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Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco

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Abstract

The conserved late element (CLE) was originally identified as an evolutionarily conserved DNA sequence present in geminiviral intergenic regions. CLE has subsequently been observed in promoter sequences of bacterial (T-DNA) and plant origin, suggesting a role in plant and plant viral gene regulation. Synthetic DNA cassettes harboring direct repeats of the CLE motif were placed upstream from a −46 to +1 minimal CaMV 35S promoter-luciferase reporter gene and reporter activity characterized in Nicotiana species during both transient and stable expression. A single direct-repeat cassette of the element (2× CLE) enhances luciferase activity by 2-fold, independent of the element’s orientation, while multiple copies of the cassette (4–12× CLE) increases activity up to 10- to 15-fold in an additive manner. Transgenic tobacco lines containing synthetic CLE promoter constructs enhance luciferase expression in leaf, cotyledon and stem tissues, but to a lesser extent in roots. Single nucleotide substitution at six of eight positions within the CLE consensus (GTGGTCCC) eliminates CLE enhancer-like activity. It has been previously reported that CLE interacts with the AC2 protein from Pepper Huasteco Virus (PHV-AC2). PHV-AC2 (also called AL2 or C2) is a member of the transcriptional activator protein, or TrAP, gene family. In transient and stable expression systems PHV-AC2 expression was found to result in a 2-fold increase in luciferase activity, irrespective of the presence of CLE consensus sequences within the reporter’s promoter. These data suggests that the PHV-AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of post-transcriptional gene silencing.

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Abbreviations

AC2:

geminivirus encoded TrAP protein

Agst:

agropine synthase terminator

AYVV :

Ageratum yellow veinvirus

bar :

phosphinothricin acetyl transferase

BGMV:

Bean golden mosaic virus

CaMV:

Cauliflower mosaic virus

CaMV35S:

Cauliflower mosaic virus 35S promoter

CLCuV:

Cotton leaf curl virus

DTT:

dithiothreitol

HcPro:

viral helper component Protein (suppressor of silencing)

GUSi:

intron-modified β-glucuronidase

IM:

infiltration media

Km:

kanamycin

LB:

Luria--Bertani

FiLUC:

firefly (Photinus pyralis) intron-modified luciferase

MES:

2-(N-Morpholino)ethanesulfonic acid

MiMV:

Mirabilis mosaic virus

MS:

Murashige and Skoog

Nost:

nopaline synthase terminator

Npt II :

neomycin phosphotransferase II

pAg7:

gene 7 terminator

pBS:

pBluescript II (SK+)

PClSV:

peanut chlorotic streak virus (promoter)

PGMV:

Pepper golden mosaic virus (formally Serrano golden mosaic virus)

PHV:

Pepper Huasteco virus

Pnos:

nopaline synthase promoter

PTGS:

post-transcriptional gene silencing

PVY:

Potato virus Y

R0:

primary transformant

R1:

progeny of self fertilized R0 plant

RiLUC:

Renilla reniformis (sea pansy) intron-modified luciferase

RLU:

relative light units

TBE:

Tris--Borate EDTA

T-DNA:

transferred-DNA

TGMV:

Tomato golden mosaic virus

ToLCV:

Tomato leaf curl virus

TYLCV:

Tomato yellow leaf curl virus

TrAP:

transcription activating protein

35St:

CaMV 35S terminator

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  1. USDA-ARS (United States Department of Agriculture-Agricultural Research Services), 3810 4th Street, Lubbock, TX, 79415, USA

    Christopher Ian Cazzonelli, John Burke & Jeff Velten

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  1. Christopher Ian Cazzonelli
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  2. John Burke
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Correspondence to Jeff Velten.

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Cazzonelli, C.I., Burke, J. & Velten, J. Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco. Plant Mol Biol 58, 465–481 (2005). https://doi.org/10.1007/s11103-005-6589-x

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