A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts

Introduction: Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. Objectives: To demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. Methods: To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. Results: We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Conclusions: Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.German Research Foundation, DFGPeer Reviewe

Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns. MatK is essential for plant cell survival in tobacco, and thus null mutants have not yet been isolated. We therefore attempted to over-express MatK from a neutral site in the chloroplast, placing it under the control of a theophylline-inducible riboswitch. This ectopic insertion of MatK lead to a variegated cotyledons phenotype. The addition of the inducer theophylline exacerbated the phenotype in a concentration-dependent manner. The extent of variegation was further modulated by light, sucrose and spectinomycin, suggesting that the function of MatK is intertwined with photosynthesis and plastid translation. Inhibiting translation in the transplastomic lines has a profound effect on the accumulation of several chloroplast mRNAs, including the accumulation of an RNA antisense to rpl33, a gene coding for an essential chloroplast ribosomal protein. Our study further supports the idea that MatK expression needs to be tightly regulated to prevent detrimental effects and establishes another link between leaf variegation and chloroplast translation

Stable Membrane-Association of mRNAs in Etiolated, Greening and Mature Plastids

Chloroplast genes are transcribed as polycistronic precursor RNAs that give rise to a multitude of processing products down to monocistronic forms. Translation of these mRNAs is realized by bacterial type 70S ribosomes. A larger fraction of these ribosomes is attached to chloroplast membranes. This study analyzed transcriptome-wide distribution of plastid mRNAs between soluble and membrane fractions of purified plastids using microarray analyses and validating RNA gel blot hybridizations. To determine the impact of light on mRNA localization, we used etioplasts, greening plastids and mature chloroplasts from Zea mays as a source for membrane and soluble extracts. The results show that the three plastid types display an almost identical distribution of RNAs between the two organellar fractions, which is confirmed by quantitative RNA gel blot analyses. Furthermore, they reveal that different RNAs processed from polycistronic precursors show transcript-autonomous distribution between stroma and membrane fractions. Disruption of ribosomes leads to release of mRNAs from membranes, demonstrating that attachment is likely a direct consequence of translation. We conclude that plastid mRNA distribution is a stable feature of different plastid types, setting up rapid chloroplast translation in any plastid type