Differential roles of geminivirus Rep and AC4 (C4) in the induction of necrosis in Nicotiana benthamiana (original) (raw)
Related papers
Journal of General Virology, 2008
We have devised an in planta system for functional analysis of the replication-associated protein (Rep) of African cassava mosaic virus (ACMV). Using this assay and PCR-based random mutagenesis, we have identified an ACMV Rep mutant that failed to trigger the hypersensitive response (HR), but had an enhanced ability to initiate DNA replication. The mutant Rep-green fluorescent protein (GFP) fusion protein was localized to the nucleus. Sequence analysis showed that the mutated Rep gene had three nucleotide changes (A6AT, T375AG and G852AA); only the A6AT transversion resulted in an amino acid substitution (Arg to Ser), which is at the second residue in the 358 amino acid ACMV Rep protein. Our results indicate that a single amino acid can alter the differential ability of ACMV Rep to trigger the host-mediated HR defence mechanism and to initiate viral DNA replication. The implications of this finding are discussed in the context of plant-virus interactions.
We have devised an in planta system for functional analysis of the replication-associated protein (Rep) of African cassava mosaic virus (ACMV). Using this assay and PCR-based random mutagenesis, we have identified an ACMV Rep mutant that failed to trigger the hypersensitive response (HR), but had an enhanced ability to initiate DNA replication. The mutant Rep-green fluorescent protein (GFP) fusion protein was localized to the nucleus. Sequence analysis showed that the mutated Rep gene had three nucleotide changes (A6AT, T375AG and G852AA); only the A6AT transversion resulted in an amino acid substitution (Arg to Ser), which is at the second residue in the 358 amino acid ACMV Rep protein. Our results indicate that a single amino acid can alter the differential ability of ACMV Rep to trigger the host-mediated HR defence mechanism and to initiate viral DNA replication. The implications of this finding are discussed in the context of plant-virus interactions.
Proteomics, 2013
Tomato, one of the most important crops cultivated worldwide, has been severely affected by begomoviruses such as the Tomato chlorotic mottle virus (ToCMoV). Virulence factor AC2 is considered crucial for a successful virus-plant interaction and is known to act as a transcriptional activator and in some begomoviruses to function as an RNA silencing suppressor factor. However, the exact functions of the AC2 protein of the begomovirus ToCMoV are not yet established. The aim of the present study was to identify differentially expressed proteins of the model plant Nicotiana benthamiana in response to the expression of the AC2 gene, isolated from ToCMoV. N. benthamiana plants were inoculated with Agrobacterium tumefaciens containing the viral vector Potato virus X (PVX) and with the PVX-AC2 construction. 2DE was performed and proteins were identified by MS. The results showed that the expression of ToCMoV AC2 alters the levels of several host proteins, which are important for normal plan...
2016
Heterotrimeric G-proteins, consisting of Gα, Gβ and Gγ subunits, are important signal transducers in eukaryotes. In plants, G-protein-mediated signaling contributes to defense against a range of fungal and bacterial pathogens. Here we studied response of G-protein-deficient mutants to ssRNA viruses representing two different families: Cucumber mosaic virus (CMV) (Bromoviridae) and Turnip mosaic virus (TuMV) (Potyviridae). We found that development of spreading necrosis on infected plants was suppressed in the Gβ-deficient mutant (agb1-2) compared to wild type and Gα- deficient mutant (gpa1-4). In accordance, ion leakage caused by viral infection was also significantly reduced in agb1-2 compared to wild type and gpa1-4. Nevertheless, both viruses replicated better in agb1-2 plants, while gpa1-4 was similar to wild type. Analysis of pathogenesis related genes showed that Gβ negatively regulated salicylic acid, jasmonic acid and abscisic acid marker genes during CMV and TuMV infections. Interestingly, analysis of salicylic acid deficient transgenic plants indicated that salicylic acid did not affect resistance against these viruses and did not influence the Gβ-mediated defense response. We conclude that heterotrimeric G-proteins play a positive role in defense against viral pathogens probably by promoting cell death.
The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection
Molecular Plant-Microbe Interactions®, 2019
Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane bindi...
Geminiviral Triggers and Suppressors of Plant Antiviral Immunity
Microorganisms
Geminiviruses are circular single-stranded DNA plant viruses encapsidated into geminate virion particles, which infect many crops and vegetables and, hence, represent significant agricultural constraints worldwide. To maintain their broad-range host spectrum and establish productive infection, the geminiviruses must circumvent a potent plant antiviral immune system, which consists of a multilayered perception system represented by RNA interference sensors and effectors, pattern recognition receptors (PRR), and resistance (R) proteins. This recognition system leads to the activation of conserved defense responses that protect plants against different co-existing viral and nonviral pathogens in nature. Furthermore, a specific antiviral cell surface receptor signaling is activated at the onset of geminivirus infection to suppress global translation. This review highlighted these layers of virus perception and host defenses and the mechanisms developed by geminiviruses to overcome the p...
Journal of Virology, 2003
Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replicationassociated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R 2 , R 5 , R 7 and K 11 or H 56 , L 57 and H 58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.
Virology, 1997
The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced into Nicotiana benthamiana using Agrobacterium tumefaciens. The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection.
Interaction of the Carlavirus Cysteine-Rich Protein with the Plant Defense System
Molecular Biology, 2005
The genome of viruses of the genus Carlavirus codes for a small cysteine-rich protein (CRP) with unknown functions. To study the role of CRP of the chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome containing the CVB CRP gene was constructed. Expression of CVB CRP in the PVX genetic surrounding drastically changed the character of symptoms produced by PVX in Nicotiana benthamiana. The recombinant virus caused local necrotic lesions of inoculated leaves and necrosis of apical leaves rather than asymptomatic infection and mild mosaic, which are caused by PVX in this host plant. In N. tabacum , the infection pattern depended on the plant host genotype: recombinant PVX spread systemically only in plants carrying the N gene. Agroinfiltration-mediated transient expression assays showed that CRP neither acts as an avirulence factor in N. benthamiana nor suppresses posttranscriptional gene silencing. CVB CRP was identified as a virus pathogenicity determinant that controls the interaction of the virus with the host plant in a way that depends on plant defense, which is mediated by resistance genes such as the N gene.