shaking at 150 rpm. In contrast, yeast phase, 108 conidia of strain G809, mutant, and complemented strains were cultured in 50 ml SDB at 37°C for 3 days in a shaking incubator with continuous shaking at 150 rpm. For oxidative stress investigation, the conidia, mycelium, and yeast cells were incubated for 60 min at 37°C before extracting the RNA for use as a control. To generate the stress conditions, menadione, H2O2, and NO2 were added into the SDB broth containing the conidia, mycelium, and yeast cells and incubated for 60 min at 37°C. All cultures were incubated in a shaking incubator with continuous shaking at 150 rpm. For red pigment production and laccase production experiment, the 108 conidia of wild-type, mutant, and complemented strains were collected from the cultures grown for 12 days at 25°C on ANM media. For mycelium growth, the 108 conidia of wild-type, mutant, and complemented strains were cultured in 50 ml SDB at 25°C for 3 days with continuous shaking at 150 rpm. For yeast phase, 108 conidia of each strain were cultured in 50 ml SDB at 37°C for 3 days in a shaking incubator with continuous shaking at 150 rpm. Subsequently, all strains were examined for the expression of cluster of red pigment encoding genes and laccase encoding genes by qRT-PCR. The messenger RNA (mRNA) expression levels of mutant and complemented strains were compared with wild-type strain. The cells from each experiment were harvested by centrifugation at 4500g for 5 min. RNA samples were isolated using a NucleoSpin® RNA II kit. The internal control was amplified with specific primers for the actin gene.17 PCR was performed to detect any DNA contamination in the RNA sample. RNA extraction and cDNA synthesis RNA was extracted from T. marneffei cells by using a total RNA isolation kit (NucleoSpin® RNAII, Macherey-Nagel, GmbH & Co. KG, Düren, Germany) according to the manufacturer's instructions. The quantitative and qualitative variables of extracted RNA were measured using a UV spectrophotometer (Nanodrop 2000) in the range 1.80 to 2.20 for the A260/A280 ratio and 1.80 to 2.30 for the A260/A230 ratio. The DNA contamination in the RNA sample was detected by PCR method with Act1F and Act1R primers. The complementary DNA (cDNA) was synthesized with one microgram of the total RNA isolated from each fungal form using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Burlington, Canada). Briefly, 4 μl of 5 × reaction buffer, 1 μl of random hexamer primer, 1 μg total RNA, 1 μl of ribolock RNase inhibitor, 2 μl of 10 mM dNTP, 1 μl of RevertAid M-MuLV RT reverse transcriptase, and sterile nuclease free H2O to give a final volume of 20 μl were added into the reaction mixture. The cDNA was synthesized by incubating the reaction mixture for 5 min at 25°C, followed by 60 min at 42°C, and then the reaction was terminated by heating at 70°C for 5 min. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in this study was performed using the SYBR Green qPCR mix (Thunderbird SYBR Green Chemistry, Toyobo, Japan), and the intensity of the fluorescence signal was detected by using a 7500 Real-time PCR System (Applied Biosystems, Foster City, CA). There are three major phases in the qRT-PCR reaction: denaturation, annealing, and extension. The genes of interest were amplified according to the following conditions: one cycle of 95°C, 60 s; followed by 40 cycles of 95°C, 60 s and 60°C, 60 s. The melting curve analysis was determined for the presence or absence of primer dimers, which would indicate nonoptimal primer annealing temperatures. The housekeeping gene, act (β-actin) gene, was used as an endogenous control for qRT-PCR normalization. The mRNA expression levels of interest in different strains of T. marneffei (wild-type, mutant, and complemented strains) were calculated using the 2−ΔΔct formula. To determine the expression of yapA and putative genes probably regulated by yapA gene, RNA from each sample was subjected to the qRT-PCR method. The mRNA expression levels of yapA gene and oxidative stress response genes such as cat1 (catalase encoding gene), cpe1 (catalase-peroxidase encoding gene), and sodA (copper, zinc superoxide dismutase (Cu, Zn SOD)-encoding gene) were studied. In addition, the mRNA expression levels of antioxidant groups including thioredoxin groups such as trr1/trr2 (thioredoxin reductase encoding gene), and trxA (thioredoxin encoding gene) and the glutathione group for example gcs1 (glutamate-cysteine ligase encoding gene), and glr1 (glutathione oxidoreductase encoding gene) were examined. The primers used to amplify cat1, cpe1, sodA, trr1/2, trxA, gcs1, and glr1 genes are summarized in Table 2. Table 2. The primers used to amplify yapA and genes probably regulated by yapA (cat1, cpeA, sodA, trr1/2, trxA, gcs1, and glr1 genes) in this study. Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ View Large Table 2. The primers used to amplify yapA and genes probably regulated by yapA (cat1, cpeA, sodA, trr1/2, trxA, gcs1, and glr1 genes) in this study. Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ View Large To investigate whether yapA is involved in red pigment and laccase production, the T. marneffei red pigment biosynthetic genes, including pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) and dix genes that encode for multicopper oxidase proteins including lac (extracellular dihydrogeodin oxidase/laccase), PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 were examined. The primers used to amplify red pigment and laccase encoding genes are summarized in Table 3. Table 3. The primers used to amplify yapA and genes probably regulated by yapA (red pigment, extracellular dihydrogeodin oxidase/laccase encoding gene and multicopper oxidase encoding genes). Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ View Large Table 3. The primers used to amplify yapA and genes probably regulated by yapA (red pigment, extracellular dihydrogeodin oxidase/laccase encoding gene and multicopper oxidase encoding genes). Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ View Large RNA extraction and cDNA synthesis were performed and analyzed using qRT-PCR as explained above. The data of mRNA expression levels were calculated using the 2−ΔΔct formula and normalized against the endogenous control actin mRNA level. The statistical significance of the relative expression levels of these genes were calculated using the GraphPad Prism 5 program with an analysis of variance (ANOVA). Statistical significance was accepted when the P-value was less than or equal to .05. Results Downregulation of oxidative and nitrosative stress-related genes in the ΔyapA mutant An essential role of Yap1p in the oxidative stress response is its activation of the transcription of its target genes. The Yap1p targets are those that are involved in antioxidant defense mechanism including thioredoxin system genes and glutathione system genes. To investigate the expression levels of genes probably regulated by yapA, the wild-type, mutant, and complemented strains were incubated with oxidative stressors including: H2O2, menadione, and NaNO2. Then, RNA from each sample was subjected to qRT-PCR analysis. The oxidative stress response genes such as cat1 (catalase encoding gene), cpeA (catalase-peroxidase encoding gene), and sodA (copper, zinc superoxide dismutase encoding gene) were studied. In all stress conditions, the expression levels of these genes in the wild-type and complemented strains had higher relative expression levels than the mutant in all forms of conidia, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase, which showed similar relative expression level in all of the wild-type, mutant, and complemented strains (Figs. 1–3, A–C). Figure 1. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 1. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 2. View largeDownload slide Expression data from seven tested genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were grown for 72 h, at 25°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 2. View largeDownload slide Expression data from seven tested genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were grown for 72 h, at 25°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 3. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes (cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G)). Conidia of each strain were grown for 72 h, at 37°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 3. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes (cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G)). Conidia of each strain were grown for 72 h, at 37°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). In addition, the mRNA expression levels of the antioxidant groups including thioredoxin groups such as trr1/trr2 (thioredoxin reductase encoding gene), and the trxA (thioredoxin encoding gene) and glutathione group, for example, gcs1 (glutamate-cysteine ligase encoding gene), and glr1 (glutathione oxidoreductase encoding gene) were also decreased in all growth forms of the ΔyapA mutant (Figs. 1–3, D–G). The relative expression levels of these genes of yapA mutant are summarized in Table 3. The ΔyapA mutant had decreased expression levels of oxidative stress response genes in all growth forms with various levels of significance. This data indicated that the T. marneffei yapA gene of all growth forms (conidia, mycelium, and yeast phase) could regulate the oxidative stress response genes and the genes which are members of antioxidant groups under certain conditions of stress. The yapA gene involved in red pigment production Our previous study showed that the ΔyapA mutant had increased red pigment production at 25°C when cultured for 7 days on ANM agar (Fig. 4A) or 4 days in SD synthetic broth (Fig. 4B). Recently, the red pigment, which is produced by the mould form of T. marneffei, was investigated. In 2014, Woo and colleagues reported that five genes including pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) were involved in the biosynthesis of red pigment in T. marneffei.18 Figure 4. View largeDownload slide The red pigment production of T. marneffei when cultured for 7 days on ANM agar (A) or 4 days in SD synthetic broth (B). This Figure is reproduced in color in the online version of Medical Mycology. Figure 4. View largeDownload slide The red pigment production of T. marneffei when cultured for 7 days on ANM agar (A) or 4 days in SD synthetic broth (B). This Figure is reproduced in color in the online version of Medical Mycology. To answer the question of whether yapA could regulate the expression of the gene cluster involved with red pigment production, these pigment encoding gene expressions were examined by qRT-PCR. The ΔyapA mutant possessed a significantly higher relative expression level of these five genes, including pks3, rp1, rp2, rp3, and rp4, than the wild-type and complemented strains from the mould form (Fig. 5). Thus, the yapA gene may be involved in red pigment biosynthesis by negative regulation of these five genes. Figure 5. View largeDownload slide Expression data from five genes which involved the biosynthesis of red pigment rp1, rp2, rp3, rp4 and pks3 of the conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRTPCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 5. View largeDownload slide Expression data from five genes which involved the biosynthesis of red pigment rp1, rp2, rp3, rp4 and pks3 of the conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRTPCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). The yapA gene regulated laccase encoding gene expression Because Moap1 in Magnaporthe oryzae regulated the production of extracellular peroxidases and laccases which have been known to be involved in pigment production, we sought to determine if the yapA gene in T. marneffei regulated the production of extracellular laccase. Expressions of the six genes which encoded for extracellular dihydrogeodin oxidase/laccase (lac) and multicopper oxidase proteins such as PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 were determined by qRT-PCR. The relative expression levels of these six genes decreased significantly in the ΔyapA mutant in all growth forms (conidia, mould and yeast phase) (Fig. 6). This data indicated that the T. marneffei yapA gene of all growth forms (conidia, mycelium, and yeast phase) could regulate transcription in the laccase gene cluster. Figure 6. View largeDownload slide Expression data from six genes which involved the laccase encoding genes lac, PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 of conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 6. View largeDownload slide Expression data from six genes which involved the laccase encoding genes lac, PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 of conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Discussion The aim of this study was to identify the target genes of yapA in T. marneffei using the qRT-PCR method. Our previous results revealed that yapA is an important gene involved in growth, development, and pigmentation in T. marneffei. Moreover, the yapA gene plays a role in response against oxidative stress and nitrosative stress in T. marneffei. An important role of Yap1p in the stress response is the activation of the transcription of its target genes. To discover more detail concerning yapA target genes in T. marneffei, especially in control conditions of stress response and pigmentation. The expression levels of oxidative stress response genes such as cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress were examined in all growth phases (conidia, mycelia, and yeast phase). In addition, the expression of the red pigment biosynthetic gene cluster (pks3 [polyketide synthase], rp1 [transcription activator], rp2 [β-subunit fatty acid synthase], rp3 [α-subunit fatty acid synthase], and rp4 [oxidoreductase]) and laccase gene cluster including, lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) were investigated. Results showed reduction in the expression levels of these oxidative stress response genes (cat1, cpeA, sodA, gcs1, glr1, trr1/trr2, and trxA) in the yapA mutant. This result was similar to those found in the investigation into the function of Yap1 of S. cerevisiae that regulated glr1, gpx2 (glutathione peroxidase) and gsh2 (glutathione synthetase.11 Moreover, the Δpap1 mutant was found to be unable to promote expression of trx2, trr1 (thioredoxin reductase encoding gene), ctt1 (catalase encoding gene) in response to H2O2.19 Thus, yapA could control these oxidative stress responsible genes. However, the cat1 expression levels under conditions of oxidative stress in mold phase of the mutant were similar to the wild-type and complemented strains. It had been reported that the A. fumigatus yap1 controlled the catalase 2 but not cat1.14 Therefore, the function of catalases for scavenging ROS is probably regulated by another pathway in the mould phase. Our previous study showed that the ΔyapA mutant showed increased red pigment production when cultured at 25°C (Fig. 4A–B). We demonstrated that the ΔyapA mutant possessed a significantly higher relative expression level of pks3, rp1, rp2, rp3 and rp4 than the wild-type and complemented strains in the mould form (Fig. 5). Hence, the yapA gene may be involved in red pigment biosynthesis by negative regulation of these genes. The Moap1 gene in Magnaporthe oryzae has been reported as being involved in the production of extracellular peroxidases and laccases. The Moap1 mutant, which had reduced the activity of the secreted laccase, resulted in less pigmentation in the Moap1 mutant.20 Thus, we hypothesized that the yapA gene in T. marneffei possibly regulated the production of extracellular laccase. Our result showed that the yapA gene could regulate the laccase gene cluster, including lac (extracellular dihydrogeodin oxidase/laccase) and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060). Hence, the yapA gene is involved in the production of extracellular peroxidases and laccases by regulation of these genes. Moreover, the yap1 gene was isolated as gene that confers various drug resistances. The Yap1p mediated multidrug resistance in Saccharomyces cerevisiae by regulating the 3-amino-1, 2, 4-triazole and 4-nitroquinoline-N-oxide resistance (atr) transcription in response to the 3-amino-1, 2, 4-triazole and 4-nitroquinoline-N-oxide.21 Moreover, fluconazole resistance-1 (flr1) gene, encoding a membrane transporter of the major facilitator superfamily, contains three YRE in the promoter that influenced by Yap1p when cell treated with methylmethane sulfonate, benomyl drug and other oxidizing agents.22 In S. pombe, Pap1p is involved in multiple drugs resistance, such as brefeldin A or caffeine when Pap1p overexpressed by inducing expression of several genes such as Pap1-dependent efflux pumps Hba2 and two ABC-family transporter Caf3 and Caf5.23,24 In addition, some strains of T. marneffei have been reported to resist to fluconazole.5 Hence, the role of T. marneffei yapA gene in the regulation of gene expression in response to drug stress including fluconazole resistance-1 (flr1) gene, and azole resistance gene (erg11, erg16, cyp51) should be further investigated. In conclusion, yapA is the global transcription regulator that could regulate many downstream genes. Since the mutation of yapA can cause several defects involved in the pathogenesis of T. marneffei, this gene might be useful as a new target in the future control of T. marneffei infection. Acknowledgements This work was supported by the National Research University Project, which was authorized by the Higher Education Commission of Thailand, and the Faculty of Medicine, Chiang Mai University. We would like to express our gratitude to Dr. Alex Andrianopoulos for providing the T. marneffei strain G809 for this study. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Ma BH , Ng CS , Lam R et al. Recurrent hemoptysis with Penicillium marneffei and Stenotrophomonas maltophilia in Job's syndrome . Can Respir J . 2009 ; 16 : e50 – 52 . Google Scholar CrossRef Search ADS PubMed 2. 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The Aspergillus fumigatus transcriptional regulator AfYap1 represents the major regulator for defense against reactive oxygen intermediates but is dispensable for pathogenicity in an intranasal mouse infection model . Eukaryot Cell . 2007 ; 6 : 2290 – 2302 . Google Scholar CrossRef Search ADS PubMed 15. Dankai W , Pongpom M , Youngchim S , Cooper CR , Vanittanakom N . The yapA encodes bZIP transcription factor involved in stress tolerance in pathogenic fungus Talaromyces marneffei . PLoS One . 2016 ; 10 : e0163778 . doi: 10.1371/journal.pone.0163778 . Google Scholar CrossRef Search ADS 16. Bugeja HE , Boyce KJ , Weerasinghe H et al. Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei . Fungal Genet Biol . 2012 ; 49 : 772 – 778 . Google Scholar CrossRef Search ADS PubMed 17. Dankai W , Pongpom M , Vanittanakom N . Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei . J Microbiol Methods . 2015 ; 118 : 42 – 50 . Google Scholar CrossRef Search ADS PubMed 18. Woo PC , Lam C-W , Tam EW et al. The biosynthetic pathway for a thousand-year-old natural food colorant and citrinin in Penicillium marneffei . Sci Rep . 2014 ; 4 : 6728 . Google Scholar CrossRef Search ADS PubMed 19. Toone WM , Kuge S , Samuels M , Morgan BA , Toda T , Jones N . Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1 . Genes Dev . 1998 ; 12 : 1453 – 1463 . Google Scholar CrossRef Search ADS PubMed 20. Guo M , Chen Y , Du Y et al. The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae . PLoS Pathog. 2011 ; 7 : e1001302 . doi: 10.1371/journal.ppat.1001302. pmid:21383978 . Google Scholar CrossRef Search ADS PubMed 21. Toledano MB , Delaunay A , Biteau B , Spector D , Azevedo D . Oxidative stress responses in yeast. In Hohmann S , Mager WH , eds. Yeast Stress Responses , Berlin : Springer-Verlag , 2003 : 241 – 303 . 22. Nguyen DT , Alarco AM , Raymond M . Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agen . J Biol Chem . 2001 ; 276 : 1138 – 1145 . Google Scholar CrossRef Search ADS PubMed 23. Benko Z , Fenyvesvolgyi C , Pesti M , Sipiczki M . The transcription factor Pap1/Caf3 plays a central role in the determination of caffeine resistance in Schizosaccharomyces pombe . Mol Genet Genomics . 2004 ; 271 : 161 – 170 . Google Scholar CrossRef Search ADS PubMed 24. Calvo IA , Gabrielli N , Iglesias-Baena I et al. Genome-wide screen of genes required for caffeine tolerance in fission yeast . PLoS One . 2009 ; 8 : e6619 . Google Scholar CrossRef Search ADS © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

An investigation into the possible regulation of the expression of genes by yapA in Talaromyces marneffei using the qRT- PCR method

Medical Mycology , Volume 56 (6) – Aug 1, 2018

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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1369-3786
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Abstract

Abstract The pathogenic dimorphic fungus Talaromyces marneffei is known to cause a fatal systemic mycosis in immunocompromised patients, especially in HIV patients in Southeast Asia. The basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. A prior study described that yapA was involved in the oxidative and nitrosative stress response in T. marneffei. Interestingly, an essential role of Saccharomyces cerevisiae Yap1p in the oxidative stress response is the activation of the transcription of its target genes. To identify the target genes of yapA in T. marneffei, the qRT-PCR method were used in this study. Investigation into the expression of genes which are probably regulated by yapA revealed that yapA controlled the expression of cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress conditions in all forms of conidium, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase with a similar relative expression level in all of the wild-type, mutant and complemented strains. These genes are involved in response against oxidative stress and nitrosative stress in this fungus. The data showed that they could be regulated by the yapA gene during stress conditions. Moreover, the yapA gene is also known to control red pigment production by inhibiting the regulation of the five polyketide synthase (pks) genes, pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) in the mould phase. In addition, it also regulates transcription in the laccase gene cluster including lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) in all stages of the T. marneffei lifecycle (conidia, mould, and yeast phase). This study suggests the importance of the role of the yapA gene in the stress response and virulence of T. marneffei. Talaromyces marneffei, yapA, bZIP transcription factor, stress response, qRT-PCR Introduction Talaromyces marneffei (Penicillium marneffei) is a pathogenic temperature-dependent dimorphic fungus that causes a systemic mycosis in immunocompromised patients, especially in patients diagnosed with human immunodeficiency virus (HIV) in Southeast Asia. In addition, the infection has also been reported as occurring in non–HIV-infected individuals with underlying immune defects, including patients with hyper immunoglobulin E (IgE) syndrome,1 x-link hyper IgM disease,2 SLE,3 and patients with impaired interferon γ (INF-γ) and TH17 response.4 The endemic areas of T. marneffei infection have been shown to be in Southeast Asia, particularly Thailand, Northeastern India, Hong Kong, Southern China, Vietnam, and Taiwan.5 However, there have been cases of T. marneffei infection in Ghana and Togo in patients who had never been in Asia.6,7Talaromyces marneffei is likely to be a primary pulmonary pathogen that disseminates to other internal organs via the hematogenous route. Once the conidia are inhaled into the lungs of the host, the conidia are phagocytosed and destroyed by the alveolar macrophages. However, in immunodeficient patients, the inhaled conidia could convert into yeast cells within the macrophages and spread to other organs.5,8 Resistance to stress conditions that are induced in the phagocytes is the key aspect of these fungi for intracellular survival. The basic leucine-zipper (bZip) transcription factor gene in Saccharomyces cerevisiae, namely, yap1 (yeast activating protein-1), is one of the most important determinants of the yeast's response to oxidative stress and is responsible for the transcriptional activation of various genes involved in ROS detoxification.9 An essential role of Yap1p in the oxidative stress response is the activation of transcription of its target genes. The Yap1p targets are those that are involved in the antioxidant defense mechanism including thioredoxin system genes and glutathione system genes. The yap1 target gene, thioredoxin encoding gene (trx2), is involved in the oxidative stress response. Also, the glutamylcysteine synthetase (gsh1) gene is one of several genes that are transcriptionally regulated by Yap1p.10 In addition, other glutathione genes, glutathione reductase (glr1), glutathione peroxidase (gpx2) and glutathione synthetase (gsh2), express dependently upon by the Yap1 transcriptional activator protein.11 Global genomic analysis of Δyap1 S. cerevisiae on exposure to H2O2 revealed that Yap1p could regulate the activities of the thioredoxin antioxidant system, glutathione system and phosphate pathways.12 This correlates with the inability of Δyap1 S. cerevisiae to adapt in stress conditions instigated by H2O2 and malondialdehyde.13 In the filamentous fungus Aspergillus fumigatus the Afyap1 gene regulates several defense genes against oxidative stress including the catalase gene, thioredoxin reductase, and other anti-oxidant genes.14 To date, the basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. Our previous study reported that the function of the T. marneffei yapA gene is related to the stress response and fungal growth, and it has been suggested that it may well be a potential virulence factor in T. marneffei.15 The objective of this study was to identify the genes that are transcriptionally regulated by yapA in T. marneffei under certain stress conditions, red pigment and laccase production by the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) method. Methods Fungal strains and culture conditions The genetically modified T. marneffei G809 strain (ΔligD::pyrG+niaD pyrG) modified from Talaromyces marneffei FRR2161 (CBS 334.59, ATCC18224),16 was kindly provided by Dr. Alex Andrianopoulos (Department of Genetics, The University of Melbourne, Parkville, Victoria, Australia). The strain G809, ΔyapA mutant (ΔligD pyrG− ΔyapA::niaD pyrG+) and complemented strain (ΔligD pyrG− ΔyapA::yapA niaD pyrG+) were cultured on ANM for 10 days. The genotypes of T. marneffei are described in Table 1. The conidia of wild-type, mutant and complemented strains were collected by cotton swab-scraping and resuspended in sterile normal saline containing tween 40. The suspension was filtered through sterile glass wool to remove the mycelial fragments. Table 1. Strains and genotypes of the T. marneffei in this study. Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] View Large Table 1. Strains and genotypes of the T. marneffei in this study. Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] Strain Genotype (reference) G809/ yapA+ ΔligD::pyrG+niaD pyrG+ (genetically modified strain from ATCC18224) [16] ΔyapA (ΔligD pyrG− ΔyapA::niaD pyrG+ [15] CM1 (complemented strain) ΔligD pyrG− ΔyapA::yapA niaD pyrG+ [15] View Large Culture conditions To study the expression of the genes possibly regulated by yapA, the expression patterns were determined from the asexual conidia, mycelia, and yeast phase. To investigate the asexual conidia, the conidia of wild-type, mutant, and complemented strains were collected from the cultures grown for 12 days at 25°C on ANM media. For mycelium phase, 108 conidia of wild-type, mutant, and complemented strains were cultured in 50 ml SDB at 25°C for 3 days in a shaking incubator with continuous shaking at 150 rpm. In contrast, yeast phase, 108 conidia of strain G809, mutant, and complemented strains were cultured in 50 ml SDB at 37°C for 3 days in a shaking incubator with continuous shaking at 150 rpm. For oxidative stress investigation, the conidia, mycelium, and yeast cells were incubated for 60 min at 37°C before extracting the RNA for use as a control. To generate the stress conditions, menadione, H2O2, and NO2 were added into the SDB broth containing the conidia, mycelium, and yeast cells and incubated for 60 min at 37°C. All cultures were incubated in a shaking incubator with continuous shaking at 150 rpm. For red pigment production and laccase production experiment, the 108 conidia of wild-type, mutant, and complemented strains were collected from the cultures grown for 12 days at 25°C on ANM media. For mycelium growth, the 108 conidia of wild-type, mutant, and complemented strains were cultured in 50 ml SDB at 25°C for 3 days with continuous shaking at 150 rpm. For yeast phase, 108 conidia of each strain were cultured in 50 ml SDB at 37°C for 3 days in a shaking incubator with continuous shaking at 150 rpm. Subsequently, all strains were examined for the expression of cluster of red pigment encoding genes and laccase encoding genes by qRT-PCR. The messenger RNA (mRNA) expression levels of mutant and complemented strains were compared with wild-type strain. The cells from each experiment were harvested by centrifugation at 4500g for 5 min. RNA samples were isolated using a NucleoSpin® RNA II kit. The internal control was amplified with specific primers for the actin gene.17 PCR was performed to detect any DNA contamination in the RNA sample. RNA extraction and cDNA synthesis RNA was extracted from T. marneffei cells by using a total RNA isolation kit (NucleoSpin® RNAII, Macherey-Nagel, GmbH & Co. KG, Düren, Germany) according to the manufacturer's instructions. The quantitative and qualitative variables of extracted RNA were measured using a UV spectrophotometer (Nanodrop 2000) in the range 1.80 to 2.20 for the A260/A280 ratio and 1.80 to 2.30 for the A260/A230 ratio. The DNA contamination in the RNA sample was detected by PCR method with Act1F and Act1R primers. The complementary DNA (cDNA) was synthesized with one microgram of the total RNA isolated from each fungal form using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Burlington, Canada). Briefly, 4 μl of 5 × reaction buffer, 1 μl of random hexamer primer, 1 μg total RNA, 1 μl of ribolock RNase inhibitor, 2 μl of 10 mM dNTP, 1 μl of RevertAid M-MuLV RT reverse transcriptase, and sterile nuclease free H2O to give a final volume of 20 μl were added into the reaction mixture. The cDNA was synthesized by incubating the reaction mixture for 5 min at 25°C, followed by 60 min at 42°C, and then the reaction was terminated by heating at 70°C for 5 min. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in this study was performed using the SYBR Green qPCR mix (Thunderbird SYBR Green Chemistry, Toyobo, Japan), and the intensity of the fluorescence signal was detected by using a 7500 Real-time PCR System (Applied Biosystems, Foster City, CA). There are three major phases in the qRT-PCR reaction: denaturation, annealing, and extension. The genes of interest were amplified according to the following conditions: one cycle of 95°C, 60 s; followed by 40 cycles of 95°C, 60 s and 60°C, 60 s. The melting curve analysis was determined for the presence or absence of primer dimers, which would indicate nonoptimal primer annealing temperatures. The housekeeping gene, act (β-actin) gene, was used as an endogenous control for qRT-PCR normalization. The mRNA expression levels of interest in different strains of T. marneffei (wild-type, mutant, and complemented strains) were calculated using the 2−ΔΔct formula. To determine the expression of yapA and putative genes probably regulated by yapA gene, RNA from each sample was subjected to the qRT-PCR method. The mRNA expression levels of yapA gene and oxidative stress response genes such as cat1 (catalase encoding gene), cpe1 (catalase-peroxidase encoding gene), and sodA (copper, zinc superoxide dismutase (Cu, Zn SOD)-encoding gene) were studied. In addition, the mRNA expression levels of antioxidant groups including thioredoxin groups such as trr1/trr2 (thioredoxin reductase encoding gene), and trxA (thioredoxin encoding gene) and the glutathione group for example gcs1 (glutamate-cysteine ligase encoding gene), and glr1 (glutathione oxidoreductase encoding gene) were examined. The primers used to amplify cat1, cpe1, sodA, trr1/2, trxA, gcs1, and glr1 genes are summarized in Table 2. Table 2. The primers used to amplify yapA and genes probably regulated by yapA (cat1, cpeA, sodA, trr1/2, trxA, gcs1, and glr1 genes) in this study. Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ View Large Table 2. The primers used to amplify yapA and genes probably regulated by yapA (cat1, cpeA, sodA, trr1/2, trxA, gcs1, and glr1 genes) in this study. Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ Primer Product Gene name Sequence size (bp) cat1 (XM_002151929) Cat1-Forward 5΄ ggA TCA AgC CCg ATg gAA CA 3΄ 257 This study Cat1-Reverse 5΄ CTC gCT TTg Agg CCA gAC TT 3΄ cpeA (AF537129.1) Cpe-Forward 5΄ CTT CAA AAg Cgg CAA ggg TC 3΄ 173 This study Cpe-Reverse 5΄ AAg CCT CTg CAT TTT Tgg Cg 3΄ sodA (DQ413185.1) SodA-Forward 5΄ TCT CAT ggA ACA TCA CCg gC 3΄ 239 This study SodA-Reverse 5΄ gCT CTC AgC ACC gAT CAA CT 3΄ glr1 (XM_002146024.1) Glr-Forward 5΄ TgT gAT Tgg Agg Cgg AAg 3΄ 158 This study Glr-Reverse 5΄ CAC gCT TgA AAT ggC CgT Ag 3΄ gcs1 (XM_002143483.1) Gcs-Forward 5΄ TTg AgT gTA CTg CCT CTg Cg 3΄ 175 This study Gcs-Revese 5΄ TCT CgT TCT TTC CCC TTg gC 3΄ trr1/2 (XM_002151094.1) Trr1/2-Forward 5΄ CAC TCg TgT CgA CTT CAg C 3΄ 158 This study Trr1/2 Reverse 5΄ gCA ggC CgA gAT ACC gTT TT 3΄ trxA (XM_002144941.1) TrxA-Forward 5΄ CTC TTC gCC AgT CAC AAT 3΄ 196 This study TrxA-Reverse 5΄ gTC gAC gTC AAA CTT gAC AAA 3΄ View Large To investigate whether yapA is involved in red pigment and laccase production, the T. marneffei red pigment biosynthetic genes, including pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) and dix genes that encode for multicopper oxidase proteins including lac (extracellular dihydrogeodin oxidase/laccase), PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 were examined. The primers used to amplify red pigment and laccase encoding genes are summarized in Table 3. Table 3. The primers used to amplify yapA and genes probably regulated by yapA (red pigment, extracellular dihydrogeodin oxidase/laccase encoding gene and multicopper oxidase encoding genes). Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ View Large Table 3. The primers used to amplify yapA and genes probably regulated by yapA (red pigment, extracellular dihydrogeodin oxidase/laccase encoding gene and multicopper oxidase encoding genes). Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ Primer Product Gene name Sequence size (bp) pks3 Polyketide synthase LPW9874 5΄ ggg gTA CCC CTT CTC TTT Cgg ATC TCT TC 3΄ 585 (Woo et al. 2014) LPW9875 5΄ gAA gAT CTg CCT AAT gTC AAg CTT TTC g 3΄ rp1 Transcriptional factor LPW 18208 5΄ggg gTA CCC TgC Tgg CgA TAC CgA gTT C3΄ 365 (Woo et al. 2014) LPW 18209 5΄ gAA gAT CTg CAA ggC ATC AgC TCA ATg A 3΄ rp2 Fatty acid synthase beta subunit LPW 17708 5΄ ggg gTA CCC ATg ggC TAC TCg gTT Tgg A 3΄ 384 (Woo et al. 2014) LPW 17709 5΄ gAA gAT CTg TTC gCC TTT ggA gTT CTg C 3΄ rp3 3-oxoacyl-[acyl-carrier-protein] synthase LPW 18212 5΄ ggg gTA CCg CAg TAA TCg gTT ggg TTC g 3΄ 359 (Woo et al. 2014) LPW 18213 5΄ gAA gAT CTC gCA Tgg AAC TgA Agg ATg A 3΄ rp4 Oxidoreductase LPW 18216 5΄ ggg gTA CCA AAg TCA ATg ACC CTg CCg A 3΄ 348 (Woo et al. 2014) LPW 18217 5΄ gAA gAT CTg TCA AAg ACC Tgg CTg gCA C 3΄ lac (XM_002144961.1) extracellular dihydrogeodin oxidase/laccase LacF 5΄gAT ggT gTA gCT ggC ggT AT 3΄ 227 This study LacR 5΄gTC gTC TCA AAg CgA gTT CC 3΄ PMAA_050860 multicopper oxidase PMAA_050860F 5΄ AgC CAA CAA ATg ggT CTA Cg 3΄ 173 This study PMAA_050860R 5΄ CCA ATA gCA Tgg Cgg TAT CT 3΄ PMAA_072680 multicopper oxidase PMAA_072680F 5΄ ACg AAC ACg CCA ATC TAT CC 3΄ 247 This study PMAA_072680R 5΄ gCT TTT gCg TCC AAg AgA AC 3΄ PMAA_085520 multicopper oxidase PMAA_085520F 5΄ CCC TCT TCA ACA ATC CCA gA 3΄ 189 This study PMAA_085520R 5΄ gCg ATT gAA gAA gCC gTA AG 3΄ PMAA_082010 multicopper oxidase PMAA_082010F 5΄ Tgg CAT ggC TTA CAC CAA TA 3΄ 204 This study PMAA_082010R 5΄ gAA Agg ggg TTT Tgg ATC AT 3΄ PMAA_082060 multicopper oxidase PMAA_082060F 5΄ AgA ggA gAg gCC Tgg ATA gC 3΄ 218 This study PMAA_082060R 5΄ gTT CAT gCC CTC gTT TgT TT 3΄ View Large RNA extraction and cDNA synthesis were performed and analyzed using qRT-PCR as explained above. The data of mRNA expression levels were calculated using the 2−ΔΔct formula and normalized against the endogenous control actin mRNA level. The statistical significance of the relative expression levels of these genes were calculated using the GraphPad Prism 5 program with an analysis of variance (ANOVA). Statistical significance was accepted when the P-value was less than or equal to .05. Results Downregulation of oxidative and nitrosative stress-related genes in the ΔyapA mutant An essential role of Yap1p in the oxidative stress response is its activation of the transcription of its target genes. The Yap1p targets are those that are involved in antioxidant defense mechanism including thioredoxin system genes and glutathione system genes. To investigate the expression levels of genes probably regulated by yapA, the wild-type, mutant, and complemented strains were incubated with oxidative stressors including: H2O2, menadione, and NaNO2. Then, RNA from each sample was subjected to qRT-PCR analysis. The oxidative stress response genes such as cat1 (catalase encoding gene), cpeA (catalase-peroxidase encoding gene), and sodA (copper, zinc superoxide dismutase encoding gene) were studied. In all stress conditions, the expression levels of these genes in the wild-type and complemented strains had higher relative expression levels than the mutant in all forms of conidia, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase, which showed similar relative expression level in all of the wild-type, mutant, and complemented strains (Figs. 1–3, A–C). Figure 1. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 1. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 2. View largeDownload slide Expression data from seven tested genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were grown for 72 h, at 25°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 2. View largeDownload slide Expression data from seven tested genes which function as anti-oxidant genes: cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G). Conidia of each strain were grown for 72 h, at 25°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 3. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes (cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G)). Conidia of each strain were grown for 72 h, at 37°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 3. View largeDownload slide Expression data from seven genes which function as anti-oxidant genes (cat1 (A), cpeA (B), sodA (C), gcs (D), glr (E), trr1/2 (F), and trxA (G)). Conidia of each strain were grown for 72 h, at 37°C then incubated with stressor at 37°C for 1 h. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). In addition, the mRNA expression levels of the antioxidant groups including thioredoxin groups such as trr1/trr2 (thioredoxin reductase encoding gene), and the trxA (thioredoxin encoding gene) and glutathione group, for example, gcs1 (glutamate-cysteine ligase encoding gene), and glr1 (glutathione oxidoreductase encoding gene) were also decreased in all growth forms of the ΔyapA mutant (Figs. 1–3, D–G). The relative expression levels of these genes of yapA mutant are summarized in Table 3. The ΔyapA mutant had decreased expression levels of oxidative stress response genes in all growth forms with various levels of significance. This data indicated that the T. marneffei yapA gene of all growth forms (conidia, mycelium, and yeast phase) could regulate the oxidative stress response genes and the genes which are members of antioxidant groups under certain conditions of stress. The yapA gene involved in red pigment production Our previous study showed that the ΔyapA mutant had increased red pigment production at 25°C when cultured for 7 days on ANM agar (Fig. 4A) or 4 days in SD synthetic broth (Fig. 4B). Recently, the red pigment, which is produced by the mould form of T. marneffei, was investigated. In 2014, Woo and colleagues reported that five genes including pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) were involved in the biosynthesis of red pigment in T. marneffei.18 Figure 4. View largeDownload slide The red pigment production of T. marneffei when cultured for 7 days on ANM agar (A) or 4 days in SD synthetic broth (B). This Figure is reproduced in color in the online version of Medical Mycology. Figure 4. View largeDownload slide The red pigment production of T. marneffei when cultured for 7 days on ANM agar (A) or 4 days in SD synthetic broth (B). This Figure is reproduced in color in the online version of Medical Mycology. To answer the question of whether yapA could regulate the expression of the gene cluster involved with red pigment production, these pigment encoding gene expressions were examined by qRT-PCR. The ΔyapA mutant possessed a significantly higher relative expression level of these five genes, including pks3, rp1, rp2, rp3, and rp4, than the wild-type and complemented strains from the mould form (Fig. 5). Thus, the yapA gene may be involved in red pigment biosynthesis by negative regulation of these five genes. Figure 5. View largeDownload slide Expression data from five genes which involved the biosynthesis of red pigment rp1, rp2, rp3, rp4 and pks3 of the conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRTPCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 5. View largeDownload slide Expression data from five genes which involved the biosynthesis of red pigment rp1, rp2, rp3, rp4 and pks3 of the conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRTPCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). The yapA gene regulated laccase encoding gene expression Because Moap1 in Magnaporthe oryzae regulated the production of extracellular peroxidases and laccases which have been known to be involved in pigment production, we sought to determine if the yapA gene in T. marneffei regulated the production of extracellular laccase. Expressions of the six genes which encoded for extracellular dihydrogeodin oxidase/laccase (lac) and multicopper oxidase proteins such as PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 were determined by qRT-PCR. The relative expression levels of these six genes decreased significantly in the ΔyapA mutant in all growth forms (conidia, mould and yeast phase) (Fig. 6). This data indicated that the T. marneffei yapA gene of all growth forms (conidia, mycelium, and yeast phase) could regulate transcription in the laccase gene cluster. Figure 6. View largeDownload slide Expression data from six genes which involved the laccase encoding genes lac, PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 of conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Figure 6. View largeDownload slide Expression data from six genes which involved the laccase encoding genes lac, PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060 of conidia (A), mold (B) and yeast (C) phase. RNA was isolated and then analyzed using qRT-PCR. Data are shown as the 2-ΔΔct levels calculated relative to the normal condition, normalized against endogenous control actin mRNA levels. The data are representative of three different experiments. (NS = p > 0.05, * = p ≤ 0.05, ** = p < 0.01 and *** = p < 0.001). Discussion The aim of this study was to identify the target genes of yapA in T. marneffei using the qRT-PCR method. Our previous results revealed that yapA is an important gene involved in growth, development, and pigmentation in T. marneffei. Moreover, the yapA gene plays a role in response against oxidative stress and nitrosative stress in T. marneffei. An important role of Yap1p in the stress response is the activation of the transcription of its target genes. To discover more detail concerning yapA target genes in T. marneffei, especially in control conditions of stress response and pigmentation. The expression levels of oxidative stress response genes such as cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress were examined in all growth phases (conidia, mycelia, and yeast phase). In addition, the expression of the red pigment biosynthetic gene cluster (pks3 [polyketide synthase], rp1 [transcription activator], rp2 [β-subunit fatty acid synthase], rp3 [α-subunit fatty acid synthase], and rp4 [oxidoreductase]) and laccase gene cluster including, lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) were investigated. Results showed reduction in the expression levels of these oxidative stress response genes (cat1, cpeA, sodA, gcs1, glr1, trr1/trr2, and trxA) in the yapA mutant. This result was similar to those found in the investigation into the function of Yap1 of S. cerevisiae that regulated glr1, gpx2 (glutathione peroxidase) and gsh2 (glutathione synthetase.11 Moreover, the Δpap1 mutant was found to be unable to promote expression of trx2, trr1 (thioredoxin reductase encoding gene), ctt1 (catalase encoding gene) in response to H2O2.19 Thus, yapA could control these oxidative stress responsible genes. However, the cat1 expression levels under conditions of oxidative stress in mold phase of the mutant were similar to the wild-type and complemented strains. It had been reported that the A. fumigatus yap1 controlled the catalase 2 but not cat1.14 Therefore, the function of catalases for scavenging ROS is probably regulated by another pathway in the mould phase. Our previous study showed that the ΔyapA mutant showed increased red pigment production when cultured at 25°C (Fig. 4A–B). We demonstrated that the ΔyapA mutant possessed a significantly higher relative expression level of pks3, rp1, rp2, rp3 and rp4 than the wild-type and complemented strains in the mould form (Fig. 5). Hence, the yapA gene may be involved in red pigment biosynthesis by negative regulation of these genes. The Moap1 gene in Magnaporthe oryzae has been reported as being involved in the production of extracellular peroxidases and laccases. The Moap1 mutant, which had reduced the activity of the secreted laccase, resulted in less pigmentation in the Moap1 mutant.20 Thus, we hypothesized that the yapA gene in T. marneffei possibly regulated the production of extracellular laccase. Our result showed that the yapA gene could regulate the laccase gene cluster, including lac (extracellular dihydrogeodin oxidase/laccase) and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060). Hence, the yapA gene is involved in the production of extracellular peroxidases and laccases by regulation of these genes. Moreover, the yap1 gene was isolated as gene that confers various drug resistances. The Yap1p mediated multidrug resistance in Saccharomyces cerevisiae by regulating the 3-amino-1, 2, 4-triazole and 4-nitroquinoline-N-oxide resistance (atr) transcription in response to the 3-amino-1, 2, 4-triazole and 4-nitroquinoline-N-oxide.21 Moreover, fluconazole resistance-1 (flr1) gene, encoding a membrane transporter of the major facilitator superfamily, contains three YRE in the promoter that influenced by Yap1p when cell treated with methylmethane sulfonate, benomyl drug and other oxidizing agents.22 In S. pombe, Pap1p is involved in multiple drugs resistance, such as brefeldin A or caffeine when Pap1p overexpressed by inducing expression of several genes such as Pap1-dependent efflux pumps Hba2 and two ABC-family transporter Caf3 and Caf5.23,24 In addition, some strains of T. marneffei have been reported to resist to fluconazole.5 Hence, the role of T. marneffei yapA gene in the regulation of gene expression in response to drug stress including fluconazole resistance-1 (flr1) gene, and azole resistance gene (erg11, erg16, cyp51) should be further investigated. In conclusion, yapA is the global transcription regulator that could regulate many downstream genes. Since the mutation of yapA can cause several defects involved in the pathogenesis of T. marneffei, this gene might be useful as a new target in the future control of T. marneffei infection. Acknowledgements This work was supported by the National Research University Project, which was authorized by the Higher Education Commission of Thailand, and the Faculty of Medicine, Chiang Mai University. We would like to express our gratitude to Dr. Alex Andrianopoulos for providing the T. marneffei strain G809 for this study. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Ma BH , Ng CS , Lam R et al. Recurrent hemoptysis with Penicillium marneffei and Stenotrophomonas maltophilia in Job's syndrome . Can Respir J . 2009 ; 16 : e50 – 52 . Google Scholar CrossRef Search ADS PubMed 2. 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Toledano MB , Delaunay A , Biteau B , Spector D , Azevedo D . Oxidative stress responses in yeast. In Hohmann S , Mager WH , eds. Yeast Stress Responses , Berlin : Springer-Verlag , 2003 : 241 – 303 . 22. Nguyen DT , Alarco AM , Raymond M . Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agen . J Biol Chem . 2001 ; 276 : 1138 – 1145 . Google Scholar CrossRef Search ADS PubMed 23. Benko Z , Fenyvesvolgyi C , Pesti M , Sipiczki M . The transcription factor Pap1/Caf3 plays a central role in the determination of caffeine resistance in Schizosaccharomyces pombe . Mol Genet Genomics . 2004 ; 271 : 161 – 170 . Google Scholar CrossRef Search ADS PubMed 24. Calvo IA , Gabrielli N , Iglesias-Baena I et al. Genome-wide screen of genes required for caffeine tolerance in fission yeast . PLoS One . 2009 ; 8 : e6619 . Google Scholar CrossRef Search ADS © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Medical MycologyOxford University Press

Published: Aug 1, 2018

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