Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.
Many RNA viruses have genetically diverse populations known as quasispecies. Important biological characteristics may be related to the levels of diversity in the quasispecies (quasispecies cloud size), including adaptability and host range. Previous work using Tobacco mosaic virus and Cucumber mosaic virus indicated that evolutionarily related viruses have very different levels of diversity in a common host. The quasispecies cloud size for these viruses remained constant throughout serial passages. Inoculation of these viruses on a number of hosts demonstrated that quasispecies cloud size is not constant for these viruses but appears to be dependent on the host. The quasispecies cloud size remained constant as long as the viruses were maintained on a given host. Shifting the virus between hosts resulted in a change in cloud size to levels associated with the new host. Quasispecies cloud size for these viruses is related to host-virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases.
At a recent workshop, experts discussed the benefits, risks, and research priorities associated with using genetically manipulated insects in the control of vector-borne diseases.
Genetic bottlenecks may occur in virus populationsPlant viruses are dependent on vectors for their horizontal transmission, and aphids are the most common and important group of plant virus vectors. Aphids transmit at least 275 plant viruses, and approximately 75% of these viruses are transmitted in a nonpersistent manner, or stylet borne (14). This mode of transmission is characterized by a rapid rate of virus acquisition (Ͻ1 min) and inoculation by the aphids (8). A successful aphid transmission event is dependent upon the uptake of virus, stable retention of the acquired virions, the release of retained virions from regions within the mouth parts of the vector, and their delivery to a site of infection (10).Nonpersistent transmission by aphids may have an important effect on the dynamics and evolution of virus populations. Infection of a host may start with a small number of virions; transmission events could constitute genetic bottlenecks, which by definition are stochastic events that can introduce random elements into the genetic structures of populations (13). A major cause of virus strain differentiation could be genetic drift as a result of population bottlenecks during aphid transmission. However, so far there has been no experimental evidence to show that genetic bottlenecks occur during the aphid transmission of plant viruses.Cucumber mosaic virus (CMV) is efficiently transmitted in a nonpersistent manner by more than 75 species of aphids (11). The coat protein (CP) of CMV is a primary determinant of aphid transmission (1). The Fny isolate of CMV is efficiently transmitted by both Aphis gossypii (Glover) and Myzus persicae (Sulzer) (12).In a previous study, using an artificial population of CMV consisting of 14 mutants with silent restriction enzyme marker mutations, we showed that systemic infection constituted a significant bottleneck for the CMV populations (4). To understand the role of genetic bottlenecks during aphid transmission of CMV, we mechanically inoculated 12 of the CMV mutants into squash cotyledons. A. gossypii and M. persicae were used for the transmission of CMV from inoculated source leaves to healthy squash leaves. When the newly infected squash leaves were analyzed for the presence of each of the 12 marker mutants, we found that aphid transmission induced a significant genetic bottleneck in the CMV population. MATERIALS AND METHODSVirus mutants and plant inoculation. Cotyledons of squash seedlings (Cucurbita pepo cv. Elite) were mechanically inoculated with individual or mixed viral RNAs of CMV mutants (a, b, c, d, e, f, g, h, i, j, k and l) described previously (4). Three days postinoculation, the inoculated cotyledons were used as the virus source leaves for aphid transmission to healthy squash cotyledons.Aphids and transmission assays. A. gossypii was reared on healthy squash or cotton seedlings, and M. persicae was raised on healthy Chinese cabbage or turnips. Before acquisition, aphids were starved for 2 to 3 h on a moistened filter paper in a petri dish. Individual aphids ...
The levels of population diversity of three related Sindbis-like plant viruses, Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus (CCMV), in infections of a common host, Nicotiana benthamiana, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in N. benthamiana. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.The error-prone replication, large populations, and rapid replication times associated with RNA viruses result in the potential for genetically diverse populations, termed quasispecies, arising within a single host (16). Developed as a model for early forms of life (14), the theoretical quasispecies describes a steady-state collection of genetic mutants that vary around a consensus sequence. Viral quasispecies are complex and dynamic distributions where the level of population variation (quasispecies cloud size) reacts to changes in selection pressures (8). There are a number of biological and evolutionary implications associated with highly diverse populations (7). Maintaining a large quasispecies cloud size could allow a virus ready access to a pool of mutants which could become the selectively advantaged dominant RNA species in a shift to a new environment. Conversely, highly diverse populations that are subjected to repeated bottlenecks have been shown to lose fitness through a process known as Muller's ratchet (4,9,10,25,26).Both DNA (15, 39) and RNA plant viruses (6,23,30,32) can maintain highly diverse populations in collections of field isolates, but the quasispecies variation of single plant virus isolates has not been examined. Plant viruses can use several mechanisms to generate quasispecies diversity, including replication error, recombination, and, for multipartite viruses, reassortment (for a review see reference 33). However, the extent of population variation is limited by selection pressure for variants that interact successfully with different host and viral proteins necessary for completion of the infection cycle.The Sindbis-like virus group includes a number of plant and animal viruses with similarities in their genome organizations and nonstructural proteins. The three virus species chosen for this study were Tobacco mosaic virus (TMV; genus Tobamovirus), Cucumber mosaic virus (CMV; genus Cucumovirus, family Bromoviridae), and Cowpea chlorotic mottle virus (CCMV; genus Bromovirus, family Bromoviridae). TMV is a monopartite rod-shaped virus, and CMV and CCMV are tripar...
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