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

Liu et al. BMC Plant Biology 2014, 14:302 http://www.biomedcentral.com/1471-2229/14/302 RESEARCH ARTICLE Open Access Altered expression of Arabidopsis genes in response to a multifunctional geminivirus pathogenicity protein Lu Liu1, Ho Yong Chung2, Gabriela Lacatus3, Surendranath Baliji4, Jianhua Ruan1* and Garry Sunter2* Abstract 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. Keywords: Geminiviruses, Microarray, Pathogenesis, Expression, Regulatory networks Background The Geminiviridae family comprises a large and diverse group of viruses that infect a wide range of important monocotyledonous and dicotyledonous crop species and cause significant yield losses [1,2]. Viral pathogenesis depends on a series of interactions between virus, host * Correspondence: ; 1 Department of Computer Science, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX, USA 2 Department of Biology, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX, USA Full list of author information is available at the end of the article and insect vector. As very few viral proteins are encoded by geminiviruses, they rely, in large part, on the replication and transcription machinery of the host. One consequence of this host dependence is that geminiviruses are useful models for providing novel insights into the control of both plant and animal DNA replication and transcription. The circular single-stranded DNA (ssDNA) genome of geminiviruses is amplified in the nuclei of infected cells by rolling circle (RCR) and recombination-dependent (RDR) replication using cellular DNA polymerases [3,4]. The resulting double-stranded DNA replicative forms (RF) are used as template for generation of viral transcripts by host © 2014 Liu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. BMC Plant Biology 2014, 14:302 http://www.biomedcentral.com/1471-2229/14/302 RNA polymerase II. Geminiviruses produce small multifunctional proteins to compensate for a limited coding capacity. For example, begomoviruses including Cabbage leaf curl (CaLCuV) and Tomato golden mosaic (TGMV) virus, code for a pathogenicity protein, AC2 (Figure 1A), that modulates metabolism [5,6], regulates transcription [7,8] and suppresses RNA silencing [9-11]. AC2 (also known as AL2 and TrAP) is required for expression of the coat protein (CP) and BR1 movement protein genes of both CaLCuV and TGMV [12-15]. It has been shown that AC2 is capable of inducing CP expression through two distinct and independent mechanisms. In mesophyll cells AC2 activates the CP promoter, but in vascular tissue AC2 acts to derepress the promoter [7,12]. Distinct sequences mediate activation and derepression by AC2. Sequences required for activation are located within the common region upstream of the CP transcription start site [8,12], whereas sequences required for repression are located 1.2 to 1.5 kbp upstream of CP transcription start site [7,12]. Among begomoviruses, the transcription function of AC2 is not virus specific as both CaLCuV or TGMV AC2 proteins can transactivate the TGMV coat protein (CP) promoter [12,16]. AC2 does not appear to be a canonical transcription factor as it does not bind dsDNA efficiently and appears to be targeted to responsive promoters via proteinprotein interactions with cellular factors. A recent study Figure 1 Diagram of CaLCuV AC2 and SCTV C2 proteins used in over-expression studies. (A) The linear drawing represents functional domains (span of amino acids indicated) present within the full-length CaLCuV AC2 protein. The N-terminal region contains a basic region of four arginine residues and a potential nuclear localization sequence. The C-terminus contains a minimal transcription activation domain within an acidic region. A region containing conserved cysteine and histidine residues forms a putative zinc finger domain, with a high degree of homology with the SCTV C2 protein. (B) Truncated form of the CaLCuV AC2 protein lacking the C-terminal 29 amino acids containing the acidic activation domain. (C) Full-length SCTV C2 protein, which lacks an acidic activation domain, but has homology to the putative zinc finger domain in CaLCuV AC2. Page 2 of 16 has identified a plant specific DNA binding protein, PEAPOD2 (PPD2), that specifically binds to sequences known to mediate activation of the CP promoter of CaLCuV and TGMV in mesophyll cells [ (...truncated)