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Background: Geminivirus AC2 is a multifunctional protein that acts as a pathogenicity factor. Transcriptional regulation by AC2 appears to be mediated through interaction with a plant specific DNA binding protein, PEAPOD2 (PPD2), that specifically binds to sequences known to mediate activation of the CP promoter of Cabbage leaf curl virus (CaLCuV) and Tomato golden mosaic virus (TGMV). Suppression of both basal and innate immune responses by AC2 in plants is mediated through inactivation of SnRK1.2, an Arabidopsis SNF1 related protein kinase, and adenosine kinase (ADK). An indirect promoter targeting strategy, via AC2-host dsDNA binding protein interactions, and inactivation of SnRK1.2-mediated defense responses could provide the opportunity for geminiviruses to alter host gene expression and in turn, reprogram the host to support virus infection. The goal of this study was to identify changes in the transcriptome of Arabidopsis induced by the transcription activation function of AC2 and the inactivation of SnRK1.2., Results: Using full-length and truncated AC2 proteins, microarray analyses identified 834 genes differentially expressed in response to the transcriptional regulatory function of the AC2 protein at one and two days post treatment. We also identified 499 genes differentially expressed in response to inactivation of SnRK1.2 by the AC2 protein at one and two days post treatment. Network analysis of these two sets of differentially regulated genes identified several networks consisting of between four and eight highly connected genes. Quantitative real-time PCR analysis validated the microarray expression results for 10 out of 11 genes tested., Conclusions: It is becoming increasingly apparent that geminiviruses manipulate the host in several ways to facilitate an environment conducive to infection, predominantly through the use of multifunctional proteins. Our approach of identifying networks of highly connected genes that are potentially co-regulated by geminiviruses during infection will allow us to identify novel pathways of co-regulated genes that are stimulated in response to pathogen infection in general, and virus infection in particular.

The begomovirus AL2 protein is a transcriptional activator, a silencing suppressor, and inhibitor of basal defense. AL2 forms a complex at the CP promoter, through interaction with a plant-specific DNA-binding protein, Arabidopsis PEAPOD2 (also known as TIFY4B). AtTIFY4B has three domains (PPD, TIFY and CCT_2) conserved between homologs from different plant species. We confirmed that the AL2 protein from Tomato golden mosaic virus and Cabbage leaf curl virus interacts with TIFY4B from Arabidopsis, tomato and Nicotiana benthamiana in the nucleus of plant cells. Bimolecular Fluorescence Complementation demonstrated that the interaction involves both the TIFY and CCT_2 domains. Surprisingly, amino acids 84-150 can prevent AtTIFY4B from localizing to the nucleus, and interaction with AL2 results in some of the protein re-entering the nucleus. When AtTIFY4B is over-expressed, we observe an increase in mean latent period, where systemic symptoms are detected on average, 4 days later than in mock treated plants. This appears to be a consequence of reduced viral DNA titers, possibly related to the role of TIFY4B in cell cycle arrest. Our results point to a potential role for TIFY4B in host defense against geminiviruses. Expression of TIFY4B in N. benthamiana increases in response to geminivirus infection, which would result in suppression of proliferation, reducing viral replication. Geminiviruses may counter this defense response through an interaction between AL2 and TIFY4B, which would inhibit TIY4B function. The consequence of this inhibition would be failure to arrest the cell cycle, providing an environment conducive for geminivirus replication.

We generated transgenic lines of Arabidopsis thaliana with an RNA interference construct that expressed hairpin double-stranded RNA for DET2:DWF4:SMT2 to induce sequence-specific RNA silencing. In transgenic plants, expressions of DET2, DWF4, and SMT2 were simultaneously reduced, and the campesterol content was increased by up to 420% compared to the level in the wild-type plant. Triple knock-down of the DET2, DWF4, and SMT2 enzymes also resulted in reduction of brassinosteroid (BR)-specific biosynthesis intermediates. Transgenic plants harboring the RNA interference construct displayed a semi-dwarf phenotype due to altered development. Our findings indicate that redesigning of plant architecture is possible through simultaneous suppression of multiple genes involved in BR biosynthesis.