Transfection of Diaporthe perjuncta with Diaporthe RNA Virus

Abstract

Diaporthe perjuncta is a pathogen of grapevines worldwide. A positive-strand RNA virus, Diaporthe RNA virus (DaRV), occurs in hypovirulent isolates of this fungus. A virus-free isolate from a South African grapevine was transfected with in vitro-transcribed positive strands of DaRV. Based on reverse transcription-PCR and partial sequence analysis, the transfected virus was identified as DaRV. The in vitro-transcribed RNA transcripts used to transfect fungal spheroplasts contained parts of the vector at their distal ends. These vector sequences were separated from the DaRV genome during replication in the new host. The transfected isolate had morphological features that differed from those of the isogenic virus-free strain, including production of a yellow pigment, a decreased growth rate, and lack of sporulation. An apple-based pathogenicity test did not reveal any differences in virulence between the virus-free and DaRV-transfected isolates. This study showed that virus-free fungal hosts can be successfully transfected with viruses other than the Cryphonectria parasitica hypovirus.Mycoviruses from many species of fungi have been observed and characterized at the genomic level (6,22,30). The fungal hosts include several well-known plant pathogens, including Cryphonectria parasitica (17,19,20,35),5,14,23,24,25), Helminthosporium victoriae (34), Sphaeropsis sapinea (32, 38), and Diaporthe perjuncta (28,31,37). In C. parasitica, the viruses CHV1-EP713 and CHV1-Euro7 mediate hypovirulence to various degrees (7,11,13). Vegetative compatibility groups restrict the spread of such viruses and have reduced the success of biological control through hypovirulence in the United States (1,2,3,26).The interest in viruses that infect fungi is strongly linked to the potential use of these viruses as biological control agents of plant-pathogenic fungi. An emerging area of interest is the use of mycoviruses to study basic genetic processes that lead to fungal pathogenesis (29). The fungal reaction, commonly manifested as hypovirulence-associated traits, is not a general response of the fungus to hypovirus infection but rather is a response triggered by molecular cues encoded by specific viral sequences (10, 12). Reverse genetics developed for the C. parasitica hypovirus has shown that specific mutations and deletions of specific regions of the hypovirus are associated with specific responses in the pathogen. For example, deletion of the papain-like protease, p29, from CHV1-EP713 encoded by open reading frame A restores orange pigmentation and moderately increases sporulation in transfected isolates (15).Although several mycoviral genomes have been sequenced and characterized, only the C. parasitica hypoviruses have been used in transfection and transformation studies (16). Thus, the progress in developing mycoviruses as possible biological control agents has been slow. In order to advance the field further, the reverse genetics developed for C. parasitica hypoviruses must be extended to mycoviruses with properties different from ...

Effect of Diaporthe RNA virus 1 (DRV1) on growth and pathogenicity of different Diaporthe species

“…Fungal spheroplasts were prepared by digesting tenday-old fungal mycelium with a mixture of chitinase (Fluka, Buchs, Switerland) and 6 cellulase (Sigma, St. Louis, MO) as described previously (Moleleki et al, 2003). The spheroplasts were pulsed three times at 1.8kV with the viral transcripts using multiporator (Eppendorf, Hamburg, Germany).…”

“…Synthetic DRV1 RNA transcripts were in vitro-t r a n s c r i b e d u s i n g T 7 R N A polymerase (Roche Diagnostics, Mannheim, Germany) from SalI-linearized pDV3 as described by Moleleki et al (2003). Fungal spheroplasts were prepared by digesting tenday-old fungal mycelium with a mixture of chitinase (Fluka, Buchs, Switerland) and 6 cellulase (Sigma, St. Louis, MO) as described previously (Moleleki et al, 2003).…”

“…(5¢-TCCATCTCACCGGAGCGGCAG-3¢) were used to reverse-transcribe the 5¢ and 3¢, respectively (Moleleki et al, 2003). After polyadenylating these ends, a nested PCR was used to amplify the 5¢ end using Oli78 (5¢-CCTGGGTGACGGTTGTTACAC-3¢) and the 3¢ end using Oli81 (5¢-TTGAACGATGGGTGTAGGTGG-3¢) as described previously (Moleleki et al, 2003).…”

“…The nucleic acids were purified sequentially using phenol, phenol/chloroform and chloroform. RT-PCR was done using the DRV1-specific primer pair, Oli64 (5¢-GTCGCATCTCACAGCCGAGCG-3¢) and Oli80 (5¢-CTCACCAGCCTCCAACCG-3¢) using the Titan One Tube RT-PCR System (Roche Diagnostics, Mannheim, Germany) (Moleleki et al, 2003). Double stranded RNA was denatured for 3 min at 98°C.…”

“…The development of full-length cDNA clones of other mycoviruses is likely to provide additional knowledge not available from the Cryphonectria-hypovirus system. In an effort to develop transfection protocols for mycoviruses other than the Cryphonectria hypovirus, we have developed a full-length cDNA clone of Diaporthe RNA virus 1 (DRV1) and used the in vitro-produced RNA from it to transfect a virus-free D. perjuncta isolate (Moleleki et al, 2003). In a related study, Esteban and Fujimura (2003) transformed yeast cells negative for 23S RNA virus and showed transmission of the virus in daughter cells.…”