Members of the Geminiviridae replicate small circular ssDNA genomes in the nucleus through dsDNA intermediates that associate with cellular histones to form minichromosomes. These relatively simple viruses do not encode polymerases, and must recruit host machinery to express viral mRNAs and replicate viral DNA. Thus, geminiviruses are valuable model systems for studying mechanisms and regulation of these processes. In addition, because viral chromatin is formed de novo in infected cells, geminiviruses are also unique models to study pathways involved in the establishment of epigenetic marks, including cytosine methylation and histone post-translational modifications (PTMs).
Our work employs members of the Begomovirus genus, including Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus (CaLCuV), and the type member of the genus Curtovirus, Beet curly top virus (BCTV). We are particularly interested in the AL2 protein (also called AC2) encoded by begomoviruses, and the related L2 protein (also called C2) found in curtoviruses. Both AL2 and L2 are pathogenicity factors, and AL2 in addition is a transcription factor that activates the expression of viral genes needed late in infection, including capsid protein.
Viral chromatin methylation as an epigenetic defense against geminiviruses
We use geminiviruses as models to study small RNA-directed DNA and histone methylation in the model plant Arabidopsis thaliana. Chromatin methylation is a fundamental epigenetic process that is usually repressive and results in chromatin compaction and transcriptional gene silencing (TGS). Repressive methylation is important for proper regulation of gene expression during development, and also is critical for the control of endogenous invasive DNAs such as transposons. It also leads to the establishment of heterochromatin.
Our work has shown that plants use repressive methylation to limit geminivirus replication and transcription, and that geminivirus AL2 and L2 proteins counter this defense by inhibiting methylation-mediated TGS. One suppression mechanism relies on the ability of AL2 and L2 to bind and inactivate adenosine kinase (ADK), an enzyme that phosphorylates adenosine to produce AMP. In addition to its role in adenosine salvage, ADK is also required for efficient production of the essential methyltransferase co-factor and methyl group donor, S-adenosyl methionine.
Current projects are focused on host EML proteins, which are "chromatin readers" that bind specific histone PTMs on viral chromatin. We are also characterizing non-coding RNAs transcribed from geminivirus DNA by RNA polymerases IV, and V, which play important roles in establishing repressive DNA and histone methylation. We are also using the multifunctional AL2 and L2 proteins to probe methylation pathways.
Current projects are investigating the possibility that SnRK1:ADK complexes are part of a signaling cascade that is activated by pathogen attack. In addition, we are studying a novel mechanism that may allow SnRK1 to interfere with translation of viral proteins.
Jackel, J.N., Storer, J.M., Coursey, T., and Bisaro, D.M. (2016) Arabidopsis RNA polymerases IV and V are required to establish H3K9 methylation, but not cytosine methylation, on geminivirus chromatin. Journal of Virology 90: 7529-7540.
Baltes, N.J., Hummel, A.W., Konecna, E., Cegan, R., Bruns, A.N., Bisaro, D.M., and Voytas, D.F. (2015) Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system. Nature Plants 1:15145, DOI: 10.1038/NPlants.2015.145
Jackel, J.N., Buchmann, R.C., Singhal, U., and Bisaro, D.M. (2015) Analysis of geminivirus AL2 and L2 proteins reveals a novel AL2 silencing suppressor activity. Journal of Virology 89: 3176-3187.
Raja, P., Jackel, J.N., Li, S., Heard, I.M., and Bisaro, D.M. (2014) Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses. Journal of Virology 88: 2611-2622.
Mohannath, G., Jackel, J.N., Lee, Y.H., Buchmann, R.C., Wang, H., Patil, V., Adams, A.K., and Bisaro, D.M. (2014) A complex containing SNF1-related kinase (SnRK1) and adenosine kinase in Arabidopsis. PLoS One 9: e87592.
Canizares, M.C., Lozano-Duran, R., Canto, T., Bejarano, E.R., Bisaro, D.M., Navas-Castillo, J., and Moriones, E. (2013) Effects of crinivirus coat protein-interacting plant protein SAHH on post-transcriptional RNA silencing and its suppression. Molecular Plant-Microbe Interactions 26: 1004-1015.
Zhu, Y., Cherukuri, N.C., Jackel, J.N., Wu, Z., Crary, M., Buckley, K., Bisaro, D.M., and Parris, D.S. (2012) Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells. Journal of Virology 86: 3038-3049.
Yang, X., Xie, Y., Raja, P., Li, S., Wolf, J.N., Shen, Q., Bisaro, D.M., and Zhou, X. (2011) Suppression of methylation-mediated transcriptional gene silencing by βC1 protein-SAHH interaction during geminivirus-betasatellite infection. PLoS Pathogens 7:e1002329.
Raja, P., Wolf, J.N., and Bisaro, D.M. (2010) RNA silencing directed against geminiviruses: Post-transcriptional and epigenetic components. Biochimica and Biophysica Acta (Gene Regulatory Mechanisms) 1799: 337-351.
Buchmann, R.C., Asad, S., Wolf, J.N., Mohannath, G., and Bisaro, D.M. (2009) Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation. Journal of Virology 83: 5005-5013.
Golenberg, E.M., Sather, D.N., Hancock, L.C., Buckley, K.J., Villafranco, N., and Bisaro, D.M. (2009) Development of a gene silencing DNA vector derived from a broad host range geminivirus. BMC Plant Methods: 5:9 doi:10.1186/1746-4811-5-9.
Raja, P., Sanville, B.C., Buchmann, R.C., and Bisaro, D.M. (2008) Viral genome methylation as an epigenetic defense against geminiviruses. Journal of Virology 82: 8997-9007.
Yang, X., Baliji, S. Woody, S., Buchmann, R.C., Wang, H., Lindbo, J., Sunter, G., and Bisaro, D.M. (2007) Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction. Journal of Virology 81: 11972-11981.
Wang, H., Buckley, K., Yang, X.Y., Buchmann, C., and Bisaro, D.M. (2005) Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins. Journal of Virology 79: 7410-7418.
Wang, H., Hao. L., Sunter, G., and Bisaro, D.M. (2003) Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins. Plant Cell 15: 3020-3032.
Hao, L., Wang, H., Sunter, G., and Bisaro, D.M. (2003) Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell 15: 1034-1048.