Silencing suppressor activity of a begomovirus DNA ß encoded protein and its effect on heterologous helper virus replication.

DNA ß satellites are circular single-stranded molecules associated with some monopartite begomoviruses in the family Geminiviridae. They co-infect with their helper viruses to induce severe disease in economically important crops. The ßC1 protein encoded by DNA ß is a pathogenicity determinant and has been reported to suppress post-transcriptional gene silencing (PTGS). The ßC1 proteins from various DNA ß molecules show low levels of amino acid sequence conservation. We show here that the ßC1 from DNA ß associated with Cotton leaf curl Multan virus (CLCuMV) is a suppressor of systemic PTGS. When this DNA ß satellite co-inoculated with a heterologous helper virus, Tomato leaf curl virus (ToLCV), reduced the level of ToLCV siRNA and this was associated with a higher level of virus accumulation in infected tobacco plants. This may be a mechanism by which ßC1 protects a heterologous virus from host gene silencing.

A novel shaggy-like kinase interacts with the Tomato leaf curl virus pathogenicity determinant C4 protein.

Tomato leaf curl virus-Australia (ToLCV) C4 protein has been shown to be associated with virus pathogenesis. Here, we demonstrate that C4 acts as a suppressor of gene silencing. To understand the multifunctional role of C4, a novel shaggy-like kinase (SlSK) from tomato, which interacts with ToLCV C4 in a yeast two-hybrid assay, was isolated and interaction between these proteins was confirmed in vitro and in planta. Using deletion analysis of C4, a 12 amino acid region in the C-terminal part of C4 was identified which was shown to be essential for its binding to SlSK. We further demonstrate that this region is not only important for the interaction of C4 with SlSK, but is also required for C4 function to suppress gene silencing activity and to induce virus symptoms in a PVX system. The potential significance of ToLCV C4 and SlSK interaction is discussed.

Interaction with a host ubiquitin-conjugating enzyme is required for the pathogenicity of a geminiviral DNA beta satellite.

DNA beta is a single-stranded satellite DNA which encodes a single gene, betaC1. To better understand the role of betaC1 in the pathogenicity of DNA beta, a yeast two-hybrid screen of a tomato cDNA library was carried out using betaC1 from Cotton leaf curl Multan virus (CLCuMV) DNA beta as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between betaC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the betaC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-beta-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing betaC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of betaC1 with SlUBC3 is required for DNA-beta-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA beta satellite.

DNA beta is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA beta contains a single gene, betaC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the beta-glucuronidase reporter gene driven by a betaC1 promoter-deletion series of the DNA beta associated with cotton leaf curl Multan virus identified a 68 nt region (between -139 and -207) which is important for betaC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the betaC1 start codon. Mutation of the G-box resulted in a significant reduction in betaC1 promoter activity and DNA beta replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA beta.

Identification and characterization of a host reversibly glycosylated peptide that interacts with the Tomato leaf curl virus V1 protein.

Monopartite geminiviruses of the genus Begomovirus have two virion-sense genes, V1 and V2. V2 encodes the viral coat protein, but the function of V1 is largely unknown, although some studies suggest that it may play a role in cell-to-cell movement. Yeast two-hybrid technology was used to identify possible host binding partners of V1 from Tomato leaf curl virus (TLCV) to better understand its function. A protein closely related to a family of plant reversibly glycosylated peptides, designated SlUPTG1, was found to interact with V1 in yeast and in vitro. SlUPTG1 may function endogenously in the synthesis of cell wall polysaccharides, since a bacterially expressed form of the protein acted as an autocatalytic glycosyltransferase in vitro, a SlUPTG1:GFP fusion protein localized to the cell wall, and expression of SlUPTG1 appeared to be highest in actively dividing tissues. However, expression of SlUPTG1 in a transient TLCV replication assay increased the accumulation of viral DNA, suggesting that this host factor also plays a role in viral infection. Together, these data provide new insight into the role of V1 in TLCV infection and reveal another host pathway which geminiviruses may manipulate to achieve an efficient infection.

A NAC domain protein interacts with tomato leaf curl virus replication accessory protein and enhances viral replication.

Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.

Evidence that the coactivator CBP/p300 is important for phenobarbital-induced but not basal expression of the CYP2H1 gene.

We have previously identified an upstream 556-bp enhancer domain for the chicken CYP2H1 gene that responds to phenobarbital and binds several transcription factors, including the orphan chicken xenobiotic receptor (CXR). By contrast, the promoter lacks a CXR site and is not inducible by phenobarbital. Although it has been established that CXR can interact with the coactivator SRC-1, there are no reports as to whether other coactivators may be important for phenobarbital-mediated inducibility. Our studies using the adenovirus E1A wild-type protein, which inhibits the coactivators cAMP response element binding protein (CBP) and CBP associated factor (p/CAF), provide evidence for the involvement of one or both of these coactivators at the enhancer but not at the promoter of the CYP2H1 gene. The observations that mutant E1A proteins did not affect the enhancer activity and that inhibition by wild-type E1A was reversed by CBP and p/CAF confirmed the involvement of these coactivators in the induction process. We propose that the intrinsic histone acetyl transferase activity of one or both of these coactivators participates in chromatin remodeling thereby stimulating drug induction of the promoter. This proposal was supported by experiments with the histone deacetylase inhibitor, trichostatin A, which resulted in the superinduction of the drug response but had little effect on basal expression of the CYP2H1 gene. The work provides evidence for the first time for the involvement of the coactivators CBP and p/CAF in the phenobarbital-mediated induction of the CYP2H1 gene.