Novel hypovirulence-associated RNA mycovirus in the plant-pathogenic fungus Botrytis cinerea: molecular and biological characterization.
ABSTRACT: Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A -1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.
Project description:Two novel double-stranded RNA (dsRNA) mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), were isolated from the plant pathogenic fungus, Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps), are both predicted to contain two discontinuous open reading frames (ORFs), ORF1 and ORF2. A hypothetical polypeptide (P1) and a RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236) homologs were detected downstream of the RdRp domain (RdRp_4; pfam02123) of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC) were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed -1 ribosomal frameshifting (-1 PRF). Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together, with a comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species.
Project description:Botrytis cinerea is a necrotrophic fungus causing disease on many important agricultural crops. Two novel mycoviruses, namely Botrytis cinerea hypovirus 1 (BcHV1) and Botrytis cinerea fusarivirus 1 (BcFV1), were fully sequenced. The genome of BcHV1 is 10,214 nt long excluding a poly-A tail and possesses one large open reading frame (ORF) encoding a polyprotein possessing several conserved domains including RNA-dependent RNA polymerase (RdRp), showing homology to hypovirus-encoded polyproteins. Phylogenetic analysis indicated that BcHV1 may belong to the proposed genus Betahypovirus in the viral family Hypoviridae. The genome of BcFV1 is 8411 nt in length excluding the poly A tail and theoretically processes two major ORFs, namely ORF1 and ORF2. The larger ORF1 encoded polypeptide contains protein domains of an RdRp and a viral helicase, whereas the function of smaller ORF2 remains unknown. The BcFV1 was phylogenetically clustered with other fusariviruses forming an independent branch, indicating BcFV1 was a member in Fusariviridae. Both BcHV1 and BcFV1 were capable of being transmitted horizontally through hyphal anastomosis. Infection by BcHV1 alone caused attenuated virulence without affecting mycelial growth, significantly inhibited infection cushion (IC) formation, and altered expression of several IC-formation-associated genes. However, wound inoculation could fully rescue the virulence phenotype of the BcHV1 infected isolate. These results indicate the BcHV1-associated hypovirulence is caused by the viral influence on IC-formation-associated pathways.
Project description:Here, we characterized a negative single-stranded (-ss)RNA mycovirus, Botrytis cinerea mymonavirus 1 (BcMyV1), isolated from the phytopathogenic fungus Botrytis cinerea. The genome of BcMyV1 is 7863 nt in length, possessing three open reading frames (ORF1?3). The ORF1 encodes a large polypeptide containing a conserved mononegaviral RNA-dependent RNA polymerase (RdRp) domain showing homology to the protein L of mymonaviruses, whereas the possible functions of the remaining two ORFs are still unknown. The internal cDNA sequence (10-7829) of BcMyV1 was 97.9% identical to the full-length cDNA sequence of Sclerotinia sclerotiorum negative stranded RNA virus 7 (SsNSRV7), a virus-like contig obtained from Sclerotinia sclerotiorum metatranscriptomes, indicating BcMyV1 should be a strain of SsNSRV7. Phylogenetic analysis based on RdRp domains showed that BcMyV1 was clustered with the viruses in the family Mymonaviridae, suggesting it is a member of Mymonaviridae. BcMyV1 may be widely distributed in regions where B. cinerea occurs in China and even over the world, although it infected only 0.8% of tested B. cinerea strains.
Project description:A new mycovirus was found in the Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ''Unirnaviridae''. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe.
Project description:A new mycovirus was identified in Trichoderma harzianum strain 137 isolated in Xinjiang province, China. The whole genome sequence of the mycovirus was determined by metagenomic sequencing, RT-PCR, and RACE cloning. The mycovirus contained two genomic segments. The first segment was 2088 bp long and contained a single ORF (ORF1) encoding the RNA-dependent RNA polymerase (RdRP) (72.26 kDa). The second segment was 1634 bp long and also contained a single ORF (ORF2) encoding a hypothetical protein of 37.472 kDa. We named this novel mycovirus "Trichoderma harzianum bipartite mycovirus 1" (ThBMV1). Phylogenetic analysis showed that ThBMV1 clusters with other unclassified dsRNA mycoviruses.
Project description:BACKGROUND: Botrytis cinerea CCg378 is a wild-type strain infected with two types of double-stranded RNA (dsRNA) mycoviruses and which presents hypovirulence-associated traits. The objectives of the present study were to characterize the mycoviruses and investigate their relationship with the low virulence degree of the fungal host. RESULTS: B. cinerea CCg378 contains five dsRNA molecules that are associated with two different types of isometric viral particles of 32 and 23 nm in diameter, formed by structural polypeptides of 70-kDa and 48-kDa, respectively. The transfection of spheroplasts of a virus-free strain, B. cinerea CKg54, with viral particles purified from the CCg378 strain revealed that the 2.2-kbp dsRNAs have no dependency on the smaller molecules for its stable maintenance in the fungal cytoplasm, because a fungal clone that only contains the 2.2-kbp dsRNAs associated with the 32-nm particles was obtained, which we named B. cinerea CKg54vi378. One of the 2.2 kbpdsRNA segments (2219 bp) was sequenced and corresponds to the gene encoding the capsid protein of B. cinerea CCg378 virus 1 (Bc378V1), a putative new member of the Partitiviridae family. Furthermore, physiological parameters related to the degree of virulence of the fungus, such as the sporulation rate and laccase activity, were lower in B. cinerea CCg378 and B. cinerea CKg54vi378 than in B. cinerea CKg54. Additionally, bioassays performed on grapevine leaves showed that the CCg378 and CKg54vi378 strains presented a lower degree of invasiveness on the plant tissue than the CKg54 strain. CONCLUSIONS: The results show that B. cinerea CCg378 is coinfected by two mycoviruses and that the 2.2-kbp dsRNAs correspond to the 32-nm mycovirus genome, which would be a new member of the Partitiviridae family as it has the typical pattern of partitiviruses. On the other hand, the results suggest that the hypovirulence of B. cinerea CCg378 could be conferred by both mycoviruses, since the fungal clone B. cinerea CKg54vi378 presents an intermediate virulence between the CKg54 and CCg378 strains. Therefore, the putative partitivirus would be partially contributing to the hypovirulence phenotype of the CCg378 strain.
Project description:The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.
Project description:Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.
Project description:Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.
Project description:Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.