Double-Stranded RNA Mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.     

Transmission of Fusarium boothii mycovirus via protoplast fusion causes hypovirulence in other phytopathogenic fungi

There is increasing concern regarding the use of fungicides to control plant diseases, whereby interest has increased in the biological control of phytopathogenic fungi by the application of hypovirulent mycoviruses as a possible alternative to fungicides. Transmission of hypovirulence-associated double-stranded RNA (dsRNA) viruses between mycelia, however, is prevented by the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi. We determined whether protoplast fusion could be used to transmit FgV1-DK21 virus, which is associated with hypovirulence on F. boothii (formerly F. graminearum strain DK21), to F. graminearum, F. asiaticum, F. oxysporum f. sp. lycopersici, and Cryphonectria parasitica. Relative to virus-free strains, the FgV1-DK21 recipient strains had reduced growth rates, altered pigmentation, and reduced virulence. These results indicate that protoplast fusion can be used to introduce FgV1-DK21 dsRNA into other Fusarium species and into C. parasitica and that FgV1-DK21 can be used as a hypovirulence factor and thus as a biological control agent.     

A 971-bp insertion in the rns gene is associated with mitochondrial hypovirulence in a strain of Cryphonectria parasitica isolated from nature

In the chestnut-blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes frequently are elicited by double-stranded RNA (dsRNA) virus infections. However, some strains manifest cytoplasmically transmissible hypovirulence traits without containing any mycovirus. In this study, we describe an altered form of mtDNA that is associated with hypovirulence and senescence in a virus-free strain of C. parasitica, KFC9, which was obtained from nature and has an elevated level of cyanide-resistant respiration. In this strain, a 971-bp DNA element, named InC9, has been inserted into the first exon of the mitochondrial small-subunit ribosomal RNA (rns) gene. Sequence analysis indicates that InC9 is a type A1 group II intron that lacks a maturase-encoding ORF. RT-PCR analyses showed that the InC9 sequence is spliced inefficiently from the rRNA precursor. The KFC9 strain had very low amounts of mitochondrial ribosomes relative to virulent strains, thus most likely is deficient in mitochondrial protein synthesis and lacks at least some of the components of the cyanide-sensitive, cytochrome-mediated respiratory pathway. The attenuated-virulence trait and the splicing-defective intron are transferred asexually and concordantly by hyphal contact from hypovirulent donor strains to virulent recipients, confirming that InC9 causes hypovirulence.     

Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents.     

Molecular characterization of a dsRNA mycovirus, Fusarium graminearum virus-DK21, which is phylogenetically related to hypoviruses but has a genome organization and gene expression strategy resembling those of plant potex-like viruses

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.     

Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination

Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polylmerase when the mitochondrial code (UGA?=?Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae.     

Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica : ascospore inheritance and implications for mitochondrial recombination

Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polylmerase when the mitochondrial code (UGA = Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae. Received: 5 May 1997 / Accepted: 10 July 1997     

Transmissible mitochondrial hypovirulence in a natural population of Cryphonectria parasitica

A cytoplasmically transmissible hypovirulence syndrome has been identified in virus-free strains of the chestnut blight fungus Cryphonectria parasitica isolated from healing cankers on American chestnut trees in southwestern Michigan. The syndrome is associated with symptoms of fungal senescence, including a progressive decline in the growth potential and abundance of conidia, and elevated levels of respiration through the cyanide-insensitive alternative oxidase pathway. Conidia from senescing mycelia exhibited varying degrees of senescence ranging from normal growth to death soon after germination. Cytoplasmic transmission of hypovirulence between mycelia occurred by hyphal contact and coincided with the transfer of a specific restriction fragment length polymorphism from the mitochondrial DNA (mtDNA) of the donor strains into the mtDNA of virulent recipients. The transmission of the senescence phenotype was observed not only among vegetatively compatible strains but also among incompatible strains. Hypovirulence was present in isolates from the same location with different nuclear genotypes as identified by DNA fingerprinting. This study confirms that mitochondrial hypovirulence can occur spontaneously and spread within a natural population of a phytopathogenic fungus.     

Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.     

Differential accumulation of poly(A)+ RNA between virulent and double-stranded RNA-induced hypovirulent strains of Cryphonectria (Endothia) parasitica.

The double-stranded RNA responsible for transmissible hypovirulence in Cryphonectria (Endothia) parasitica was found to affect the accumulation of specific poly(A)+ RNA. Using differential hybridization techniques, two genes were isolated, Vir1 and Vir2, which were specifically expressed as poly(A)+ RNAs in the virulent cells. The highly expressed RNA sequences from these genes were not found in total RNA isolated from either American or European hypovirulent strains, although the genes were present in their genomes. Other virulence- and hypovirulence-specific RNA sequences were also detected. One isolated hypovirulence-specific RNA sequence was expressed in both virulent and hypovirulent cells, but in a two- to fourfold-higher concentration in the hypovirulent cells. The results show that hypovirulence is associated with concurrent changes in a few highly expressed poly(A)+ RNAs, which suggests a specific effect of the double-stranded RNA on fungal gene expression.     

Horizontal transfer and hypovirulence associated with double-stranded RNA in Beauveria bassiana

Beauveria bassiana strains from different hosts and geographic origins were assayed for the presence of double-stranded RNA (dsRNA). Two of them (15.4 %) showed extra bands, with approximately 4.0–3.5 kb and 2–0.7 kb, respectively, after electrophoretic separation of undigested nucleic acids. Virus-like particles were approximately 28–30 nm diam. The dsRNA was maintained after conidiogenesis (vertical transmission) and was transmitted horizontally by hyphal anastomosis. Strains purged of dsRNA obtained after cycloheximide treatment showed increased conidial production when compared with strains carrying dsRNA particles. Bioassays demonstrated hypovirulence associated with dsRNA. The mean mortality against the insect Euschistus heros was reduced in strains containing dsRNA when compared with the isogenic dsRNA-free ones.     

Double-stranded RNAs associated with Fusarium proliferatum mitochondria

Many fungi harbor double-stranded (ds) RNA molecules, which can have phenotypic effects such as hypovirulence, altered colony morphology, and pigmentation. In some species of Fusarium, dsRNA molecules are found in every strain examined. We examined 100 F. proliferatum strains collected primarily from maize and sorghum in the United States, but found only four that carried dsRNAs. Each strain harbored a distinct set of dsRNAs, which ranged in size from approximately 0.7–3.1 kb. A single dsRNA band was observed from one strain, but multiple bands were observed from the other three. The strains with multiple dsRNAs transmitted these dsRNAs as sets at a high frequency (≥ 97 %) to vegetatively produced microconidia, but the single dsRNA of the fourth strain was only rarely (≤ 3 %) transmitted in this manner. None of these dsRNAs could be transmitted through sexual crosses in which the dsRNA-containing strain served as the male parent. Transmission through the female parent could not be tested as the field strains and dsRNA-free derivatives of these strains were female sterile. The dsRNAs from the strains with multiple dsRNAs were present in and protected against ribonuclease A digestion in crude mitochondrial preparations. The high transmission rate to single-conidiospore cultures, the lack of transmission through the male parent of sexual crosses, and the protection against ribonuclease A digestion are all consistent with a mitochondrial localization of the dsRNAs from the strains carrying multiple dsRNAs. dsRNAs often function as viruses in fungi, and the three F. proliferatum strains reported here join strains of Ophiostoma novo-ulmi, Rhizoctonia solani, and Cryphonectria parasitica as the only fungi known to carry dsRNAs associated with the mitochondria. Contribution number 02-495-J from the Kansas Agricultural Experiment Station (Manhattan).     

DsRNA-free transmissible hypovirulence associated with formation of intra-hyphal hyphae in Botrytis cinerea

A spontaneous mutant CanBc-3HV and its parental strain CanBc-3 of Botrytis cinerea were investigated in terms of pathogenicity, colony morphology, hypovirulence transmissibility, presence of double-stranded RNA (dsRNA), and formation of intra-hyphal hyphae (IH). Results showed that inoculation of CanBc-3HV on detached leaves of Brassica napus did not produce any visible necrotic lesions (20°C, 72h), whereas inoculation of CanBc-3 caused necrotic leaf lesions. Compared to CanBc-3, CanBc-3HV grew slowly, formed numerous mycelial sectors, sporulated sporadically and failed to produce sclerotia on potato dextrose agar (PDA) (20°C, 15d). Hypovirulence and the abnormal cultural characteristics of CanBc-3HV were transmissible from CanBc-3HV to CanBc-3 in pair cultures on PDA. However, the transmission was unsuccessful from CanBc-3HV to another virulent strain CanBc-2 of B. cinerea. These results suggest that transmission of the hypovirulence and the abnormal cultural characteristics of CanBc-3HV are strain-specific. No dsRNA was detected in mycelia of either CanBc-3HV or CanBc-3, implying that the hypovirulence of CanBc-3HV is caused by a transmissible element (TE) of non-RNA mycoviral origin. Formation of IH through self-infection was observed in CanBc-3HV, CanBc-3T1 (a hypovirulent derivative of CanBc-3 trans-infected by TE in CanBc-3HV), but was not observed in CanBc-3, suggesting that IH formation is associated with the hypovirulence of CanBc-3HV. To our knowledge, this is the first report of dsRNA-free transmissible hypovirulence associated with IH formation in B. cinerea.     

A viral gene confers hypovirulence-associated traits to the chestnut blight fungus.

A viral double-stranded (ds)RNA associated with reduced virulence (hypovirulence) and the accompanying biological control of the chestnut blight fungus, Cryphonectria parasitica, was shown recently to contain two contiguous coding domains designated ORF A and ORF B. We report here that transformation of an isogenic virulent, dsRNA-free C. parasitica strain with a cDNA copy of ORF A conferred traits similar to those exhibited by the dsRNA-containing hypovirulent strain: characteristics included reduced pigmentation, reduced laccase accumulation and suppressed conidiation. However virulence was not reduced, indicating an apparent uncoupling of associated traits from hypovirulence. These results establish a direct cause and effect relationship between a viral dsRNA genetic element present in a hypovirulent C. parasitica strain and specific phenotypic traits. They demonstrate further that these traits are not the result of a general reaction of the fungus to the presence of the replicating viral RNA, but are caused by a specific viral coding domain.     

Hypovirus papain-like protease p29 functions in trans to enhance viral double-stranded RNA accumulation and vertical transmission

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to approximately 50% for Deltap29, and 10 to 20% for Deltap69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Deltap29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Deltap69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys(70) or Cys(72), strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.     

Cutinase in Cryphonectria parasitica, the chestnut blight fungus: suppression of cutinase gene expression in isogenic hypovirulent strains containing double-stranded RNAs.

Plant-pathogenic fungi produce cutinase, an enzyme required to degrade plant cuticles and facilitate penetration into the host. The absence of cutinase or a decrease in its production has been associated with a decrease in pathogenicity of the fungus. A set of isogenic strains of Cryphonectria parasitica, the chestnut blight fungus, was tested for the presence and amounts of cutinase activity. The virulent strain of C. parasitica produced and secreted significantly higher amounts of cutinase than the hypovirulent strains. Use of both nucleic acid and polyclonal antibody probes for cutinase from Fusarium solani f. sp. pisi showed that cutinase in C. parasitica is 25 kDa in size and is coded by a 1.1-kb mRNA. Both mRNA and protein were inducible by cutin hydrolysate, while hypovirulence agents suppressed the level of mRNA and the enzyme. Since all the strains had the cutinase gene, the suppression of expression was due to the hypovirulence agents. The data presented are the first report indicating that hypovirulence agents in C. parasitica regulate a gene associated with pathogenicity in other plant-pathogenic fungi.     

产黄青霉ds RNA病毒通过原生质体融合的转移

将国内青霉素产生菌(Penicillium chrysogenum)的黄孢子系统及绿孢子(包括淡绿,灰绿)系统的十多个菌株,经过病毒提取、电镜观察、奥氏免疫双扩散、凝胶电泳及放射免疫测定,证明黄孢子系统的菌株含有不同滴定度的、直径40nm的球形病毒,而绿孢子系统中检查不出病毒。从营养要求、孢子颜色不同的带病毒和无病毒菌体中分离原生质体,进行不同组合的原生质体的融合杂交,获得营养互补融合的异核体。异核体1中,病毒通过胞质融合转移到原来无病毒的灰绿孢子菌株及细胞核融合后的杂合二倍体中。灰绿孢子的病毒量接近二倍体的1/3。二倍体菌落生长稳定,低温保存二年后经0.01—0.02M对氟苯丙氨酸(PFA)诱发和分离,产生亲本类型的分离子,分离子及二倍体仍然含有病毒。异核体2作亲本性分离,黄孢子仍有病毒,淡绿孢子及细胞核融合后产生的二倍体均无病毒,表明非感染性为显性。此种淡绿孢子的突变体中存在非感病菌系,它不支持病毒的复制。提取各杂交组二倍体内的病毒所特有的dsRNA时,可看出dsRNA的存在和病毒的存在一致。多数杂合二倍体的青霉素产量比亲本高。