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JXB Advance Access originally published online on June 6, 2008
Journal of Experimental Botany 2008 59(10):2815-2829; doi:10.1093/jxb/ern143
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

Maddalena de Virgilio1 *, Francesca De Marchis2 *, Michele Bellucci2, Davide Mainieri1, Marika Rossi1, Eugenio Benvenuto3, Sergio Arcioni2 and Alessandro Vitale1,{dagger}

1Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, via Bassini 15, 20133 Milano, Italy, EU
2Istituto di Genetica Vegetale, Consiglio Nazionale delle Ricerche, Articolazione Territoriale di Perugia, via della Madonna Alta 130, 06128 Perugia, Italy, EU
3ENEA-BIOTEC Sezione Genetica e Genomica Vegetale, C.R. Casaccia, 00060 Roma, Italy, EU

{dagger} To whom correspondence should be addressed. E-mail: vitale{at}ibba.cnr.it

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein {gamma}-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of {gamma}-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same {gamma}-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its {gamma}-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

Key words: Endoplasmic reticulum, protein accumulation, protein bodies, plant factories, zein

* These authors contributed equally to this work.

Received 9 February 2008; Revised 31 March 2008 Accepted 28 April 2008


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