Effects of Defective Interfering RNA on Symptom Induction by, and Replication of, a Novel Partitivirus from a Phytopathogenic Fungus, Rosellinia necatrix

Abstract

A novel mycovirus termed Rosellinia necatrix partitivirus 2 (RnPV2), isolated from a phytopathogenic fungus, Rosellinina necatrix strain W57, was molecularly and biologically characterized in both natural and experimental host fungi. Three doublestranded RNA (dsRNA) segments, dsRNA1, dsRNA2, and defective interfering dsRNA1 (DI-dsRNA1), whose sizes were approximately 2.0, 1.8, and 1.7 kbp, respectively, were detected in W57. While the dsRNA2 sequence, encoding the coat protein, was reported previously, dsRNA1 and DI-dsRNA1 were shown to encode competent and defective (truncated) RNA-dependent RNA polymerase, respectively. Artificial introduction of RnPV2 into an RNA silencing-defective, Dicer-like 2 knockout mutant (⌬dcl-2) of a nonnatural host, Cryphonectria parasitica (chestnut blight fungus), resulted in successful infection by the DI-dsR-NA1-carrying and -free RnPV2. The DI-dsRNA1-free RnPV2 strain was characterized by a higher ratio of accumulation of the intact dsRNA1 to dsRNA2, enhanced replication and severer symptom expression, compared with the DI-carrying strain. These findings confirmed the nature of DI-dsRNA1 as a DI-RNA. Both viral strains replicated to higher levels in a ⌬dcl-2 mutant than in a wild-type C. parasitica fungal strain (EP155) and induced severe symptoms in the ⌬dcl-2 mutant but subtle symptoms in EP155, indicating that the host RNA silencing targets the partitivirus. No obvious phenotypic effects of infection by either virus strain were detected in the natural host fungus. These combined results represent the first example of a partitivirus with DI-RNA that alters viral symptom induction in a host-dependent manner.

A Novel Victorivirus from a Phytopathogenic Fungus, Rosellinia necatrix, Is Infectious as Particles and Targeted by RNA Silencing

“…RNA samples were obtained from mycelia using the method of Chiba et al (17), and Northern blot analysis was conducted according to the method of Suzuki et al (50). After phenolchloroform extraction of fungal homogenate, total RNA was obtained by ethanol precipitation, whereas ssRNA was obtained by lithium chloride precipitation.…”

“…It was anticipated that a low transfection efficiency in EP155 might be responsible for this failure. Therefore, we switched the fungal strain to the RNA silencing-defective ⌬dcl-2 strain (a dicer-like gene deletion mutant of the EP155 strain) which had previously shown higher susceptibility to a heterologous partitivirus RnPV2 than the EP155 strain (17). This mutant strain was successfully transfected with the heterologous victorivirus RnVV1.…”

“…Among these, dcl-2 and agl-2 are required for antiviral defense (26,27). The RNA silencing pathway is able to suppress replication of hypoviruses, mycoreoviruses (26,27), and also a heterologous partitivi-rus (family Partitiviridae) (17). Furthermore, such RNA silencing is required for the generation and maintenance of defective interfering (DI)-RNAs of the prototype hypovirus, Cryphonectria hypovirus 1/EP713 (CHV1-EP713) (27,28).…”

“…However, reproducible transfection protocols with purified particles have been established for several other dsRNA viruses (see, for example, references 11, 12, and13) and also a single-stranded DNA (ssDNA) virus (14). The establishment of transfection protocols has led to better understanding of the etiology of mycovirus infection, exploration of mycovirus-host and virus-virus interactions, and the determination of experimental host ranges (15)(16)(17). These developments encouraged us to test a victorivirus from a filamentous fungus for infectivity as particles.…”

“…Due to the lack of effective control measures, virocontrol (biological control) of the disease using mycoviruses that attenuate the fungal virulence is an attractive option (13,29,30). To this end, an extensive search of a large number of field isolates was conducted allowing the detection of diverse dsRNA viruses such as a bipartite dsRNA virus, Rosellinia necatrix megabirnavirus 1 (RnMBV1), a quadrivirus, Rosellinia necatrix quadrivirus 1 (RnQV1), the reovirus mycoreovirus 3 (MyRV3), and the Rosellinia necatrix partitiviruses 1 and 2 (RnPV1 and RnPV2) (13,17,(31)(32)(33)(34)(35). Some of these viruses were shown to be able to confer hypovirulence under laboratory conditions.…”