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. 2019 Feb 6;85(4):e02459-18.
doi: 10.1128/AEM.02459-18. Print 2019 Feb 15.

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

Ding-Yi Wang  1 , Bo Fu  2 , Sen-Miao Tong  3   2 , Sheng-Hua Ying  1 , Ming-Guang Feng  3
Affiliations

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

Ding-Yi Wang et al. Appl Environ Microbiol. .

Abstract

Fungal conidia serve as active ingredients of fungal insecticides but are sensitive to solar UV irradiation, which impairs double-stranded DNA (dsDNA) by inducing the production of cytotoxic cyclobutane pyrimidine dimers (CPDs) and (6-4)-pyrimidine-pyrimidine photoproducts (6-4PPs). This study aims to elucidate how CPD photolyase (Phr1) and 6-4PP photolyase (Phr2) repair DNA damage and photoreactivate UVB-inactivated cells in Beauveria bassiana, a main source of fungal insecticides. Both Phr1 and Phr2 are proven to exclusively localize in the fungal nuclei. Despite little influence on growth, conidiation, and virulence, singular deletions of phr1 and phr2 resulted in respective reductions of 38% and 19% in conidial tolerance to UVB irradiation, a sunlight component most harmful to formulated conidia. CPDs and 6-4PPs accumulated significantly more in the cells of Δphr1 and Δphr2 mutants than in those of a wild-type strain under lethal UVB irradiation and were largely or completely repaired by Phr1 in the Δphr2 mutant and Phr2 in the Δphr1 mutant after optimal 5-h exposure to visible light. Consequently, UVB-inactivated conidia of the Δphr1 and Δphr2 mutants were much less efficiently photoreactivated than were the wild-type counterparts. In contrast, overexpression of either phr1 or phr2 in the wild-type strain resulted in marked increases in both conidial UVB resistance and photoreactivation efficiency. These findings indicate essential roles of Phr1 and Phr2 in photoprotection of B. bassiana from UVB damage and unveil exploitable values of both photolyase genes for improved UVB resistance and application strategy of fungal insecticides.IMPORTANCE Protecting fungal cells from damage from solar UVB irradiation is critical for development and application of fungal insecticides but is mechanistically not understood in Beauveria bassiana, a classic insect pathogen. We unveil that two intranuclear photolyases, Phr1 and Phr2, play essential roles in repairing UVB-induced dsDNA lesions through respective decomposition of cytotoxic cyclobutane pyrimidine dimers and (6-4)-pyrimidine-pyrimidine photoproducts, hence reactivating UVB-inactivated cells effectively under visible light. Our findings shed light on the high potential of both photolyase genes for use in improving UVB resistance and application strategy of fungal insecticides.

Keywords: DNA damage repair; UV resistance; biological control potential; entomopathogenic fungi; gene expression and regulation; photolyase; photoreactivation.

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Figures

FIG 1
FIG 1
Transcriptional profiles and subcellular localization of three CPF homologs in B. bassiana. (A and B) Relative transcript (RT) levels of three CPF genes in the WT cultures grown at 25°C for 2 to 7 days at L:D 12:12 with respect to the standard at the end of day 2 and exposed to white light for 4 to 16 h with respect to the standard not exposed to light after 3-day dark incubation, respectively. Different lowercase letters marked on the bars of each group denote significant differences (Tukey's HSD test, P < 0.05). Error bars denote the SD from the results from three cDNA samples (replicates) assessed in qPCR experiments. (C to F) LSCM images (scale bars = 5 μm) for the signals of Phr1::GFP, Phr2::GFP, and CryD::GFP fusion proteins expressed in the hyphal cells and conidia of transgenic strains, respectively. The hyphal cells were collected from the 2-day-old cultures of a conidial suspension in SDB shaken at 25°C under continuously illuminated or full-dark conditions and stained with the nucleus-specific dye DAPI (shown in blue), followed by visualization through LSCM. The conidia were collected from the SDAY cultures near the respective ends of light and dark exposures during conidiation at L:D 12:12 and stained with DAPI. Note that the green signal of GFP-tagged Phr1 and Phr2 fusion proteins merges well with the stained color in the nuclei but is absent in the cytoplasm, in which the GFP-tagged CryD fusion proteins are present, irrespective of the hyphal cells and conidia exposed to light (C and E) or dark (D and F).
FIG 2
FIG 2
Roles of Phr1, Phr2, and CryD in UVB resistance and antioxidant response of B. bassiana. (A and B) Conidial survival trends of targeted gene deletion mutants and control strains over the doses of UVB irradiation and LD50 estimates for their UVB resistance, respectively. (C) Relative growth inhibition (RGI) of fungal colonies by 0.02 M menadione (MND) or 2 mM H2O2 after 8 days of cocultivation at 25°C on CDA plates, on which 1-μl aliquots of a 106 conidia ml−1 suspension were spotted for initiation of colony growth. (D) Total activities of SODs and catalases in the protein extracts isolated from 3-day-old SDAY cultures. Different lowercase letters marked on the bars of each group denote significant differences (Tukey’s HSD test, P < 0.05). Error bars denote the standard error (SE) (A) or SD (B to D) from the results from three replicates.
FIG 3
FIG 3
Factors affecting photoreactivation of B. bassiana conidia inactivated at the lethal UVB dose of 0.5 J cm−2. (A) Germination percentages of UVB-inactivated conidia reactivated by time course exposure to white light. All conidia were collected from the 8-day-old cultures grown at 25°C and L:D 12:12, irradiated at the UVB dose, reactivated for 30 to 300 min under white light, and then incubated for 19 to 23.5 h at 25°C in darkness. (B) Microscopic images for the germination status of irradiated conidia at the end of a 19-h dark incubation after a 5-h incubation under white light (photoreactivation) or in darkness (NER) at 25°C. The conidia not irradiated at the UVB dose (left column) were incubated as controls for 24 h in darkness. (C) Germination percentages of irradiated conidia after 5 h of photoreactivation and 19 h of incubation in darkness at 25°C. Conidia were collected from the 8-day-old cultures grown under continuously illuminated or full-dark conditions. Different lowercase letters marked on the bars of each group denote significant differences (Tukey’s HSD test, P < 0.05). Error bars denote the SD from the results from three replicates of each strain.
FIG 4
FIG 4
Activities of Phr1 and Phr2 in photorepair of B. bassiana DNA lesions induced by UVB irradiation. (A) ELISA readings for the amounts of UVB-induced CPDs and 6-4PPs probed by anti-CPD and 6-4PP antibodies in the impaired DNAs of two Δphr mutants and control strains. Hyphal cells from 3-day-old SDB cultures were suspended in a 0.8-mm layer of 0.01% yeast extract, irradiated at the UVB dose of 0.5 J cm−2, and incubated for 5 h under white light (photoreactivation) or in darkness (NER), followed by DNA extraction. The DNA samples extracted from the irradiated cells not exposed to white light or incubated in darkness were used as controls. (B) Relative transcript (RT) levels of partner CPF genes in the 3-day-old SDB cultures of phr mutants with respect to the WT standard. The asterisked bars in each bar group differ significantly from those that are unmarked (Tukey’s HSD test, P < 0.05). Error bars denote the SD from three DNA (A) or cDNA (B) samples of each strain.
FIG 5
FIG 5
Impact of light spectra on photoreactivation of UVB-inactivated conidia by Phr1 and Phr2 in B. bassiana. Microscopic images (A) and germination percentages (B) were taken from the conidia irradiated at the lethal UVB dose of 0.5 J cm−2, reactivated for 5 h under the lights of different spectra (425 to 580 nm), white light (positive control), and full-dark conditions (negative control), and incubated for 19 h in darkness at 25°C. The asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD test, P < 0.05). Error bars denote the SD from the results from three replicates of each strain.
FIG 6
FIG 6
Conidial UVB resistance and photoreactivation efficiency enhanced by upregulated expression of phr1 or phr2 in B. bassiana. (A) Relative transcript levels (RTL) of phr1 and phr2 in three OEphr1 and OEphr2 strains versus WT. (B and C) Conidial survival trends of WT and OEphr strains over the doses of UVB irradiation and LD50 estimates for their UVB resistance, respectively. (D) Germination percentages of the conidia exposed to white light for a 1- or 3-h photoreactivation and then incubated for 23 or 21 h in full darkness at 25°C after UVB irradiation at the doses of 0.5, 0.6, 0.7, and 0.8 J cm−2, respectively. The irradiated conidia not reactivated under white light were incubated for 24 h in darkness and used as controls. Different lowercase letters marked on the bars of each denote significant differences (Tukey’s HSD, P < 0.05). Error bars denote the SD from the results from three replicates.
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