Transcriptomic analysis revealed distinct transcriptional responses in N. benthamiana upon infiltration with...

Transient plant expression, which relies on Agrobacterium carrying T-DNA with target genes infiltrated into leaves for transient nuclear expression, is crucial for gene function research and the rapid production of pharmaceutical proteins. Commonly used laboratory Agrobacterium strains differ in their genetic backgrounds and modified helper Ti plasmids, which may induce varying degrees and durations of host immune responses and transcriptional reprogramming. However, the differential responses of N. benthamiana, the most commonly used host, to these strains remain unclear. In this study, we for the first time systematically investigated the transcriptional defense responses of N. benthamiana to six common laboratory Agrobacterium strains (Agrobacterium tumefaciens C58C1, GV3101, LBA4404, AGL-1, EHA105, and the Rhizobium rhizogene strain ArA4). Transcriptome profiling of tobacco leaves was conducted at 3 and 7 dpi to clarify the conserved and divergent regulation of tobacco immune pathways and stress responses by different strains. Additionally, we selected genes significantly upregulated in all strain-infiltrated groups and verified three novel defence-related genes, PAR1, WRKY81, and DMR6, that negatively regulate recombinant protein expression efficiency. We also analysed the expression patterns of previously reported immune-related genes that regulate recombinant protein yields, revealing the differential regulatory characteristics of tobacco in response to infiltration by different Agrobacterium strains. The results show that different strains have divergent activation patterns of immune genes and may adopt distinct colonization modes. We provide an important basis for selecting Agrobacterium strains for transient expression in N. benthamiana, optimizing tobacco transient expression systems, and constructing high-efficiency plant bioreactors.