Two Photolyases Repair Distinct DNA Lesions and Reactivate UVB-Inactivated Conidia of an Insect Mycopathogen under Visible Light

Bioinformatic analysis of CPF homologs in B. bassiana.Three CPF homologs were located in the genomic database of B. bassiana (30) under the NCBI RefSeq accession no. NZ_ADAH00000000 by Blast analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) based on the queries of all CPF sequences of Aspergillus fumigatus and U. maydis in the NCBI protein databases. The sequences of located Phr1, Phr2, and CryD (GenBank accession numbers EJP69699, EJP70165, and EJP68422, respectively) were aligned for structural comparison by means of the SMART program (http://smart.embl-heidelberg.de). The phylogenetic relationships of each with the corresponding homologs of some other entomopathogenic or nonentomopathogenic fungi were analyzed using a neighbor-joining method in the MEGA7 software (http://www.megasoftware.net).

Subcellular localization of CPF homologs in B. bassiana.The open reading frames (ORFs) of phr1, phr2, and cryD and the promoter region of the homogenous gene tef encoding translation elongation factor 1 alpha (GenBank accession no. EJP68386) were amplified from the WT cDNA and genomic DNA with paired primers (Table S1), respectively. The vector pAN52-C-bar in which the C cassette 5′-PmeI-SpeI-EcoRV-EcoRI-BamHI-3′ follows the Aspergillus nidulans promoter PtrpC (34) was digested with BglII/NcoI for a replacement of PtrpC by Ptef. The resultant cassette was digested with EcoRI/BamHI and ligated to GFP (GenBank accession no. U55763). The resultant vector pAN52-C-GFP-bar was digested with EcoRV. Each amplified ORF was then inserted into the N terminus of GFP in the linearized vector using a one-step cloning kit (Vazyme, Nanjing, China), forming pAN52-x::GFP-bar (x = phr1, phr2, or cryD). The final plasmid was transformed into WT through blastospore transformation (44). Putative transformants were screened by the bar resistance to phosphinothricin (200 μg ml⁻¹) and examined under a fluorescence microscope. For each transformation, a strain showing maximal green signal was grown on SDAY (4% glucose, 1% peptone, and 1.5% agar plus 1% yeast extract) for full conidiation. The conidia were suspended in SDB (i.e., agar-free SDAY) and incubated at 25°C for 2 days on a shaking (150 rpm) bed under continuously illuminated and full-dark conditions, respectively. Hyphal cells harvested from each culture were stained with the nucleus-specific dye DAPI (4′,6′-diamidine-2′-phenylindole dihydrochloride) and visualized for subcellular localization of each GFP-tagged fusion protein through LSCM.

Construction of phr1, phr2, and cryD mutants.Our previous backbone plasmids and protocols (32, 45) were used to delete each of the CPF-coding genes from the WT strain by homogeneous recombination of its 5′ and 3′ coding/flanking fragments separated by the bar marker (Fig. S3A) and rescue it in the corresponding deletion mutant by ectopic integration of a cassette consisting of its full-length coding/flanking sequence and sur marker. Briefly, the 5′ and 3′ fragments of each target gene were amplified from the genomic DNA of WT with paired primers (Table S1) and inserted into p0380-bar at appropriate enzyme sites using the one-step cloning kit (Vazyme), forming p0380-5′x-bar-3′x (x = phr1, phr2, or cryD) for targeted gene deletion. The full-length sequence of each target gene with flanking regions was amplified from the WT DNA and inserted into p0380-sur-gateway to exchange for the gateway fragment under the action of Gateway BP Clonase II enzyme mix (Invitrogen, Shanghai, China), yielding p0380-sur-x for targeted gene complementation. The resultant plasmids were propagated in Escherichia coli TOP10 or E. coli DH5a (Invitrogen) and transformed into the WT strain or the corresponding deletion mutant via Agrobacterium-mediated transformation. Putative mutants were screened by the bar resistance to phosphinothricin (200 μg ml⁻¹) or the sur resistance to chlorimuron ethyl (15 μg ml⁻¹) in a selective medium and identified through PCR and Southern blot analyses with paired primers and designed probes (Table S1). All genomic DNAs used for Southern blotting of phr1, phr2, and cryD were digested with EcoRV, XhoI, and HindIII, respectively. Positive Δx mutants (Fig. S3B to D) and control strains (WT and Δx::x mutants) were used in the following experiments of three replicates.

Phenotypic experiments.For each fungal strain, 1-μl aliquots of a 10⁶ conidia ml⁻¹ suspension were centrally spotted on the plates of SDAY, CDA (3% sucrose, 0.3% NaNO₃, 0.1% K₂HPO₄, 0.05% KCl, 0.05% MgSO₄, and 0.001% FeSO₄ plus 1.5% agar), and CDAs amended with different carbon (3% of glucose, trehalose, glycerol, or sodium acetate) or nitrogen (0.3% of NH₄Cl, NaNO₂, or NH₄NO₃) sources. The spotted plates were incubated for 8 days at the optimal regime of 25°C and L:D 12:12, followed by estimation of the diameter of each colony as an index of growth rate, with two measurements taken perpendicular to each other across its center. The same spotting method was used to initiate colony growth at the same regime on CDA alone (control) or supplemented with a sensitive concentration of H₂O₂ (2 mM) or menadione (0.02 mM) for oxidative stress. After an 8-day incubation, the diameter of each colony was estimated as mentioned above. Relative growth inhibition of each strain by each oxidant was computed as (S_c – S_t)/S_c × 100, where S_c and S_t denote the areas of colonies grown in the control and each treatment, respectively. In addition, an SOD activity assay kit (Sigma-Aldrich, St. Louis, MO, USA) and catalase activity assay kit (Jiancheng Biotech, Nanjing, China) were used to quantify the total activities of SODs and catalases, respectively, in the protein extracts isolated from 3-day-old SDAY cultures, as described previously (46). One unit of enzymatic activity was defined as the SOD amount required for inhibition of 50% pyrogallol autoxidation rate or 1 mM H₂O₂ consumed per min. The total SOD or catalase activity was expressed as units per milligram of protein extract.

To assess conidiation capacity, 100-μl aliquots of a 10⁷ conidia ml⁻¹ suspension were evenly spread on SDAY plates (9-cm diameter), followed by 8-day incubation at 25°C and L:D 12:12 for full conidiation. Three plugs were then taken from each plate culture using a cork borer (5-mm diameter). The conidia on each plug were released into 1 ml of 0.02% Tween 80 via ultrasonic vibration. The conidial concentration in the suspension was assessed using a hemocytometer and converted to the number of conidia per unit area of plate culture.

Conidia harvested from the SDAY cultures of each strain were assessed for their viability and resistance to UVB irradiation. Conidial viability was quantified as GT₅₀ (in hours) for 50% germination on the plates of a germination medium (GM; 2% sucrose, 0.5% peptone, and 1.5% agar) at optimal temperature of 25°C (46). Conidial UVB resistance was quantified according to our previous protocols (6, 7). Briefly, 80-μl aliquots of a 10⁷ conidia ml⁻¹ suspension were evenly smeared onto GM plates (7-cm diameter), followed by ∼10 min of air-drying. The uncovered plates were placed in the sample tray of a Bio-Sun⁺⁺ UV irradiation chamber (Vilber Lourmat, Marne-la-Vallée, France) and irradiated with a weighted wavelength of 312 nm at the UVB doses of 0.1 to 0.6 J cm⁻² or not irradiated (control). The UVB irradiation of the weighted wavelength was emitted from two 30-W fluorescent tubes (T-20.M) above the sample tray and automatically adjusted four times per second for an error control of no more than 1 μJ cm⁻² at a fixed dose (according to the manufacturer’s guide). The irradiated plates were immediately covered with lids and incubated for 24 h at 25°C. The germination percentage on each plate was assessed with the counts of germinated and nongerminated conidia in three fields of microscopic view (×100 magnification). The LD₅₀ (J cm⁻²) indicative of UVB resistance was estimated from the fitted survival trends of conidia over the applied doses (6).

The conidial virulence of each strain was assayed on G. mellonella larvae (∼300 mg each) through two infection modes. Briefly, cohorts of ∼35 larvae were immersed for ∼10 s in 40-ml aliquots of a 10⁷ conidia ml⁻¹ suspension for normal cuticle infection. Alternatively, 5 μl of a 10⁵ conidia ml⁻¹ suspension was injected into the hemocoel of each larva in each cohort (i.e., ∼500 conidia injected per larva). All the treated cohorts were separately maintained in plastic containers for 10 days at 25°C and monitored daily for survival/mortality records. The resultant time-mortality trends were subjected to probit analysis, yielding estimates of LT₅₀ as an index of fungal virulence.

Assays for photorepair activities of Phr1 and Phr2 against CPDs and 6-4PPs.Hyphal cells were collected from the 3-day-old cultures of Δphr1 mutant, Δphr2 mutant, and control strains initiated with 10⁶ conidia ml⁻¹ SDB, rinsed repeatedly with distilled water, and resuspended in 0.01% yeast extract. Aliquots of a 3-ml cell suspension were poured into 7-cm-diameter petri dishes, forming a suspension layer of 0.8 mm in depth. The dishes were irradiated at the high UVB dose of 0.5 J cm⁻² in the UV chamber for generation of CPDs and 6-4PPs in the DNA of each strain. The irradiated dishes were immediately covered with lids and incubated at 25°C for 5 h under white light (photoreactivation) or in full darkness (NER treatment), followed by the collection of hyphal cells through centrifugation. The white light was controlled in a 280-liter illuminated growth chamber (Laifu Tech Co., Ningbo, China), which is installed vertically with eight 26-W 95-cm-long white fluorescent tubes in each side and presents a white-light intensity of 12,000 lx (see the manufacturer's guide). The dishes for NER treatment were wrapped with two layers of light-proof foil and incubated in the same growth chamber. Subsequently, total DNA of each strain was extracted from the hyphal cells of either treatment using the Biospin fungus genomic DNA extraction kit (Bioer Technology Co. Ltd., Hangzhou, China). The DNA immediately isolated from the UVB-irradiated cells without photoreactivation or NER treatment was used as a control for each strain. ELISAs with anti-CPD and anti-6-4PP antibodies were performed to quantify photorepair activities against the UVB-induced CPDs and 6-4PP in three DNA samples diluted to 0.4 and 4 μg ml⁻¹, respectively, following the user’s guides of the CPD and 6-4PP ELISA kits (Cosmo Bio Co. Ltd., Japan). The absorbance of each DNA dilution at 492 nm was used to represent the amount of CPDs or 6-4PPs as an index of photorepair activity.

Transcriptional profiling of three CPF genes.To profile the transcript levels of three CPF genes during normal incubation or in response to the time of light exposure, the WT strain was grown by spreading 100-μl aliquots of a 10⁷ conidia ml⁻¹ suspension on cellophane-overlaid SDAY plates and incubated at 25°C for 2 to 7 days in the fixed L:D 12:12 cycle or 3 days in full darkness. The dark cultures were then exposed to white light for 4, 8, and 16 h or not exposed (control). Total RNAs were extracted daily from the SDAY cultures during a 7-day incubation or from the 3-day-old dark cultures exposed to light for different hours under the action of an RNAiso Plus reagent (TaKaRa, Dalian, China) and reversely transcribed into cDNAs with a PrimeScript RT reagent kit (TaKaRa) at 37°C. To reveal the impact of each phr deletion on the expression of other CPF genes, total RNAs were extracted from the 3-day-old SDAY cultures of Δphr1 mutant, Δphr2 mutant, and control strains grown at the regime of 25°C and L:D 12:12 and transcribed into cDNAs, as mentioned above. The qPCR analysis with paired primers (Table S1) was performed under the action of SYBR Premix Ex Taq (TaKaRa) to assess transcript levels of phr1, phr2, and cryD in three cDNA samples (with a content standardized by dilution) derived from the WT cultures of each treatment (time length of incubation or light exposure) or from the 3-day-old cultures of each strain using the β-actin gene as an internal standard. The 2^−ΔΔCT method (47) was used to compute the relative transcript level of each CPF gene in the WT strain during the 7-day period of cultivation with respect to the standard on day 2 or over the time of light exposure with respect to the standard not exposed to light after continuous dark incubation and in each Δphr mutant or complementary strain with respect to the WT standard.

Assays for photoreactivation of UVB-impaired conidia.Conidia were collected from the 8-day-old SDAY cultures of deletion mutants and control strains grown at the optimal regime of 25°C and L:D 12:12, spread on the GM plates, and irradiated at the high UVB dose of 0.5 J cm⁻² in the aforementioned UV chamber. To optimize the time of light exposure, the irradiated plates were immediately covered with lids and incubated at 25°C for reactivation of 0, 10, 30, 60, 120, and 300 min under white light in the illuminated growth chamber, as mentioned above. After light exposure, all of the plates were transferred to a fully dark growth chamber for incubation of 19 to 24 h at 25°C. To assess conidial responses to different lights during photoreactivation, the irradiated dishes were covered with the lids, to which transmission spectrum-specific filters (425, 450, 480, 510, 540, and 580 nm [fluctuating ∼15 nm around each central transmission spectrum]; Jinhezhong Co., Beijing, China) were attached, and the lower parts (bottoms) of the dishes were tightly wrapped with light-proof foil to prevent lateral light from transmission but to not affect aeration, as described previously (48). The dishes attached with the filters or not attached for the white-light control and dark control (dishes completely wrapped with foil) were photoreactivated at 25°C for the optimal time of 5 h in the illuminated chamber, followed by a 19-h incubation in full darkness. At the end of the dark incubation, the germination percentage of irradiated conidia on each plate was estimated using microscopic counts, as mentioned above. To evaluate the impact of conidiation conditions on photoreactivation, conidia from the 8-day-old SDAY cultures of the tested strains grown at 25°C under continuous illumination and dark conditions were also irradiated at the same UVB dose, followed by estimation of their germination percentages after a 5-h photoreactivation and 19-h dark incubation.

Validation for enhancement of UVB resistance and photoreactivation efficiency by phr1 or phr2 overexpression.The strong promoter Ptef was ligated into the XmaI site of p0380-bar digested with XmaI, forming the backbone plasmid p0380-Ptef-bar for targeted gene overexpression. Subsequently, the ORFs of phr1 and phr2 amplified from the WT cDNA were inserted into the XmaI/BamHI sites of the backbone plasmid digested by XmaI and BamHI, respectively. The resultant plasmids were propagated in E. coli DH5a (Invitrogen) and transformed into the WT strain via Agrobacterium-mediated transformation. Putative OEphr colonies were screened by bar resistance to phosphinothricin (200 μg ml⁻¹) and sequentially identified through PCR and qPCR with paired primers (Table S1). Three OEphr1 or OEphr2 strains were selected based on upregulated expression levels of phr1 or phr2 and evaluated in parallel with WT for conidial UVB resistance and photoreactivation efficiency, as described in the experiments of the Δphr mutant and control strains. In photoreactivation assays, particularly, the conidia of each strain were irradiated at the UVB doses of 0.5, 0.6, 0.7, and 0.8 J cm⁻², followed by 1 or 3 h of reactivation under white light and 23 or 21 h of dark incubation for germination at 25°C. The irradiated conidia not exposed to light were used as a control.

Statistical analysis.All data from the experiments of three replicates were subjected to one-factor (strain) analysis of variance, followed by Tukey’s honestly significant difference (HSD) test for the difference of means between the deletion mutants and control strains.