The preparation of <i>Agrobacterium tumefaciens</i> cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these, underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale, requiring large quantities of <i>A. tumefaciens</i> culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth amongst the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask vs. bioreactors and provides novel insights into fundamental biological processes of <i>A. tumefaciens</i> influenced by such growth conditions.
Agroinfiltration is used to treat plants with modified strains of <i>Agrobacterium tumefaciens</i> for the purpose of transient <i>in planta</i> expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently-expressed target protein products as prolonged exposure to agroinfiltration medium suggests remodeling of the bacterial proteins towards enhanced infection of plant cells.
Perennial ryegrass (Lolium perenne) is the most cultivated cool-season grass worldwide with crucial roles in carbon fixation, turfgrass applications, and fodder for livestock. Lolium perenne forms a mutualism with the strictly vertically transmitted fungal endophyte, Epichloë festucae var lolii. The fungus produces alkaloids that protect the grass from herbivory, as well as conferring protection from drought and nutrient stress. The rising concentration of atmospheric CO2, a proximate cause of climatic change, is known to have many direct and indirect effects on plant growth. There is keen interest in how the nature of this plant–fungal interaction will change with climate change. Lolium perenne is an obligately outcrossing species, meaning that the genetic profile of the host is constantly being reshuffled. Meanwhile, the fungus is asexual implying both a relatively constant genetic profile and the potential for incompatible grass–fungus pairings. In this study, we used a single cultivar, “Alto”, of L. perenne. Each plant was infected with one of four strains of the endophyte: AR1, AR37, NEA2, and Lp19 (the “common strain”). We outcrossed the Alto mothers with pollen from a number of individuals from different ryegrass cultivars to create more genetic diversity in the hosts. We collected seed such that we had replicate maternal half-sib families. Seed from each family was randomly allocated into the two levels of the CO2 treatment, 400 and 800 ppm. Elevated CO2 resulted in an c. 18% increase in plant biomass. AR37 produced higher fungal concentrations than other strains; NEA2 produced the lowest fungal concentrations. We did not find evidence of genetic incompatibility between the host plants and the fungal strains. We conducted untargeted metabolomics and quantitative proteomics to investigate the grass-fungus interactions between and within family and treatment groups. We identified a number of changes in both the proteome and metabalome. Taken together, our data set provides new understanding into the intricacy of the interaction between endophyte and host from multiple molecular levels and suggests opportunity to promote plant robustness and survivability in rising CO2 environmental conditions through application of bioprotective epichloid strains.
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