Satish C. Dogra

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.

Molecular Regulation of Heme Biosynthesis in Higher Vertebrates

Publisher Summary The regulation of heme biosynthesis in animals has been a topic of interest for many years. It is generally agreed that the first enzyme of the heme pathway, 5-aminolevulinate synthase, determines the rate of heme biosynthesis and the regulation of this enzyme is, therefore, a major focus of this chapter. The heme biosynthetic pathway is present in all cell types except mature erythrocytes. The final step of heme biosynthesis occurs in mitochondria and the heme is then utilized for the formation of different hemoproteins located in mitochondria, microsomes, peroxisomes, the cytosol, and probably the nucleus. Heme can control the biosynthesis of some proteins. In erythroid cells, heme controls the translation of proteins, notably α- and β-globin chains, by modulating the activity of a specific kinase. All nucleated animal cells must synthesize heme for incorporation into respiratory cytochromes, but erythroid and liver cells have the highest rates of heme synthesis. Erythroid cells synthesize about 90% of the total heme in the body for assembly into hemoglobin. Although the bulk of heme in the liver is made in situ , the liver may also obtain some heme from serum haptoglobin–hemoglobin and heme–hemopexin complexes, following intravascular hemolysis. All enzymes of the heme biosynthetic pathway, except for protoporphyrinogen oxidase, have been cloned from higher vertebrates. The genes encoding these enzymes are located on different chromosomes.

Transcriptional activation of cytochrome P450 genes by different classes of chemical inducers.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes.

Interaction with a Host Ubiquitin-Conjugating Enzyme Is Required for the Pathogenicity of a Geminiviral DNA β 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.

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.

Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA β 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 duplicated HNF-3 binding site in the CYP2H2 promoter underlies the weak phenobarbital induction response

We are investigating induction of chicken cytochrome P450 genes by the sedative phenobarbital in chick embryo hepatocytes. The steady-state level of induced mRNA for the gene CYP2H1 is about 10-fold higher than that of a second gene, CYP2H2. Here, we show that a difference in drug-responsive enhancer activity does not underlie the differential response of these genes to phenobarbital since upstream enhancer regions are identical in these genes. The first 198 bp of CYP2H2 promoter sequence is identical to the CYP2H1 gene promoter, except that the functional HNF-3 binding site in the CYP2H1 promoter is replaced with a duplicated HNF-3 sequence in the CYP2H2 promoter. Transient expression analysis established that the promoter activity of the CYP2H2 gene was about ninefold lower than the CYP2H1 gene. Mutagenesis of either of the partially overlapping HNF-3 sites in the CYP2H2 gene substantially induced drug induction. Gel-shift analysis established that each of these HNF-3 sites bound HNF-3, most likely HNF-3beta. In-vitro footprint analysis demonstrated that all the identified sites in the CYP2H2 promoter bound protein except the duplicated HNF-3 region. However, protein binding was observed by in-vitro footprint analysis if either of the HNF-3 sites was mutated in the CYP2H2 promoter. Hence, duplication of the HNF-3 site in the CYP2H2 promoter does not allow binding of HNF-3 in the promoter context and may be predominantly, if not exclusively, responsible for the poor response of the CYP2H2 gene to phenobarbital.