Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein derived from plants—is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.

Plant aspartic proteases (APs; E.C.3.4.23) are a group of proteolytic enzymes widely distributed among different species characterized by the conserved sequence Asp-Gly-Thr at the active site. With a broad spectrum of biological roles, plant APs are suggested to undergo functional specialization and to be crucial in developmental processes, such as in both biotic and abiotic stress responses. Over the last decade, an increasing number of publications highlighted the APs’ involvement in plant defense responses against a diversity of stresses. In contrast, few studies regarding pathogen-secreted APs and AP inhibitors have been published so far. In this review, we provide a comprehensive picture of aspartic proteases from plant and pathogenic origins, focusing on their relevance and participation in defense and offense strategies in plant–pathogen interactions.

No abstract

Medicago truncatula is an established model for studying legume biology. More recently, it has also been exploited as a Molecular Farming platform for the production of recombinant proteins, with the successful expression of fungal and human proteins in plants and cell suspension cultures of this species. One of the challenges that now must be overcome is the degradation of final products during production and downstream processing stages. In the M. truncatula genome, there are more than 400 putative protease-encoding genes, but to date, the proteolytic content of Medicago cell cultures has not been studied. In this report, the proteolytic activities that can potentially hamper the successful production of recombinant proteins in this system are evaluated. The potential proteases responsible for the degradation of target proteins are identified. Interestingly, the number of proteases found in Medicago spent medium is considerably lower than that of the well-established tobacco bright yellow 2 (BY-2) system. Papain-like cysteine proteases are found to be the major contributors to recombinant protein degradation in Medicago. This knowledge is used to engineer a cell line with reduced endogenous protease activity by expressing a selective protease inhibitor, further improving this expression platform.

Grapevine is one of the most relevant crops in the world being used for economically important products such as wine. However, relevant grapevine cultivars are heavily affected by diseases such as the downy mildew disease caused by Plasmopara viticola. Improvements on grapevine resistance are made mainly by breeding techniques where resistance traits are introgressed into cultivars with desired grape characteristics. However, there is still a lack of knowledge on how resistant or tolerant cultivars tackle the P. viticola pathogen. In this study, using a shotgun proteomics LC-MS/MS approach, we unravel the protein modulation of a highly tolerant grapevine cultivar, Vitis vinifera “Regent”, in the first hours post inoculation (hpi) with P. viticola. At 6 hpi, proteins related to defense and to response to stimuli are negatively modulated while at 12 hpi there is an accumulation of proteins belonging to both categories. The co-occurrence of indicators of effector-triggered susceptibility (ETS) and effector-triggered immunity (ETI) is detected at both time-points, showing that these defense processes present high plasticity. The results obtained in this study unravel the tolerant grapevine defense strategy towards P. viticola and may provide valuable insights on resistance associated candidates and mechanisms, which may play an important role in the definition of new strategies for breeding approaches.