Altered expression of Arabidopsis genes in response to a multifunctional geminivirus pathogenicity protein.

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.

Geminivirus AL2 protein induces expression of, and interacts with, a calmodulin-like gene, an endogenous regulator of gene silencing.

RNA silencing is an innate cellular response involved in antiviral defense. Arabidopsis calmodulin-like protein 39 (At-rgsCaM) is related to known regulators of RNA silencing in tomato and Nicotiana tabacum. Geminivirus AL2 protein functions to suppress post-transcriptional and transcriptional gene silencing, possibly through induction of an endogenous regulator. In support of this, the At-rgsCaM promoter responds to Tomato golden mosaic virus (TGMV) AL2 in protoplasts and geminivirus infection increases rgsCaM expression in Arabidopsis and Nicotiana benthamiana. Further, over-expression of rgsCaM leads to increased susceptibility to infection, as a consequence of increased viral DNA loads. It has been shown that rgsCaM may target silencing suppressors of RNA viruses for degradation via the autophagy pathway. This interaction occurs within the cytoplasm, but AL2 interacts with rgsCaM in the nucleus. It is tempting to speculate that AL2 may act to sequester rgsCaM in the nucleus to prevent targeting of AL2 for degradation.

The interaction between geminivirus pathogenicity proteins and adenosine kinase leads to increased expression of primary cytokinin-responsive genes.

Pathogenicity proteins (AL2/C2) of begomo- and curtoviruses suppress silencing through inhibition of the methyl cycle, as a consequence of inhibiting adenosine kinase (ADK). ADK phosphorylates cytokinin nucleosides, helping maintain a pool of bioactive cytokinins through interconversion of free-bases, nucleosides and nucleotides. We provide evidence that inhibiting ADK affects expression of primary cytokinin-responsive genes. Specifically, we demonstrate increased activity of a primary cytokinin-responsive promoter in adk mutant Arabidopsis plants, and in response to silencing ADK expression or inhibiting ADK activity in transient assays. Similar changes in expression are observed in geminivirus infected tissue and when AL2/C2 are over-expressed. Increased cytokinin-responsive promoter activity may therefore be a consequence of an ADK/AL2/C2 interaction. Application of exogenous cytokinin increases susceptibility to geminivirus infection, characterized by a reduced mean latent period and enhanced viral replication. Thus, ADK appears to be a high value target of geminiviruses that includes increasing expression of primary cytokinin-responsive genes.

The Arabidopsis PEAPOD2 transcription factor interacts with geminivirus AL2 protein and the coat protein promoter.

The TrAP protein of bipartite begomoviruses activates the coat protein (CP) promoter in mesophyll and derepresses the promoter in vascular tissue through two sequences, located 60-125 bp and 1.2-1.5 kbp respectively, upstream of the CP gene. TrAP does not, however, exhibit specific binding to either sequence directly. We have identified a plant-specific DNA-binding protein, Arabidopsis PEAPOD2 (PPD2), that specifically binds sequences mediating activation of the CP promoter of Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus in mesophyll. This protein does not however, bind sequences required for TrAP-mediated derepression in phloem. TGMV TrAP interacts with the PPD2/CP promoter complex in electrophoretic mobility shift assays. PPD2 is associated with the nucleus, as expected for a transcription factor, but is not capable of activating transcription directly. Thus, geminivirus TrAP is likely targeted to the CP promoter through interaction with PPD2, leading to activation of CP gene expression.

Functional analysis of bipartite begomovirus coat protein promoter sequences.

We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters.