Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus Replication-associated protein promoter in transgenic plants

Abstract

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep^459::Rep) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep^459::Rep showed an expression activity 10–40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep^459::Rep (pRep⁴⁵⁹, pRep³²⁴, pRep²⁰³, pRep¹⁴⁵, pRep¹³² and pRep¹¹⁵), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep¹¹⁵ version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.

Introduction

The Geminiviridae is a plant virus family whose members are classified into four genera (Mastrevirus, Curtovirus, Topocuvirus and Begomovirus) based on their genome organization, insect vector and host range (van Regenmortel et al., 2000). The genomes of theses viruses consist of one or two single-stranded DNA molecules of approximately 2.7 kb that are replicated via a rolling-circle mechanism in the nuclei of the infected cells (Saunders et al., 1991, Stenger et al., 1991). The DNA-A component of the bipartite begomoviruses codes for proteins required for replication (Replication-associated protein, Rep and Replication enhancement protein, REn), transcription (transactivator, TrAP), and the capsid protein (CP). The DNA-B component codes the nuclear shuttle and movement proteins (NSP and MP), which are involved in the translocation of the viral DNA from the nucleus of an infected cell to adjacent cells. Intermediary double-stranded DNA molecules are the virus replicative forms that are transcriptionally active. Transcription in geminiviruses is complex and temporally regulated. Geminivirus genomes are transcribed in a bidirectional manner from the intergenic region leading to the synthesis of mRNAs that correspond to both virion and complementary sense genes (Hanley-Bowdoin et al., 2000). Replication and transcription-involved genes are expressed earlier than cp and nsp genes. Rep protein down-regulates its own expression through interaction with the positive-strand DNA replication origin, which overlaps the rep promoter (Eagle et al., 1994, Eagle and Hanley-Bowdoin, 1997, Lazarowitz et al., 1992). Expression of the late genes, cp and nsp, require the presence of TrAP (Sunter and Bisaro, 1991). A minimal sequence necessary for Tomato golden mosaic virus (TGMV) cp promoter activation by TrAP have been recently identified (Sunter and Bisaro, 2003). Another cis-acting sequence possibly involved in TrAP recruitment has been previously described for Pepper huasteco virus (PHV) (Ruiz-Medrano et al., 1999).

Activity of a number of geminivirus promoters have been examined in transgenic plants (Dry et al., 2000, Haley et al., 1992, Mazithulela et al., 2000). Some of them are able to drive a near constitutive expression in all cell types, whereas others are functional in few plant tissues. For example, coat protein promoter from the bipartite begomovirus TGMV is active in unorganized calli, and in both phloem and mesophyl cells of the plant, but in these latest cases its activation requires the presence of TrAP. Besides, certain truncated version of this promoter is active in phloem cells in the absence of the viral transactivator, which suggested the presence of a cis-acting repressor elements capable of masking the TrAP-independent activity of this promoter (Sunter and Bisaro, 1997); promoters from the monopartite begomovirus Tomato leaf curl virus (ToLCV) display an expression pattern in transgenic tobacco plants that ranges from constitutive (e.g. Rep promoter) to vascular-specific (e.g. TrAP and REn promoters) (Dry et al., 2000). In the case of the mastrevirus Maize streak virus, the CP transcription unit exhibits an expression pattern restricted to the vascular tissue of transgenic rice plants (Mazithulela et al., 2000). Some conserved motif such as G box and conserved late elements, have been identified in promoters from several geminiviruses (Arguello-Astorga et al., 1994). However, a connection between the presence or arrangement of these elements and the expression pattern conferred by a given promoter has not been found. Therefore, tissue-specific expression provided by geminivirus promoters seems to be controlled through multiple and various unknown factors and new studies need to be carried out to know the expression pattern directed by a particular geminivirus promoter.

In order to achieve a better understanding of the expression of the Tomato mottle Taino virus (ToMoTV) rep gene, in this study we have assessed the activity of its potential promoter sequence (pRep) in transgenic solanaceous species. ToMoTV is a typical bipartite geminivirus that affects tomato and potato crops in Cuba (Cordero et al., 2003, Ramos et al., 1997, Ramos et al., 2003). This work also aims to provide further details about geminivirus gene expression during plant tissue differentiation. In addition, it was demonstrated that a 132 nt pRep version contains the minimal sequence necessary for promoter activity and that it also harbors those elements sufficient to provide an expression pattern restricted to the vascular tissue.