The HIV-1 Pr55gag polyprotein binds to plastidial membranes and leads to severe impairment of chloroplast biogenesis and seedling lethality in transplastomic tobacco plants

Abstract

Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass

“…In particular, they showed chloroplasts with irregular shape and a severely defective ultrastructure characterized by rudimentary and unstacked internal membranes dispersed in an electron-dense matrix, resembling to proplastids. Similar results were also observed in our laboratory for tobacco transplastomic plants expressing HIV-1/Pr55 gag [ 52 ].…”

Chloroplast proteome response to drought stress and recovery in tomato (Solanum lycopersicum L.)

“…Pigments content was spectrophotometrically determined at 665, 649 and 470 nm using a Perkin Elmer Lambda25 UV/VIS spectrometer. Calculations were based on formulas described elsewhere [ 32 ]. Three biological and three technical replicates were used to evaluate each experimental condition.…”

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

“…So far, only the expression of a dengue virus serotype 3 premembrane and envelope polyprotein has been reported in plastids (Kanagaraj et al 2011). We demonstrate that dengue virus envelop protein domain III-based antigens can be expressed in tobacco chloroplasts and that the expression of challenging proteins via the ethanol-inducible expression system offers a possibility to avoid deleterious phenotypes associated with overexpression (Hennig et al 2007;Petersen and Bock 2011;Scotti et al 2015). The transplastomic plant lines with the transgene expression cassette controlled by the strong constitutive ribosomal RNA operon promoter showed distinct growth retardations and mild pigment deficiency.…”

“…The transplastomic plant lines with the transgene expression cassette controlled by the strong constitutive ribosomal RNA operon promoter showed distinct growth retardations and mild pigment deficiency. In many cases, plastid expression of recombinant proteins does not result in abnormal phenotypes, but an increasing number of studies has reported phenotypic alterations in transplastomic plants (Lössl et al 2003;Magee et al 2004;Scotti et al 2015;Tissot et al 2008;Tregoning et al 2003;Waheed et al 2011). The two main reasons underlying pigment deficiency or a delay in plant development are toxicity of the transgene product due to interference of the recombinant protein with essential processes in the chloroplast (e.g.…”

“…However, high-level accumulation of recombinant proteins in chloroplasts can also have a negative impact on plant growth (Hennig et al 2007;Petersen and Bock 2011;Scotti et al 2015). Although, most foreign proteins are non-toxic to chloroplasts, in some cases, abnormal phenotypes like chlorosis of the leaves, male sterility and growth retardation have been reported (Lössl et al 2003;Tregoning et al 2003;Waheed et al 2011).…”