RNA-Seq reveals virus-virus and virus-plant interactions in nature.
ABSTRACT: As research on plant viruses has focused mainly on crop diseases, little is known about these viruses in natural environments. To understand the ecology of viruses in natural systems, comprehensive information on virus-virus and virus-host interactions is required. We applied RNA-Seq to plants from a natural population of Arabidopsis halleri subsp. gemmifera to simultaneously determine the presence/absence of all sequence-reported viruses, identify novel viruses and quantify the host transcriptome. By introducing the criteria of read number and genome coverage, we detected infections by Turnip mosaic virus (TuMV), Cucumber mosaic virus and Brassica yellows virus Active TuMV replication was observed by ultramicroscopy. De novo assembly further identified a novel partitivirus, Arabidopsis halleri partitivirus 1 Interestingly, virus reads reached a maximum level that was equivalent to that of the host's total mRNA, although asymptomatic infection was common. AhgAGO2, a key gene in host defence systems, was upregulated in TuMV-infected plants. Multiple infection was frequent in TuMV-infected leaves, suggesting that TuMV facilitates multiple infection, probably by suppressing host RNA silencing. Revealing hidden plant-virus interactions in nature can enhance our understanding of biological interactions and may have agricultural applications.
Project description:Persistent infection, wherein a pathogen is continually present in a host individual, is widespread in virus-host systems. However, little is known regarding how seasonal environments alter virus-host interaction during such metastability. We observed a lineage-to-lineage infection of the host plant Arabidopsis halleri with Turnip mosaic virus for 3 years without severe damage. Virus dynamics and virus-host interactions within hosts were highly season dependent. Virus accumulation in the newly formed leaves was temperature dependent and was suppressed during winter. Transcriptome analyses suggested that distinct defence mechanisms, i.e. salicylic acid (SA)-dependent resistance and RNA silencing, were predominant during spring and autumn, respectively. Transcriptomic difference between infected and uninfected plants other than defence genes appeared transiently only during autumn in upper leaves. However, the virus preserved in the lower leaves is transferred to the clonal offspring of the host plants during spring. In the linage-to-linage infection of the A. halleri-TuMV system, both host clonal reproduction and virus transmission into new clonal rosettes are secured during the winter-spring transition. How virus and host overwinter turned out to be critical for understanding a long-term virus-host interaction within hosts under temperate climates, and more generally, understanding seasonality provides new insight into ecology of plant viruses.
Project description:Turnip mosaic virus (TuMV, family Potyviridae) and cauliflower mosaic virus (CaMV, family Caulimoviridae) are transmitted by aphid vectors. They are the only viruses shown so far to undergo transmission activation (TA) immediately preceding plant-to-plant propagation. TA is a recently described phenomenon where viruses respond to the presence of vectors on the host by rapidly and transiently forming transmissible complexes that are efficiently acquired and transmitted. Very little is known about the mechanisms of TA and on whether such mechanisms are alike or distinct in different viral species. We use here a pharmacological approach to initiate the comparison of TA of TuMV and CaMV. Our results show that both viruses rely on calcium signaling and reactive oxygen species (ROS) for TA. However, whereas application of the thiol-reactive compound N-ethylmaleimide (NEM) inhibited, as previously shown, TuMV transmission it did not alter CaMV transmission. On the other hand, sodium azide, which boosts CaMV transmission, strongly inhibited TuMV transmission. Finally, wounding stress inhibited CaMV transmission and increased TuMV transmission. Taken together, the results suggest that transmission activation of TuMV and CaMV depends on initial calcium and ROS signaling that are generated during the plant's immediate responses to aphid manifestation. Interestingly, downstream events in TA of each virus appear to diverge, as shown by the differential effects of NEM, azide and wounding on TuMV and CaMV transmission, suggesting that these two viruses have evolved analogous TA mechanisms.
Project description:Virulence evolution may have far-reaching consequences for virus epidemiology and emergence, and virologists have devoted increasing effort to understand the modulators of this process. However, still little is known on the mechanisms and determinants of virulence evolution in sterilizing viruses that, as they prevent host reproduction, may have devastating effects on host populations. Theory predicts that sterilizing parasites, including viruses, would evolve towards lower virulence and absolute host sterilization to optimize the exploitation of host resources and maximize fitness. However, this hypothesis has seldom been analyzed experimentally. We investigated the evolution of virulence of the sterilizing plant virus Turnip mosaic virus (TuMV) in its natural host Arabidopsis thaliana by serial passage experiments. After passaging, we quantified virus accumulation and infectivity, the effect of infection on plant growth and development, and virulence of the ancestral and passaged viral genotypes in A. thaliana. Results indicated that serial passaging increased the proportion of infected plants showing absolute sterility, reduced TuMV virulence, and increased virus multiplication and infectivity. Genomic comparison of the ancestral and passaged TuMV genotypes identified significant mutation clustering in the P1, P3, and 6K2 proteins, suggesting a role of these viral proteins in the observed phenotypic changes. Our results support theoretical predictions on the evolution of virulence of sterilizing parasites and contribute to better understand the phenotypic and genetic changes associated with this process.
Project description:Narcissus plants (Narcissus tazetta var. chinensis) showing mosaic or striping leaves were collected from around Japan, and tested for virus infections using potyvirus-specific primers. Many were found to be infected with a macluravirus and mixtures of different potyviruses, one third of them narcissus yellow stripe virus (NYSV)-like viruses. Genomes of nine of the NYSV-like viruses were sequenced and, together with four already published, provided data for phylogenetic and pairwise identity analyses of their place in the turnip mosaic virus (TuMV) phylogenetic group. Using existing ICTV criteria for defining potyvirus species, the narcissus viruses in TuMV group were found to be from five species; the previously described NLSYV, and four new species we call narcissus virus 1 (NV-1) and narcissus yellow stripe-1 to -3 (NYSV-1, NYSV-2 and NYSV-3). However, as all are from a single host species, and natural recombinants with NV-1 and NYSV-3 'parents have been found in China and India, we also conclude that they could be considered to be members of a single mega-species, narcissus virus; the criteria for defining such a potyvirus species would then be that their polyprotein sequences have greater than 69% identical nucleotides and greater than 75% identical amino acids.
Project description:Turnip mosaic virus (TuMV), a potyvirus, is a flexible filamentous plant virus that displays a helical arrangement of coat protein copies (CPs) bound to the ssRNA genome. TuMV is a bona fide representative of the Potyvirus genus, one of most abundant groups of plant viruses, which displays a very wide host range. We have studied by cryoEM the structure of TuMV virions and its viral-like particles (VLPs) to explore the role of the interactions between proteins and RNA in the assembly of the virions. The results show that the CP-RNA interaction is needed for the correct orientation of the CP N-terminal arm, a region that plays as a molecular staple between CP subunits in the fully assembled virion.
Project description:Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes, including responses to pathogen attack. Here, we report the identification of Glycine max (soybean) endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a bottom-up proteomics approach. Knockdown of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B in Arabidopsis thaliana (AtDRP2A, AtDRP2B), homologs of GmSDL5A, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both A. thaliana drp2a (atdrp2a) and atdrp2b knockout mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI, and 6K2. Of these viral proteins, VPg, CP, and CI are essential for viral intercellular movement, and 6K2, VPg, and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together, these data suggest that dynamin-like proteins are novel host factors of potyviruses and that endocytic processes are involved in potyvirus infection.IMPORTANCE It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.
Project description:Increased light intensity has been predicted as a major consequence of climate change. Light intensity is a critical resource involved in many plant processes, including the interaction with viruses. A central question to plant-virus interactions is understanding the determinants of virus dispersal among plants. However, very little is known on the effect of environmental factors on virus transmission, particularly through seeds. The fitness of seed-transmitted viruses is highly dependent on host reproductive potential, and requires higher virus multiplication in reproductive organs. Thus, environmental conditions that favor reduced virus virulence without controlling its level of within-plant multiplication (i.e., tolerance) may enhance seed transmission. We tested the hypothesis that light intensity conditions that enhance plant tolerance promote virus seed transmission. To do so, we challenged 18 Arabidopsis thaliana accessions with Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV) under high and low light intensity. Results indicated that higher light intensity increased TuMV multiplication and/or plant tolerance, which was associated with more efficient seed transmission. Conversely, higher light intensity reduced plant tolerance and CMV multiplication, and had no effect on seed transmission. This work provides novel insights on how environmental factors modulate plant virus transmission and contributes to understand the underlying processes.
Project description:Cauliflower mosaic virus (CaMV; family Caulimoviridae) responds to the presence of aphid vectors on infected plants by forming specific transmission morphs. This phenomenon, coined transmission activation (TA), controls plant-to-plant propagation of CaMV. A fundamental question is whether other viruses rely on TA. Here, we demonstrate that transmission of the unrelated turnip mosaic virus (TuMV; family Potyviridae) is activated by the reactive oxygen species H2O2 and inhibited by the calcium channel blocker LaCl3 H2O2-triggered TA manifested itself by the induction of intermolecular cysteine bonds between viral helper component protease (HC-Pro) molecules and by the formation of viral transmission complexes, composed of TuMV particles and HC-Pro that mediates vector binding. Consistently, LaCl3 inhibited intermolecular HC-Pro cysteine bonds and HC-Pro interaction with viral particles. These results show that TuMV is a second virus using TA for transmission but using an entirely different mechanism than CaMV. We propose that TuMV TA requires reactive oxygen species (ROS) and calcium signaling and that it is operated by a redox switch.IMPORTANCE Transmission activation, i.e., a viral response to the presence of vectors on infected hosts that regulates virus acquisition and thus transmission, is an only recently described phenomenon. It implies that viruses contribute actively to their transmission, something that has been shown before for many other pathogens but not for viruses. However, transmission activation has been described so far for only one virus, and it was unknown whether other viruses also rely on transmission activation. Here we present evidence that a second virus uses transmission activation, suggesting that it is a general transmission strategy.
Project description:Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb.
Project description:All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection.