Research: Virus-Host Interaction
Our research efforts are on two major areas: cassava mosaic geminivirus (CMG) research and plant-based vaccine production. In CMG research, we focus on the role of AC4 in virus pathogenesis and engineering of cassava for resistance to CMGs; this work is supported by the National Science Foundation. In Plant-based vaccine production, we investigate the use of subcellular signal peptides derived from geminivirus proteins in optimal vaccine production.
The AC4 Project
EACMCV encoded AC4 is a membrane protein
The EACMCV AC4 protein is a membrane protein and binds especially to the plasma membrane due to its encoded N-terminal myristoylation signal peptide. This is evident from laser confocal scanning microscope still images of AC4–GFP fusion. High magnification fusions of these images show bright punctate patterns with exclusion from microtubules. Myristoylated proteins undergo cotranslational modification with the attachment of a 14-carbon saturated fatty acid, myristic acid to Gly-2, a mechanism catalyzed by N-myristoyltransferase (NMT). Many of the myristoylated proteins play key roles in regulating cellular structure and function. We have characterized the AC4 N-myristoylation motif. First, we showed that because the requirement for Gly-2 in myristoylation motif is absolute, we substituted it with Ala and fused the mutant (AC4 G2A) to GFP. Confocal images showed that the fusion, AC4G2A–GFP relocated to the chloroplast. Chloroplast targeting by AC4G2A–GFP is supported by structural similarities between its N-terminal sequence and other chloroplast-targeting signal peptides.
Furthermore, PM binding requires energy from a posttranslational and reversible attachment of a second acylate, the 16-carbon palmitate, to Cys-3, thus when we replaced Cys-3 with Ala to obtain the AC4C3A and fused the mutant to GFP, the fusion (AC4C3A–GFP) localized to internal membranes, especially the endoplasmic reticulum. This is because of the lack of sufficient energy to target and bind to the PM.
AC4 suppresses virus induced gene silencing (VIGS)
Posttranscriptional gene silencing (PTGS), also known as RNA silencing, is a host defense mechanism whereby mRNA is degraded in a sequence specific manner in plants. This is used by plants as a defense against viruses and transposons. A similar mechanism, termed RNA interference (RNAi) occurs in many other organisms including Caenorhabditis elegans, Drosophila melanogaster and mammals. PTGS or RNAi is triggered by dsRNA, which is not normally found in eukaryotic cells, leading to degradation of homologous RNAs after transcription. The dsRNA is cleaved into short dsRNA fragments, by a ds-specific ribonuclease called Dicer, which occurs in plants as a family of four enzymes with different functions. In all cells where PTGS is active, are found these small sequence-specific sense and antisense RNAs of 21-25 nucleotides known as short interfering RNAs (siRNAs). siRNAs are incorporated into the RNA-induced silencing complex (RISC). RISC is a complex of proteins that are involved in the PTGS mechanism. The siRNA in the RISC guides the complex and strand-specifically pairs with a complementary mRNA molecule and induces an endonucleolytic cleavage of mRNAs (by the enzyme argonaute) at a site determined by complementary siRNAs. This cleavage leads to rapid degradation of the message, resulting in a corresponding reduction in the level of the encoded protein. Other enzymes in the complex are involved in the epigenetic modification of histone and DNA methylation thus limiting gene transcription. Virus-induced gene silencing (VIGS) is a type of RNA silencing that is initiated by a virus. When plant transgenes are targeted by VIGS, sequence-specific methylation of the viral DNA occurs resulting in low transcript production. The recovery exhibited by plants infected by ACMV is due, at least partially, to VIGS. To be able to by-pass this host defense mechanism, many viruses encode at least one protein that blocks gene silencing at a point on the pathway. These proteins are known as suppressors of RNA silencing. At least three suppressors of RNA silencing have been identified in CMGs including TrAP, AC4, and AV2. AC4 is very efficient at suppressing RNA silencing in the systemic phase. We have shown that for AC4 to suppress RNA silencing, it must be localized on a cell membrane, thus the involvement of the N-myristoylation motif in silencing suppression.
Ongoing AC4 research activities
Because of the increasing evidence of a role for AC4 in geminivirus pathogenicity, we are investigating host factors that interact with it. Since AC4 is a membrane protein, use of the conventional nucleus based yeast two-hybrid assay to screen for AC4-interacting proteins will likely not identify important partners to AC4. Therefore, we have used the split-ubiquitin yeast two-hybrid assay—a membrane protein based procedure—to screen an Arabidopsis cDNA library. In this assay, ubiquitin, a conserved protein of 76 amino acids, attachs to the N terminus of proteins as a signal for their degradation. The ubiquitin moiety is recognized by ubiquitin-specific proteases (UBP), resulting in the cleavage of the attached protein. Ubiquitin can be divided and expressed as N-terminal (Nub) and C-terminal moieties of ubiquitin (Cub), to which a reporter protein is attached. The two moities—Nub and Cub-reporter—assemble in the cell and form split-ubiquitin and is recognized by UBP, resulting in the cleavage and liberation of the reporter protein attached to Cub. The 3rd amino acid in ubiquitin is isoleucine and replacement of Ile-3 with glycine (NubG) blocks the affinity between Nub and Cub. This affinity can be restored by attaching interacting proteins to NubG and Cub-reporter, respectively (Fig. 1). In our system, the bait is fused to the N-terminus of Cub and the Herpes simplex VP16 transactivator is fused to the C-terminus. The bait vector carries the LEU2 gene for auxotrophic selection. A transmembrane prey protein is fused to the NubG moiety and both Cub and NubG are located on the cytoplasmic face of the membrane. The prey vector carries the TRP1 gene for auxotrophic selection.
Using this system, we have isolated several putative AC4-interacting proteins from an Arabidopsis cDNA library. Homologues of these proteins will then be identified and used in investigating their role in cassava resistance to CMGs. We have identified a previously unknown AC4 interacting protein in our screen and confirmed with fluorescence resonance energy transfer (FRET) procedure (Fig. 2).
Production of geminivirus resistant Cassava
Cassava is a major staple crop in the world especially in tropical Africa, Latin America, and the Indian subcontinent. According to Food and Agriculture Organization (FAO) estimates, over 420 million people relied on it for 50% of their calorie requirements. Despite these obvious advantages, cassava research has only recently attracted attention, due especially to the cassava mosaic disease epidemic that occurred in eastern Africa, especially Uganda in the early 1990s. This epidemic has continued to spread westward and now occurs in western Africa. Because these geminivirus epidemics characteristically involves more than one virus, our approach is to produce transgenic cassava with the potentially to simultaneously resist a broad range of these viruses. We are producing embryogenic cultures from virus-free cassava apical meristems and immature leaf lobes and gene transfer is through Agrobacterium.
