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

Abstract

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.

Photoreactivation Activities of Rad5, Rad16A and Rad16B Help Beauveria bassiana to Recover from Solar Ultraviolet Damage

“…In contrast, their photoreactivation activities under the regime of L/D 3:9 or 5:7 were able to recover sublethal or lethal UVB dose-impaired conidia. According to the differences in photoreactivation rates between the DM and control strains in the L/D treatments, Rad5 and Rad16B exhibited photoreactivation activities close to those of Rad1, Rad2, Rad10, Rad14 and Rad23 in B. bassiana [ 54 , 57 , 60 ] and those of Rad1, Rad2, Rad4A, Rad10, Rad14 and Rad26 in M. robertsii [ 56 , 58 , 59 ]. Rad16A was inferior to Rad5 or Rad16B in the photoreactivation in vivo.…”

“…Finally, the solar UVB dose accumulated daily on the sunny days of summer is several times above conidial tolerance [1], within which UVB damage is recoverable via photoreactivation in insect-pathogenic fungi [50,[52][53][54][55][56][57][58][59][60]. More than 90% of the solar UVB dose accumulates prior to 3:00 p.m., contrasting with an accumulation of ~0.2 J/cm 2 after 3:00 p.m. [1].…”

“…To explore potential factors to interact with Rad5, Rad16A or Rad16B from photolyases; photolyase regulators; and some other anti-UV proteins with high photoreactivation activities, Y2H assays were carried out as described previously [56][57][58][59][60]. In brief, the open reading frames of rad5, rad16A, rad16B, phr1, phr2, wc1, wc2, rad1, rad10, rad2, rad6, rad18, rad14, rad4A, rad23, mms2, elc1, cul3A and cul3B (see Table S1 for tag loci and used primers) were cloned from the WT cDNA as aforementioned, followed by the ligation of each one to pGADT7 (prey vector, AD) and pGBKT7 (bait vector, BD), respectively.…”

“…Unlike the Rad4-Rad23-Rad33 complex in the yeast, the insect pathogens have two Rad4 paralogs (Rad4A/B) but lack Rad33. An interaction between Rad4A and Rad23 endows them with high UVB resistance but different activities in the photoreactivation of UVB-impaired conidia despite the functional redundancy of Rad4B in B. bassiana and M. robertsii [57,58]. Rad2, Rad14 and Rad26 orthologs also display extraordinarily high photoreactivation activities in the insect pathogens due to the interactions of each with WC1, WC2, Rad1 and/or Rad10 in M. robertsii [59], and with WC1, WC2, Rad10 and RFA1 (a subunit of the DNA-binding protein, RFA) in B. bassiana [60].…”