RESUMO
Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.
Assuntos
Nicotiana , Nicotiana/virologia , Nicotiana/metabolismo , Potyviridae/genética , Potyviridae/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Permeabilidade da Membrana Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas Viroporinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Doenças das Plantas/virologia , Potássio/metabolismoRESUMO
BACKGROUND: Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are components of the wheat streak mosaic virus disease complex in the Great Plains region of the U.S.A. and elsewhere. Co-infection of wheat with WSMV and TriMV causes synergistic interaction with more severe disease symptoms compared to single infections. Plants are equipped with multiple antiviral mechanisms, of which regulation of microRNAs (miRNAs) is a potentially effective constituent. In this investigation, we have analyzed the total and relative expression of miRNA transcriptome in two wheat cultivars, Arapahoe (susceptible) and Mace (temperature-sensitive-resistant), that were mock-inoculated or inoculated with WSMV, TriMV, or both at 18 °C and 27 °C. RESULTS: Our results showed that the most abundant miRNA family among all the treatments was miRNA166, followed by 159a and 168a, although the order of the latter two changed depending on the infections. When comparing infected and control groups, twenty miRNAs showed significant upregulation, while eight miRNAs were significantly downregulated. Among them, miRNAs 9670-3p, 397-5p, and 5384-3p exhibited the most significant upregulation, whereas miRNAs 319, 9773, and 9774 were the most downregulated. The comparison of infection versus the control group for the cultivar Mace showed temperature-dependent regulation of these miRNAs. The principal component analysis confirmed that less abundant miRNAs among differentially expressed miRNAs were strongly correlated with the inoculated symptomatic wheat cultivars. Notably, miRNAs 397-5p, 398, and 9670-3p were upregulated in response to WSMV and TriMV infections, an observation not yet reported in this context. The significant upregulation of these three miRNAs was further confirmed with RT-qPCR analysis; in general, the RT-qPCR results were in agreement with our computational analysis. Target prediction analysis showed that the miRNAs standing out in our analysis targeted genes involved in defense response and regulation of transcription. CONCLUSION: Investigation into the roles of these miRNAs and their corresponding targets holds promise for advancing our understanding of the mechanisms of virus infection and possible manipulation of these factors for developing durable virus resistance in crop plants.
Assuntos
MicroRNAs , Potyviridae , MicroRNAs/genética , Doenças das Plantas/genética , Potyviridae/genéticaRESUMO
BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.
Assuntos
Coinfecção , Euphorbia , Ácidos Nucleicos , Vírus de Plantas , Potyviridae , Vírus de RNA , Inosina Trifosfatase , Filogenia , Vírus de RNA/genética , Nucleotídeos/genética , Potyviridae/genética , Vírus de Plantas/genética , Plantas/genética , RNA Viral/genética , Genoma ViralRESUMO
Triticum mosaic virus (TriMV; genus Poacevirus; family Potyviridae) is an economically important virus in the Great Plains region of the United States. TriMV is transmitted by the wheat curl mite (Aceria tosichella) Type 2 genotype but not by Type 1. Helper component-proteinase (HC-Pro) is a vector transmission determinant for several potyvirids, but the role of HC-Pro in TriMV transmission is unknown. In this study, we examined the requirement of the HC-Pro cistron of TriMV for wheat curl mite (Type 2) transmission through deletion and point mutations and constructing TriMV chimeras with heterologous HC-Pros from other potyvirids. TriMV with complete deletion of HC-Pro failed to be transmitted by wheat curl mites at detectable levels. Furthermore, TriMV chimeras with heterologous HC-Pros from aphid-transmitted turnip mosaic virus and tobacco etch virus, or wheat curl mite-transmitted wheat streak mosaic virus, failed to be transmitted by wheat curl mites. These data suggest that heterologous HC-Pros did not complement TriMV for wheat curl mite transmission. A decreasing series of progressive nested in-frame deletions at the N-terminal region of HC-Pro comprising amino acids 3 to 125, 3 to 50, 3 to 25, 3 to 15, 3 to 8, and 3 and 4 abolished TriMV transmission by wheat curl mites. Additionally, mutation of conserved His20, Cys49, or Cys52 to Ala in HC-Pro abolished TriMV transmissibility by wheat curl mites. These data suggest that the N-terminal region of HC-Pro is crucial for TriMV transmission by wheat curl mites. Collectively, these data demonstrate that the HC-Pro cistron of TriMV is a viral determinant for wheat curl mite transmission.
Assuntos
Ácaros , Doenças das Plantas , Potyviridae , Triticum , Proteínas Virais , Triticum/virologia , Animais , Doenças das Plantas/virologia , Ácaros/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Potyviridae/genética , Potyviridae/fisiologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismoRESUMO
The infection of young winter barley (Hordeum vulgare L.) root system in winter by barley yellow mosaic virus (BaYMV) can lead to high yield losses. Resistance breeding is critical for managing this virus, but there are only a few reports on resistance genes that describe how the genes control BaYMV propagation and the systemic movement from the roots to the leaves. Here we report a real-time quantitative PCR analysis of the virus in barley roots and leaves carrying BaYMV resistance genes (rym1 to rym15 and an unknown gene) to elucidate the molecular mechanisms underlying the barley response to BaYMV. The resistance mechanism directly targets the virus. Moreover, the resistance genes/cultivars were classified into the following three groups according to their BaYMV titer: (i) immune (BaYMV was undetectable in the roots or leaves), (ii) partially immune (BaYMV was detected in the roots but not in the leaves), and (iii) susceptible (BaYMV was detected in the roots and leaves). Our results clarified the functions of the resistance genes in barley roots and leaves following a BaYMV infection. We anticipate our analysis to be a starting point for more understanding of the correspondence between resistance genes of Triticeae and the soil-borne viruses.
Assuntos
Resistência à Doença , Hordeum , Doenças das Plantas , Folhas de Planta , Raízes de Plantas , Hordeum/virologia , Hordeum/genética , Doenças das Plantas/virologia , Doenças das Plantas/imunologia , Doenças das Plantas/genética , Raízes de Plantas/virologia , Raízes de Plantas/genética , Folhas de Planta/virologia , Resistência à Doença/genética , Replicação Viral/genética , Genes de Plantas/genética , Potyviridae/fisiologia , Potyviridae/genéticaRESUMO
Triticum mosaic virus (TriMV), the type species of the genus Poacevirus in the family Potyviridae, is an economically important wheat curl mite-transmitted wheat-infecting virus in the Great Plains region of the USA. In this study, the functional genomics of helper component-proteinase (HC-Pro) encoded by TriMV was examined using a reverse genetics approach. TriMV with complete deletion of HC-Pro cistron elicited systemic infection in wheat, indicating that HC-Pro cistron is dispensable for TriMV systemic infection. However, TriMV lacking HC-Pro caused delayed systemic infection with mild symptoms that resulted in little or no stunting of plants with a significant reduction in the accumulation of genomic RNA copies and coat protein (CP). Sequential deletion mutagenesis from the 5' end of HC-Pro cistron in the TriMV genome revealed that deletions within amino acids 3 to 25, except for amino acids 3 and 4, elicited mild symptoms with reduced accumulation of genomic RNA and CP. Surprisingly, TriMV with deletion of amino acids 3 to 50 or 3 to 125 in HC-Pro elicited severe symptoms with a substantial increase in genomic RNA copies but a drastic reduction in CP accumulation. Additionally, TriMV with heterologous HC-Pro from other potyvirids produced symptom phenotype and genomic RNA accumulation similar to that of TriMV without HC-Pro, suggesting that HC-Pros of other potyvirids were not effective in complementing TriMV in wheat. Our data indicate that HC-Pro is expendable for replication of TriMV but is required for efficient viral genomic RNA amplification and symptom development. The availability of TriMV with various deletions in the HC-Pro cistron will facilitate the examination of the requirement of HC-Pro for wheat curl mite transmission.
Assuntos
Potyviridae , Triticum , Potyviridae/genética , Fenótipo , RNA , Aminoácidos/genética , Doenças das PlantasRESUMO
Sugarcane streak mosaic virus (SCSMV) is one of the major pathogens of sugarcane in the world. Molecular studies and disease management of SCSMV are hindered by the lack of efficient infectious clones. In this study, we successfully constructed Agrobacterium infiltration based infectious clone of SCSMV with different variants. Infectious clones of wild type SCSMV could efficiently infect Nicotiana benthamiana and sugarcane plants resulting in streak and mosaic symptoms on systemic leaves which were further confirmed with RT-PCR and serological assays. SCSMV variants of less adenylation displayed attenuated pathogenicity on N.benthamiana. SCSMV-based recombinant heterologous EGFP protein vector was also developed. The EGFP-tagged recombinant SCSMV could highly expressed in vegetative organs including roots. These infectious clones of SCSMV could be further developed for platform tools for both biotechnological studies and management of SCSMV disease.
Assuntos
Potyviridae , Saccharum , Doenças das Plantas , Filogenia , Potyviridae/genéticaRESUMO
To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.
Assuntos
Técnicas de Diagnóstico Molecular , Vírus do Mosaico , Técnicas de Amplificação de Ácido Nucleico , Potyviridae , Triticum , Extratos VegetaisRESUMO
Wheat is an essential cereal crop for the economy and food safety of Kazakhstan. In the present work, a screening of wheat and barley from different regions of Kazakhstan was conducted using newly developed specific primers for reverse transcription PCR and loop-mediated isothermal amplification (LAMP) assays. In total, 82 and 19 of 256 samples of wheat and barley tested positive for wheat streak mosaic virus (WSMV) and barley stripe mosaic virus (BSMV), respectively. A phylogenetic analysis using two independent methods revealed that most of the analyzed isolates had a European origin. Molecular data on the distribution and diversity of cereal viruses in Kazakhstan were obtained for the first time and will help lay a foundation for the implementation of genetics and genomics in wheat phyto-epidemiology in the country.
Assuntos
Hordeum , Vírus de Plantas , Potyviridae , Cazaquistão , Filogenia , Grão ComestívelRESUMO
Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.
Assuntos
Manihot , Doenças das Plantas , Potyviridae , Recombinases , Manihot/virologia , Doenças das Plantas/virologia , Potyviridae/genética , Potyviridae/isolamento & purificação , Recombinases/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Folhas de Planta/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Transcrição Reversa , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The ipomoviruses (family Potyviridae) that cause cassava brown streak disease (cassava brown streak virus [CBSV] and Uganda cassava brown streak virus [UCBSV]) are damaging plant pathogens that affect the sustainability of cassava production in East and Central Africa. However, little is known about the rate at which the viruses evolve and when they emerged in Africa - which inform how easily these viruses can host shift and resist RNAi approaches for control. We present here the rates of evolution determined from the coat protein gene (CP) of CBSV (Temporal signal in a UCBSV dataset was not sufficient for comparable analysis). Our BEAST analysis estimated the CBSV CP evolves at a mean rate of 1.43 × 10-3 nucleotide substitutions per site per year, with the most recent common ancestor of sampled CBSV isolates existing in 1944 (95% HPD, between years 1922 - 1963). We compared the published measured and estimated rates of evolution of CPs from ten families of plant viruses and showed that CBSV is an average-evolving potyvirid, but that members of Potyviridae evolve more quickly than members of Virgaviridae and the single representatives of Betaflexiviridae, Bunyaviridae, Caulimoviridae and Closteroviridae.
Assuntos
Proteínas do Capsídeo , Evolução Molecular , Manihot , Filogenia , Doenças das Plantas , Potyviridae , Potyviridae/genética , Doenças das Plantas/virologia , Manihot/virologia , Proteínas do Capsídeo/genéticaRESUMO
In early spring 2018, significant mosaic disease symptoms were observed for the first time on barley leaves (Hordeum vulgare L., cv. New Sachiho Golden) in Takanezawa, Tochigi Prefecture, Japan. This cultivar carries the resistance gene rym3 (rym; resistance to yellow mosaic). Through RNA-seq analysis, Barley yellow mosaic virus (BaYMV-Takanezawa) was identified in the roots of all five plants (T01-T05) in the field. Phylogenetic analysis of RNA1, encompassing known BaYMV pathotypes I through V, revealed that it shares the same origin as isolate pathotype IV (BaYMV-Ohtawara pathotype). However, RNA2 analysis of isolates revealed the simultaneous presence of two distinct BaYMV isolates, BaYMV-Takanezawa-T01 (DRR552862, closely related to pathotype IV) and BaYMV-Takanezawa-T02 (DRR552863, closely related to pathotype III). The amino acid sequences of the BaYMV-Takanezawa isolates displayed variations, particularly in the VPg and N-terminal region of CP, containing mutations not found in other domains of the virus genome. Changes in the CI (RNA1 amino acid residue 459) and CP (RNA1 amino acid residue 2138) proteins correlated with pathogenicity. These findings underscore the importance of monitoring and understanding the genetic diversity of BaYMV for effective disease management strategies in crop breeding.
Assuntos
Resistência à Doença , Hordeum , Filogenia , Doenças das Plantas , Hordeum/virologia , Doenças das Plantas/virologia , Japão , Resistência à Doença/genética , RNA Viral/genética , PotyviridaeRESUMO
Cassava is a key source of calories for smallholder farmers in sub-Saharan Africa but its role as a food security crop is threatened by the cross-continental spread of cassava brown streak disease (CBSD) that causes high yield losses. In order to mitigate the impact of CBSD, it is important to minimise the delay in first detection of CBSD after introduction to a new country or state so that interventions can be deployed more effectively. Using a computational model that combines simulations of CBSD spread at both the landscape and field scales, we model the effectiveness of different country level survey strategies in Nigeria when CBSD is directly introduced. We find that the main limitation to the rapid CBSD detection in Nigeria, using the current survey strategy, is that an insufficient number of fields are surveyed in newly infected Nigerian states, not the total number of fields surveyed across the country, nor the limitation of only surveying fields near a road. We explored different strategies for geographically selecting fields to survey and found that early and consistent CBSD detection will involve confining candidate survey fields to states where CBSD has not yet been detected and where survey locations are allocated in proportion to the density of cassava crops, detects CBSD sooner, more consistently, and when the epidemic is smaller compared with distributing surveys uniformly across Nigeria.
Assuntos
Simulação por Computador , Manihot , Doenças das Plantas , Nigéria/epidemiologia , Manihot/virologia , PotyviridaeRESUMO
Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.