Institute of Microbiology, College of Life Sciences, Zhejiang University , Hangzhou, Zhejiang, China.
Appl Environ Microbiol. 2023 Sep 28;89(9):e0099423. doi: 10.1128/aem.00994-23. Epub 2023 Sep 1.
The anti-ultraviolet (UV) role of a Rad4-Rad23-Rad33 complex in budding yeast relies on nucleotide excision repair (NER), which is mechanistically distinct from photorepair of DNA lesions generated under solar UV irradiation but remains poorly known in filamentous fungi. Here, two nucleus-specific Rad4 paralogs (Rad4A and Rad4B) and nucleocytoplasmic shuttling Rad23 ortholog are functionally characterized by multiple analyses of their null mutants in , an entomopathogenic fungus lacking Rad33. Rad4A was proven to interact with Rad23 and contribute significantly more to conidial UVB resistance (90%) than Rad23 (65%). Despite no other biological function, Rad4A exhibited a very high activity in photoreactivation of UVB-impaired/inactivated conidia by 5-h light exposure due to its interaction with Rad10, an anti-UV protein clarified previously to have acquired a similar photoreactivation activity through its interaction with a photolyase in . The NER activity of Rad4A or Rad23 was revealed by lower reactivation rates of moderately impaired conidia after 24-h dark incubation but hardly observable at the end of 12-h dark incubation, suggesting an infeasibility of its NER activity in the field where nighttime is too short. Aside from a remarkable contribution to conidial UVB resistance, Rad23 had pleiotropic effect in radial growth, aerial conidiation, antioxidant response, and cell wall integrity but no photoreactivation activity. However, Rad4B proved redundant in function. The high photoreactivation activity of Rad4A unveils its essentiality for 's fitness to solar UV irradiation and is distinct from the yeast homolog's anti-UV role depending on NER. IMPORTANCE Resilience of solar ultraviolet (UV)-impaired cells is crucial for the application of fungal insecticides based on formulated conidia. Anti-UV roles of Rad4, Rad23, and Rad33 rely upon nucleotide excision repair (NER) of DNA lesions in budding yeast. Among two Rad4 paralogs and Rad23 ortholog characterized in lacking Rad33, Rad4A contributes to conidial UVB resistance more than Rad23, which interacts with Rad4A rather than functionally redundant Rad4B. Rad4A acquires a high activity in photoreactivation of conidia severely impaired or inactivated by UVB irradiation through its interaction with Rad10, another anti-UV protein previously proven to interact with a photorepair-required photolyase. The NER activity of either Rad4A or Rad23 is seemingly extant but unfeasible under field conditions. Rad23 has pleiotropic effect in the asexual cycle but no photoreactivation activity. Therefore, the strong anti-UV role of Rad4A depends on photoreactivation, unveiling a scenario distinct from the yeast homolog's NER-reliant anti-UV role.
紫外线（UV）对芽殖酵母中 Rad4-Rad23-Rad33 复合物的拮抗作用依赖于核苷酸切除修复（NER），这在机制上与太阳 UV 照射下产生的 DNA 损伤的光修复不同，但在丝状真菌中仍知之甚少。在这里，两个核特异性 Rad4 同源物（Rad4A 和 Rad4B）和核质穿梭 Rad23 直系同源物通过对缺乏 Rad33 的昆虫病原真菌中的其缺失突变体进行多项分析来进行功能表征。Rad4A 被证明与 Rad23 相互作用，并比 Rad23（65%）更显著地促进分生孢子对 UVB 的抗性（90%）。尽管没有其他生物学功能，但 Rad4A 在 5 小时光照暴露下对 UVB 损伤/失活分生孢子的光复活表现出非常高的活性，这是由于其与 Rad10 的相互作用所致，Rad10 是一种先前已被证明通过与 在 中发现的光裂合酶相互作用而获得类似光复活活性的抗 UV 蛋白。Rad4A 或 Rad23 的 NER 活性通过中度受损分生孢子在 24 小时黑暗孵育后的再激活率较低而揭示出来，但在 12 小时黑暗孵育结束时几乎观察不到，这表明在夜间过短的情况下，其 NER 活性不可行。除了对分生孢子对 UVB 抗性的显著贡献外，Rad23 在径向生长、气生分生孢子形成、抗氧化反应和细胞壁完整性方面具有多效性效应，但没有光复活活性。然而，Rad4B 在功能上被证明是多余的。Rad4A 的高光复活活性揭示了它对 适应太阳 UV 照射的必要性，并且与酵母同源物依赖 NER 的抗 UV 作用不同。重要性 基于配方分生孢子的真菌杀虫剂的应用，对太阳能 UV 损伤细胞的恢复力至关重要。芽殖酵母中 Rad4、Rad23 和 Rad33 的抗 UV 作用依赖于 DNA 损伤的核苷酸切除修复（NER）。在缺乏 Rad33 的 中表征的两个 Rad4 同系物和 Rad23 直系同源物中，Rad4A 比 Rad23 更有助于分生孢子对 UVB 的抗性，Rad23 与 Rad4A 相互作用，而不是与功能冗余的 Rad4B 相互作用。Rad4A 通过与另一种抗 UV 蛋白 Rad10 的相互作用获得了对严重受损或失活的分生孢子进行光复活的高活性，Rad10 先前已被证明与需要光修复的光裂合酶相互作用。无论 Rad4A 还是 Rad23 的 NER 活性似乎都存在，但在田间条件下不可行。Rad23 在无性周期中具有多效性效应 ，但没有光复活活性。因此，Rad4A 的强抗 UV 作用取决于光复活，揭示了与酵母同源物依赖 NER 的抗 UV 作用不同的情况。
