Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana
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We infected a transgenic Arabidopsis line (GxA), containing an amplicon-silenced 35S : : GFP transgene, with cauliflower mosaic virus (CaMV), a plant pararetrovirus with a DNA genome. Systemically infected leaves showed strong GFP fluorescence and amplicon transcripts were detectable in Northern blots, indicating that silencing of GFP had been suppressed during CaMV-infection. Transgenic Arabidopsis lines expressing CaMV protein P6, the major genetic determinant of symptom severity, were crossed with GxA. Progeny showed strong GFP fluorescence throughout and amplicon transcripts were detectable in Northern blots, indicating that P6 was suppressing local and systemic silencing. However, levels of 21 nt siRNAs derived from the GFP transgene were not reduced. In CaMV-infected plants, the P6 transgene did not reduce levels of CaMV leader-derived 21 and 24 nt siRNAs relative to levels of CaMV 35S RNA. These results demonstrate that CaMV can efficiently suppress silencing of a GFP transgene, and that P6 acts as a silencing suppressor.RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode countersilencing proteins (Voinnet, 2001;Moissiard & Voinnet, 2004). The trigger for silencing is the presence of doublestranded (ds) RNA. For viruses with single-stranded RNA genomes, dsRNAs comprise an essential replication intermediate. dsRNAs are not obligate replication intermediates for viruses with DNA genomes but may nevertheless accumulate during infection (Blevins et al., 2006). Several geminiviruses undergo silencing, and several also encode silencing suppressors (Bisaro, 2006;Cui et al., 2005;Trinks et al., 2005). Cauliflower mosaic virus (CaMV), the type member of the family Caulimoviridae, is a pararetrovirus with an 8 kbp dsDNA genome and replicates by reverse transcription (Haas et al., 2002). In Brassica napus, CaMV undergoes silencing (Al Kaff et al., 1998), and CaMVinfected Arabidopsis accumulate siRNAs that are derived mainly from the viral 35S RNA leader (Moissiard & Voinnet, 2006;Blevins et al., 2006).To test whether CaMV is also capable of suppressing silencing, we infected a transgenic Arabidopsis line in which expression of GFP is silenced (Dalmay et al., 2000). GxA (obtained from Professor D. C. Baulcombe, Sainsbury Laboratory, Norwich, UK) contains both a 35S : : GFP transgene and one expressing a potato virus X (PVX) amplicon from a phloem-specific promoter. The amplicon, which is silenced, contains GFP sequences and these cosuppress the 35S : : GFP transgene. GxA allows analysis of both local silencing in the vascular tissue (where the amplicon is expressed), and systemic movement of the silencing signal into the remaining leaf tissue (Dalmay et al., 2000).Groups of approximately ten plants were inoculated with CaMV, either manually at the two-leaf stage (Cecchini et al., 1998), or by particle bombardment at the eight-leaf stage (Love et al., 2007). Inoculated plants began to show typical systemic symptoms of infection (distortion and stunting of the central ...
We infected a transgenic Arabidopsis line (GxA), containing an amplicon-silenced 35S : : GFP transgene, with cauliflower mosaic virus (CaMV), a plant pararetrovirus with a DNA genome. Systemically infected leaves showed strong GFP fluorescence and amplicon transcripts were detectable in Northern blots, indicating that silencing of GFP had been suppressed during CaMV-infection. Transgenic Arabidopsis lines expressing CaMV protein P6, the major genetic determinant of symptom severity, were crossed with GxA. Progeny showed strong GFP fluorescence throughout and amplicon transcripts were detectable in Northern blots, indicating that P6 was suppressing local and systemic silencing. However, levels of 21 nt siRNAs derived from the GFP transgene were not reduced. In CaMV-infected plants, the P6 transgene did not reduce levels of CaMV leader-derived 21 and 24 nt siRNAs relative to levels of CaMV 35S RNA. These results demonstrate that CaMV can efficiently suppress silencing of a GFP transgene, and that P6 acts as a silencing suppressor.RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode countersilencing proteins (Voinnet, 2001;Moissiard & Voinnet, 2004). The trigger for silencing is the presence of doublestranded (ds) RNA. For viruses with single-stranded RNA genomes, dsRNAs comprise an essential replication intermediate. dsRNAs are not obligate replication intermediates for viruses with DNA genomes but may nevertheless accumulate during infection (Blevins et al., 2006). Several geminiviruses undergo silencing, and several also encode silencing suppressors (Bisaro, 2006;Cui et al., 2005;Trinks et al., 2005). Cauliflower mosaic virus (CaMV), the type member of the family Caulimoviridae, is a pararetrovirus with an 8 kbp dsDNA genome and replicates by reverse transcription (Haas et al., 2002). In Brassica napus, CaMV undergoes silencing (Al Kaff et al., 1998), and CaMVinfected Arabidopsis accumulate siRNAs that are derived mainly from the viral 35S RNA leader (Moissiard & Voinnet, 2006;Blevins et al., 2006).To test whether CaMV is also capable of suppressing silencing, we infected a transgenic Arabidopsis line in which expression of GFP is silenced (Dalmay et al., 2000). GxA (obtained from Professor D. C. Baulcombe, Sainsbury Laboratory, Norwich, UK) contains both a 35S : : GFP transgene and one expressing a potato virus X (PVX) amplicon from a phloem-specific promoter. The amplicon, which is silenced, contains GFP sequences and these cosuppress the 35S : : GFP transgene. GxA allows analysis of both local silencing in the vascular tissue (where the amplicon is expressed), and systemic movement of the silencing signal into the remaining leaf tissue (Dalmay et al., 2000).Groups of approximately ten plants were inoculated with CaMV, either manually at the two-leaf stage (Cecchini et al., 1998), or by particle bombardment at the eight-leaf stage (Love et al., 2007). Inoculated plants began to show typical systemic symptoms of infection (distortion and stunting of the central ...
Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes ( R gene) into elite crop cultivars has been the standard and most cost‐effective practice. However, R gene‐mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad‐spectrum antiviral strategies. The CRISPR ‐Cas9 (clustered regularly interspaced palindromic repeat, CRISPR ‐associated) editing system has been employed to confer resistance to human viruses and several plant single‐stranded DNA geminiviruses, pointing out the possible application of the CRISPR ‐Cas9 system for virus control. Here, we demonstrate that strong viral resistance to cauliflower mosaic virus (Ca MV ), a pararetrovirus with a double‐stranded DNA genome, can be achieved through Cas9‐mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNA s (si RNA ) are produced and mostly map to the 3′ end of single‐guide RNA s (sg RNA ), although very low levels of si RNA s map to the spacer region as well. However, these si RNA s are not responsible for the inhibited Ca MV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9‐induced DNA breaks at the sg RNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, nonfunctional coat proteins. We also recovered wild‐type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild‐type coat proteins. Our data demonstrate that the CRISPR ‐Cas9 system can be used for virus control against plant pararetroviruses with further modifications.
Inappropriate annotation of a key defence marker in Arabidopsis: will the real PR-1 please stand up?
PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two "full genome" Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as "PR-1-like". Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as " PR-1" is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.
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