| Circadian rhythms are endogenous cellular clocks that control molecular and physiological functions in most organisms. Within the domain Eukarya, the fungus Neurospora crassa is a model system for chronobiology that has helped define an autoregulatory negative feedback loop. Our project has identified putative open reading frames for orthologous proteins in Cryphonectria parasitica, a plant pathogen and causative agent of chestnut blight. We used publicly available N. crassa expressed sequences collection to identify 473 putative orthologous genes that are involved in the regulation of the biological clock within the C. parasitica genome. Included in this set of genes are principal components such as white collar-1 (wc-1), white collar-2 (wc-2), frequency (frq) and vivid (vvd) as well as six clock-controlled genes (ccg-1, ccg-4 , ccg-6, ccg-7, ccg-14 , and ccg-15), suggesting there is considerable conservation of these components between N. crassa and C. parasitica. To study light response, a film light spectrum analysis was designed to observe the development of the plant pathogen. Results demonstrated that C. parasitica reproduced normally in the presence of blue-light while simultaneously struggled to develop in red-light. We also used a confocal microscope to determine the emission spectrum of C. parasitica mycelium under various excitation wavelengths. Between 405 nm and 625 nm illumination, the spectra generated by uninfected and virus-infected cultures vary. Under UV excitation, EP155 strain emitted light around 550 nm (green) while EP155/CHV1-Euro7 emitted light around 500 nm. Yellow-green excitation demonstrated an opposite result when the hypovirulent strain had a stronger emission (600 nm), and the wild-type had a lower emission (630 nm). Red light excitation did not show any difference between both strains. Currently, knockout strains of key regulatory components are developed and transformed in C. parasitica to determine if there is a role for circadian rhythms in pathogenicity. This project is helping us to understand any potential relationship between circadian rhythms, light response and the impact of hypoviruses (virulence attenuating RNA viruses) in C. parasitica. . |
