Functional Characterization Ofpseudoidium Neolycopersiciphotolyase Reveals Mechanisms Behind The Efficacy Of Nighttime Uv On Powdery Mildew Suppression

Powdery mildews can be controlled by brief exposure to ultraviolet (UV) radiation with devastating effect on their developmental stages including conidia germination. The treatment effect can be impaired by subsequent exposure to UV-A/blue light. UV-A/blue light-activated photolyase may be responsible for this and therefore we tested the function of three cryptochrome/photolyase family (CPF)-like genes (OINE01015670_T110144, OINE01000912_T103440, and OINE01005061_T102555) identified in the obligate biotrophic fungusPseudoidium neolycopersici, the cause of tomato powdery mildew. A photolyase-deficient mutant ofEscherichia colitransformed with coding sequence of OINE01000912_T103440 and exposed to brief (UV)-C treatment (peak emission at 254 nm) showed photoreactivation and cell survival when exposed to subsequent blue light, indicating complementation of photolyase activity. In contrast, the same photolyase-deficientE. colitransformed with the coding sequences of other two CPF-like genes did not survive this treatment, even though their expression were confirmed at protein level. This confirmed that OINE01000912_T103440 is a gene encoding photolyase, here namedPnPHR1, with functionality similar to the native photolyase inE. coli, and classified as a class I cyclobutane pyrimidine dimer (CPD) photolyase. Modeling of the 634-amino acid sequence ofPnPHR1suggested that it is capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). However, spectroscopic data of the protein produced in anE. coliexpression system could only reveal the presence of a reduced form of FAD, i.e., FADH(-)as an intrinsic chromophore. Within the tested wavelength range of 365-525 nm, the survival of photolyase-deficient mutantE. colitransformed withPnPHR1 showed a broad action spectrum from 365 to 454 nm. This was very similar to the previously characterized action spectrum for survival ofP. neolycopersiciconidia that had been treated with UV-C. Quantitative RT-PCR revealed that the expression ofPnPHR1inP. neolycopersiciconidia was induced by UV-C, and peak expression occurred 4 h after brief UV-C treatment. The expression ofPnPHR1was repressed when incubated in red light after the UV-C treatment, but not when incubated in UV-A/blue light. The results may explain why the disease-reducing effect of short wavelength UV is impaired by exposure to UV-A and blue light.