Variable eIF4E-binding sites and their synergistic effect on cap-independent translation in a novel IRES of wheat yellow mosaic virus RNA2 isolates.

Some viral proteins are translated cap-independently via the internal ribosome entry site (IRES), which maintains conservative characteristic among different isolates of the same virus species. However, IRES activity showed a 7-fold variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates in this study. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and the hepta-nucleotide top loop of H2 in the LYJN isolate synergistically ensured higher IRES activity than that in the HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. WYMV RNA2 5'-UTR specifically interacted with the wheat eIF4E, accomplished by the top loop of H1 in the HC isolate or the top loop of H1 and H2 in the LYJN isolate. The high IRES activity of the WYMV RNA2 LYJN isolate was regulated by two eIF4E-binding sites, which showed a synergistic effect mediated by the proximity of the H1 and H2 top loops owing to the flexibility of the middle stem in H1. This report presents a novel evolution pattern of IRES, which altered the number of eIF4E-binding sites to regulate IRES activity.

The Regulation of the Growth and Pathogenicity of Valsa mali by the Carbon Metabolism Repressor CreA.

Carbon catabolite repression (CCR) is a very important mechanism for efficient use of carbon sources in the environment and is necessary for the regulation of fungal growth, development, and pathogenesis. Although there have been extensive studies conducted regarding this mechanism in fungi, little is yet known about the effects of CreA genes on Valsa mali. However, based on the results obtained in this study for the identification of the VmCreA gene in V. mali, it was determined that the gene was expressed at all stages of fungal growth, with self-repression observed at the transcriptional level. Furthermore, the functional analysis results of the gene deletion mutants (ΔVmCreA) and complements (CTΔVmCreA) showed that the VmCreA gene played an important role in the growth, development, pathogenicity, and carbon source utilization of V. mali.

Transcription Factor VM1G_06867: A Requirement for Growth, Pathogenicity, Development, and Maintenance of Cell Wall Integrity in Valsa mali.

Apple canker disease, caused by Valsa mali, is one of the most serious apple tree diseases in China. VmSom1 is an important transcription factor that acts on the cyclic adenosine signaling pathway (cAMP/PKA), regulating the growth, development, morphological differentiation, and pathogenic forces of the pathogen. We perform transcriptome analysis of the VmSom1 deletion mutant and the wild-type strain 11-175 and identify a significantly differentially expressed gene, VM1G_06867, a zinc finger motif transcription factor in V. mali. In this study, we obtain the VM1G_06867 gene using the single deletion mutant via homologous recombination. To determine the relationship between VmSom1 and VM1G_06867, we also obtain a double deletion mutant ΔVmSom1/06867. Compared to the wild-type strain 11-175, the single deletion mutant VM1G_06867 shows a drastic reduction in growth rate and forms more pycnidia on the PDA medium. Additionally, the growth of the mutant is inhibited by SDS, Congo red, and fluorescent brighteners. In comparison to the single deletion mutant VmSom1, the double deletion mutant ΔVmSom1/06867 shows no significant change in growth or conidiation and is unable to produce conidia. The growth rate is significantly increased in Congo red, NaCl, and Sorbitol mediums. These results demonstrate that VM1G_06867 plays important roles in growth, pathogenicity, asexual development, and maintenance of cell wall integrity. VM1G_06867 can recover osmotic stress and cell wall integrity defects caused by the deletion of VmSom1, as well as restore the loss of pathogenicity caused by the deletion of the VmSom1 gene, but not completely.

Molecular Characteristics of Subgenomic RNAs and the Cap-Dependent Translational Advantage Relative to Corresponding Genomic RNAs of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) causes severe viral diseases on many economically important plants of Solanaceae. During the infection process of TSWV, a series of 3'-truncated subgenomic RNAs (sgRNAs) relative to corresponding genomic RNAs were synthesized, which were responsible for the expression of some viral proteins. However, corresponding genomic RNAs (gRNAs) seem to possess the basic elements for expression of these viral proteins. In this study, molecular characteristics of sgRNAs superior to genomic RNAs in viral protein expression were identified. The 3' ends of sgRNAs do not cover the entire intergenic region (IGR) of TSWV genomic RNAs and contain the remarkable A-rich characteristics. In addition, the 3' terminal nucleotides of sgRNAs are conserved among different TSWV isolates. Based on the eIF4E recruitment assay and subsequent northern blot, it is suggested that the TSWV sgRNA, but not gRNA, is capped in vivo; this is why sgRNA is competent for protein expression relative to gRNA. In addition, the 5' and 3' untranslated region (UTR) of sgRNA-Ns can synergistically enhance cap-dependent translation. This study further enriched the understanding of sgRNAs of ambisense RNA viruses.

Molecular characterization of a novel endornavirus isolated from Ophiostoma bicolor associated with bark beetles.

Ophiostoma bicolor is a pathogenic fungus associated with bark beetles that can cause serious damage to host plants. In this study, a novel fungal virus, "Ophiostoma bicolor endornavirus 1" (ObEV1), was obtained from O. bicolor, and its complete genome sequence was determined. ObEV1 has a single-stranded positive-sense (+ ss) RNA genome of 10,119 nucleotides. Sequence annotation and comparison showed that the viral genome has a single large open reading frame (ORF) encoding a polyprotein of 362.48 kDa. The polyprotein contains seven conserved domains: RNA-dependent RNA polymerase (RdRp), viral RNA helicase 1 (VHel1), viral methyltransferase (VMet), DEAD-like helicase (DEXDc), gliding-GltJ (G1), large tegument protein UL36 (PHA), and YlqF-related-GTPase (Y). Sequence comparisons and phylogenetic analysis showed that ObEV1 is a novel mycovirus belonging to the genus Betaendornavirus of the family Endornaviridae. This is the first report of a mycovirus in the ophiostomatoid fungus O. bicolor.

Multiple Levels of Triggered Factors and the Obligated Requirement of Cell-to-Cell Movement in the Mutation Repair of Cucumber Mosaic Virus with Defects in the tRNA-like Structure.

Some debilitating mutations in RNA viruses are repairable; however, the triggering factors of mutation repair remain largely unknown. In this study, multiple triggering factors of mutation repair are identified based on genetic damage to the TLS in CMV. TLS mutations in different RNAs distinctively impact viral pathogenicity and present different types of mutation repair. RNA2 relative reduction level or RNA3 sequence change resulting from TLS mutation is correlated with a high rate of mutation repair, and the TLS mutation of RNA1 fails to be repaired at the high inoculum dose. However, the TLS mutation of RNA1 can be repaired at a low dose of inoculation, particularly around the dilution end-point or in the mixed inoculation with RNA2 having a pre-termination mutation of the 2b gene, an RNAi suppressor. Taken together, TLS mutations resulting in quality or quantity defects of the viral genome or TLS mutations at low doses around the dilution end-point are likely to be repaired. Different levels of TLS mutation repair necessarily require cell-to-cell movement, therefore implying its obligated effect on the evolution of low-fitness viruses and providing a new insight into Muller's ratchet. This study provides important information on virus evolution and the application of mild viral vaccines.

Comparative Genomics Reveals Evolutionary Traits, Mating Strategies, and Pathogenicity-Related Genes Variation of Botryosphaeriaceae.

Botryosphaeriaceae, as a major family of the largest class of kingdom fungi Dothideomycetes, encompasses phytopathogens, saprobes, and endophytes. Many members of this family are opportunistic phytopathogens with a wide host range and worldwide geographical distribution, and can infect many economically important plants, including food crops and raw material plants for biofuel production. To date, however, little is known about the family evolutionary characterization, mating strategies, and pathogenicity-related genes variation from a comparative genome perspective. Here, we conducted a large-scale whole-genome comparison of 271 Dothideomycetes, including 19 species in Botryosphaeriaceae. The comparative genome analysis provided a clear classification of Botryosphaeriaceae in Dothideomycetes and indicated that the evolution of lifestyle within Dothideomycetes underwent four major transitions from non-phytopathogenic to phytopathogenic. Mating strategies analysis demonstrated that at least 3 transitions were found within Botryosphaeriaceae from heterothallism to homothallism. Additionally, pathogenicity-related genes contents in different genera varied greatly, indicative of genus-lineage expansion within Botryosphaeriaceae. These findings shed new light on evolutionary traits, mating strategies and pathogenicity-related genes variation of Botryosphaeriaceae.

A Predicted Stem Loop in Coat Protein-Coding Sequence of Tobacco Vein Banding Mosaic Virus Is Required for Efficient Replication.

Potyviral coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon C574G575C576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon C574G575C576 to A574G575A576 or A574G575G576, but not C574G575A576, C574G575T576, or C574G575G576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that C574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive C-G base pairs (C576-G531, C532-G575, C574-G533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon C532G533C534 to A532G533A534 and A532G533G534, but not C532G533A534, C532G533T534, or C532G533G534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that C532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial-passage inoculations, the accumulation of spontaneous mutant viruses was restored, and codons A532G533A534, A532G533G534, A574G575A576, or A574G575G576 of mutants were each separately changed to C532G533A534, C532G533G534, C574G575A576, or C574G575G576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive C532-G575 and C574-G533 base pairs played critical roles in TVBMV replication via maintaining the stability of the stem-loop structures formed by nucleotides 531 to 576 of the CP-coding sequence.

Translation of Plant RNA Viruses.

Plant RNA viruses encode essential viral proteins that depend on the host translation machinery for their expression. However, genomic RNAs of most plant RNA viruses lack the classical characteristics of eukaryotic cellular mRNAs, such as mono-cistron, 5' cap structure, and 3' polyadenylation. To adapt and utilize the eukaryotic translation machinery, plant RNA viruses have evolved a variety of translation strategies such as cap-independent translation, translation recoding on initiation and termination sites, and post-translation processes. This review focuses on advances in cap-independent translation and translation recoding in plant viruses.

Isolation and Identification of Talaromyces sp. Strain Q2 and Its Biocontrol Mechanisms Involved in the Control of Fusarium Wilt.

Fusarium wilt is an important disease of many food crops and often causes serious damages to yield and food quality. Consequently, numerous studies mainly focused on exploring the control strategy for Fusarium oxysporum as well as the mechanism of interaction between the F. oxysporum and other beneficial soil microorganisms. In this study, we have screened and identified an efficient biocontrol strain from the soil with infection of F. oxysporum f. sp. momordica (referred to as Fom), Talaromyces purpurogenus Q2 (referred to as TpQ2), which could be effective to reduce relative abundance of the rhizospheric Fom, leading to a significant decrease of Fusarium wilt disease incidence in bitter gourd during the greenhouse and field trails. TpQ2 can reduce the relative abundance of rhizospheric Fom through inhibition of growth and development of Fom. During the co-cultivation of TpQ2 and Fom, we confirmed that TpQ2 could significantly suppress the growth and development of Fom through disturbing the normal hyphae shape and function of the cell walls of Fom via secreting cell wall-degrading enzymes and suppression of the expression of cell wall biosynthesis genes, such as FomCFEM. In the meantime, TpQ2 showed a strong negative correlation with F. oxysporum in soil and positive correlation with beneficial indigenous microorganisms that had significant negative correlation with Fusarium populations, such as Streptomycetes, Lysobacter, and Sphingobium. To summarize, TpQ2 has a good biocontrol efficacy on Fusarium wilt of bitter gourd. The biocontrol mechanisms of TpQ2 on Fusarium wilt are complex and diverse.

Genome Assembly and Annotation of Botryosphaeria dothidea sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China.

Botryosphaeria dothidea is a latent and important fungal pathogen on a wide range of woody plants. Fruit ring rot caused by B. dothidea is a major disease in China on apple. This study establishes a high-quality, nearly complete, and well-annotated genome sequence of B. dothidea strain sdau11-99. The findings of this research provide a reference genome resource for further research on the apple fruit ring rot pathogen on apple and other hosts.

A unique internal ribosome entry site representing a dynamic equilibrium state of RNA tertiary structure in the 5'-UTR of Wheat yellow mosaic virus RNA1.

Internal ribosome entry sites (IRESes) were first reported in RNA viruses and subsequently identified in cellular mRNAs. In this study, IRES activity of the 5'-UTR in Wheat yellow mosaic virus (WYMV) RNA1 was identified, and the 3'-UTR synergistically enhanced this IRES activity via long-distance RNA-RNA interaction between C80U81and A7574G7575. Within the 5'-UTR, the hairpin 1(H1), flexible hairpin 2 (H2) and linker region (LR1) between H1 and H2 played an essential role in cap-independent translation, which is associated with the structural stability of H1, length of discontinuous stems and nucleotide specificity of the H2 upper loop and the long-distance RNA-RNA interaction sites in LR1. The H2 upper loop is a target region of the eIF4E. Cytosines (C55, C66, C105 and C108) in H1 and H2 and guanines (G73, G79 and G85) in LR1 form discontinuous and alternative base pairing to maintain the dynamic equilibrium state, which is used to elaborately regulate translation at a suitable level. The WYMV RNA1 5'-UTR contains a novel IRES, which is different from reported IRESes because of the dynamic equilibrium state. It is also suggested that robustness not at the maximum level of translation is the selection target during evolution of WYMV RNA1.

Close evolutionary relationship between rice black-streaked dwarf virus and southern rice black-streaked dwarf virus based on analysis of their bicistronic RNAs.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) seriously interfered in the production of rice and maize in China. These two viruses are members of the genus Fijivirus in the family Reoviridae and can cause similar dwarf symptoms in rice. Although some studies have reported the phylogenetic analysis on RBSDV or SRBSDV, the evolutionary relationship between these viruses is scarce. METHODS: In this study, we analyzed the evolutionary relationships between RBSDV and SRBSDV based on the data from the analysis of codon usage, RNA recombination and phylogenetic relationship, selection pressure and genetic characteristics of the bicistronic RNAs (S5, S7 and S9). RESULTS: RBSDV and SRBSDV showed similar patterns of codon preference: open reading frames (ORFs) in S7 and S5 had with higher and lower codon usage bias, respectively. Some isolates from RBSDV and SRBSDV formed a clade in the phylogenetic tree of S7 and S9. In addition, some recombination events in S9 occurred between RBSDV and SRBSDV. CONCLUSIONS: Our results suggest close evolutionary relationships between RBSDV and SRBSDV. Selection pressure, gene flow, and neutrality tests also supported the evolutionary relationships.

Variable 3'polyadenylation of Wheat yellow mosaic virus and its novel effects on translation and replication.

BACKGROUND: Polyadenylation influences many aspects of mRNA as well as viral RNA. variable polyadenylation at the 3' end have been reported in RNA viruses. It is interesting to identify the characteristic and potential role of 3' polyadenylation of Wheat yellow mosaic virus (WYMV), which has been reported to contain two genomic RNAs with 3' poly(A) tails and caused severe disease on wheat in East Asia region. METHODS: 3' RACE was used to identify sequences of the 3' end in WYMV RNAs from naturally infected wheat by WYMV. In vitro translation assay was performed to analyze effect of UTRs of WYMV with or without 3'polyadenylation on translation. In vitro replication mediated by WYMV NIb protein were performed to evaluate effect of variable polyadenylation on replication. RESULTS: Variable polyadenylation in WYMV RNAs was identified via 3' RACE. WYMV RNAs in naturally infected wheat in China simultaneously present with regions of long, short, or no adenylation at the 3' ends. The effects of variable polyadenylation on translation and replication of WYMV RNAs were evaluated. 5'UTR and 3'UTR of WYMV RNA1 or RNA2 synergistically enhanced the translation of the firefly luciferase (Fluc) gene in in vitro WGE system, whereas additional adenylates had an oppositive effect on this enhancement on translation mediated by UTRs of WYMV. Additional adenylates remarkably inhibited the synthesis of complementary strand from viral genome RNA during the in vitro replication mediated by WYMV NIb protein. CONCLUSIONS: 3' end of WYMV RNAs present variable polyadenylation even no polyadenylation. 3' polyadenylation have opposite effect on translation mediated by UTRs of WYMV RNA1 or RNA2. 3' polyadenylation have negative effect on minus-strand synthesis of WYMV RNA in vitro. Variable polyadenylation of WYMV RNAs may provide sufficient selection on the template for translation and replication.

Structural alteration of a BYDV-like translation element (BTE) that attenuates p35 expression in three mild Tobacco bushy top virus isolates.

To identify the molecular effects of Tobacco bushy top virus (TBTV) evolution on the degeneration of tobacco bushy top disease, three TBTV isolates with mild virulence were compared with wild-type TBTV to assess the translation of p35, which relies on a BYDV-like translation element (BTE) in a cap-independent manner. The in vitro expression of p35 in the mild isolates was only 20% to 40% of the expression observed in wt TBTV. Based on translation data from chimeric TBTV RNA, low-level p35 expression in the three mild isolates was associated with two regions: the 5' terminal 500 nt region (RI) and the 3' internal region (RV), which included the BTE. For the RV region, low level p35 expression was mainly associated with structural alterations of the BTE instead of specific sequence mutations within the BTE based on SHAPE structural probing and mutation analysis. Additionally, structural alteration of the TBTV BTE resulted from mutations outside of the BTE, implying structural complexity of the local region surrounding the BTE. This study is the first report on the structural alteration of the 3' cap-independent translation element among different isolates of a given RNA virus, which is associated with variations in viral virulence.

Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses.

BACKGROUND: Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. METHODS: RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. RESULTS: Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5' regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. CONCLUSIONS: All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative-sense RNA viruses.

Phylogenetic and recombination analysis of Tobacco bushy top virus in China.

BACKGROUND: During the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates. METHODS: 5' and 3' RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. RESULTS: The genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3'UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations. CONCLUSIONS: The genome sizes of TBTV isolates were constant while different ORF-coding regions and 3'UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors.