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A novel robust and high-throughput method to measure cell death in Nicotiana benthamiana leaves by fluorescence imaging.

ABSTRACT: Assessing immune responses and cell death in Nicotiana benthamiana leaf agro-infiltration assays is a powerful and widely used experimental approach in molecular plant pathology. Here, we describe a reliable high-throughput protocol to quantify strong, macroscopically visible cell death responses in N. benthamiana agro-infiltration assays. The method relies on measuring the reduction of leaf autofluorescence in the red spectrum upon cell death induction and provides quantitative data suitable for straightforward statistical analysis. Two different well-established model nucleotide-binding and leucine-rich repeat domain proteins (NLRs) were used to ensure the genericity of the approach. Its accuracy and versatility were compared to visual scoring of the cell death response and standard methods commonly used to characterize NLR activities in N. benthamiana. A discussion of the advantages and limitations of our method compared to other protocols demonstrates its robustness and versatility and provides an effective means to select the best-suited protocol for a defined experiment.

SUBMITTER: Xi Y

PROVIDER: S-EPMC8578831 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5' untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5' UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5' UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.

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A novel robust and high-throughput method to measure cell death in Nicotiana benthamiana leaves by fluorescence imaging.

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