Rad2, Rad14 and Rad26 recover Metarhizium robertsii from solar UV damage through photoreactivation in vivo.

Rad2, Rad14 and Rad26 recover ultraviolet (UV) damage by nucleotide excision repair (NER) in budding yeast but their functions in filamentous fungi have not been elucidated. Here, we report mechanistically different anti-UV effects of nucleus-specific Rad2, Rad14 and Rad26 orthologs in Metarhizium robertsii, an insect-pathogenic fungus. The null mutants of rad2, rad14 and rad26 showed a decrease of ∼90% in conidial resistance to UVB irradiation. When conidia were impaired at a UVB dose of 0.15 J/cm2, they were photoreactivated (germinated) by only 6-13% through a 5-h light plus 19-h dark incubation, whereas 100%, 80% and 70% of the wild-type conidia were photoreactivated at 0.15, 0.3 and 0.4 J/cm2, respectively. The dose-dependent photoreactivation rates were far greater than the corresponding 24-h dark reactivation rates and were largely enhanced by the overexpression (OE) of rad2, rad14 or rad26 in the wild-type strain. The OE strains exhibited markedly greater activities in photoreactivation of conidia inactivated at 0.5-0.7 J/cm2 than did the wild-type strain. Confirmed interactions of Rad2, Rad14 and Rad26 with photolyase regulators and/or Rad1 or Rad10 suggest that each of these proteins could have evolved into a component of the photolyase regulator-cored protein complex to mediate photoreactivation. The interactions inhibited in the null mutants resulted in transcriptional abolishment or repression of those factors involved in the complex. In conclusion, the anti-UV effects of Rad2, Rad14 and Rad26 depend primarily on DNA photorepair-dependent photoreactivation in M. robertsii and mechanistically differ from those of yeast orthologs depending on NER.

Divergent roles of Rad4 and Rad23 homologs in Metarhizium robertsii's resistance to solar ultraviolet damage.

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 Metarhizium robertsii, 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 M. robertsii. 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 M. robertsii'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 Metarhizium robertsii 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 in vitro 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.

Efficient Photoreactivation of Solar UV-Injured Metarhizium robertsii by Rad1 and Rad10 Linked to DNA Photorepair-Required Proteins.

Nucleotide excision repair (NER) of ultraviolet (UV)-induced DNA lesions known as cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts depends on the activities of multiple anti-UV radiation (RAD) proteins in budding yeast. However, NER remains poorly known in filamentous fungi, whose DNA lesions are photorepaired by one or two photolyases, namely CPD-specific Phr1 and/or 6-4PP-specific Phr2. Previously, the white collar proteins WC1 and WC2 were proven to regulate expressions of phr2 and phr1 and photorepair 6-4PP and CDP DNA lesions, respectively, in Metarhizium robertsii, a filamentous entomopathogenic-phytoendophytic fungus. We report here high activities of orthologous Rad1 and Rad10 in 5-h photoreactivation of UVB-injured or UVB-inactivated conidia but a severely compromised capability of their reactivating those conidia via 24-h dark incubation in M. robertsii. The null mutants of rad1 and rad10 were much more compromised in conidial UVB resistance and photoreactivation capability than the previous null mutants of phr1, phr2, wc1 and wc2. Multiple protein-protein (Rad1-Rad10, Rad1-WC2, Rad10-Phr1, WC1-Phr1/2 and WC2-Phr1/2) interactions detected suggest direct/indirect links of Rad1 and Rad10 to Phr1/2 and WC1/2 and an importance of the links for their photoreactivation activities. Conclusively, Rad1 and Rad10 photoreactivate UVB-impaired M. robertsii through their interactions with the DNA photorepair-required proteins.

The Essential and the Nonessential Roles of Four Clock Elements in the Circadian Rhythm of Metarhiziumrobertsii.

FRQ (frequency protein), FRH (FRQ-interacting RNA helicase), and WC1 and WC2 (white collar proteins) are major clock elements that govern the circadian rhythm in Neurosporacrassa. However, deletion of frh is lethal for the viability of N. crassa, making it elusive whether FRH is essential or nonessential for the circadian rhythm. This needs clarification in a fungus where frh deletion is not lethal. Here, the nuclear FRH ortholog proved nonessential for the circadian rhythm of Metarhiziumrobertsii. The nucleocytoplasmic shuttling of M. robertsii FRQ, WC1, and WC2 orthologs was light-dependent. Yeast two-hybrid assay validated interactions of FRQ with FRH and WC1 instead of FRH with WC1 and WC2 or FRQ with WC2. The circadian rhythm well, shown as conidiation rings of tint and dark in 15 d-old plate cultures grown at 25 °C in a light/dark cycle of 12:12, was abolished in the absence of frq or wc1, partially disturbed in the absence of wc2, but unaffected in the absence of frh. These results indicate a requirement of either FRQ or WC1 instead of FRH for the fungal circadian rhythm. Further analyses of frq and frh mutants revealed the dispensable and the limited roles of FRQ and FRH in the insect-pathogenic lifecycle of M. robertsii, respectively.

Clinical Characteristics of 1378 Inpatients with Spinal Tuberculosis in General Hospitals in South-Central China.

In this retrospective study, charts of inpatients with spinal tuberculosis (STB) treated in large-scale general hospitals in Changsha, Hunan, China, between 2007 and 2016 were reviewed to investigate their clinical characteristics. Demographic, epidemiological and clinical features, imaging findings, treatment methods, and prognosis were summarized and analyzed. There were 1378 patients, 805 males and 573 females, with a mean age of 43.7 years. The mean interval between symptom onset and diagnosis was 16.0 months (range 15 days-240 months). The incidence of back pain, radicular pain and symptoms of systemic toxicity was 92.5%, 40.1%, and 32.1%, respectively. The rate of neurological impairment was 49.9 %. STB was present in two or more vertebrae in 91.1% of patients, with two adjacent vertebrae being involved in 67.9% of them. The lumbar segment (38.2%) was the most frequently affected, followed by the thoracic spine (35.7%). The sacrococcygeal area was the least frequently involved (0.8%). Abscesses were detected in 65.5% of patients. One thousand patients (72.6%) were managed with surgery and 378 (27.4%) with anti-TB drugs only. Cure was achieved in 1215 patients (88.2%), whereas 49 (3.5 %) had relapses. Concomitant pulmonary TB (PTB) was diagnosed in 366 patients (26.6%) and 63 (4.6%) had concomitant diabetes. Compared with the previous five years, the number of older patients, urban patients, and medical staff with STB had increased by 6.1%, 5.2%, and 1.3%, respectively in the five years studied. STB remains a severe public health problem that cannot be ignored. Most of the patients ignored early symptoms and therefore received untimely treatment. Thus, surveillance for and treatment of STB in South-central China requires strengthening. In addition to the current China-wide database of patients with PTB, a China-wide database of patients with STB should also be set up.