Novel endornaviruses infecting Phytophthora cactorum that attenuate vegetative growth, promote sporangia formation, and confer hypervirulence to the host oomycete.

Abstract

Two novel endornaviruses were found in Phytophthora cactorum isolated from severe black lesions on Boehmeria nivea (B. nivea) var. nipononivea plants in a Japanese forest. These two endornaviruses were named Phytophthora cactorum alphaendornavirus 4 (PcAEV4) and Phytophthora cactorum alphaendornavirus 5 (PcAEV5), and have site-specific nick structures in their positive RNA strands, which are hallmarks of alphaendornaviruses. Ribavirin and cycloheximide treatment of the protoplasts effectively cured the host oomycete (Kara1) of the viruses. The resultant virus-free strain (Kara1-C) displayed luxuriant mycelial growth with less zoosporangia formation as compared to the Kara1 strain. Remarkably, the Kara1-C strain exhibited reduced ability to form black lesions on B. nivea leaves, suggesting that presence of PcAEV4 and PcAEV5 in the Kara1 strain led to enhanced virulence in host plants. Under osmotic pressure and cell wall synthesis inhibition, the Kara1 strain exhibited less growth inhibition compared with the Kara1-C strain. In contrast, the Kara1 strain showed more growth inhibition to membrane-permeable surfactant compared with the Kara1-C strain, indicating that these endornaviruses can alter the susceptibility of the host oomycete to abiotic stresses. Co-localization and cell fractionation analyses showed that PcAEV4 and PcAEV5 localized to intracellular membranes, particularly the ER membrane fraction. Further, infection with these 2 endornaviruses was found to affect the hosts response to exogenous sterols, which enhanced vegetative growth and zoosporangia formation, as well as virulence of the host oomycete. These results provide deep insights into the effects of endornavirus infection in the Phytophthora spp. and also highlight protoplast-based methods in advancing Phytophthora virus studies.