Heterologous expression of site-specific DNA recombinase genes alters plant development and abiotic stress responses in creeping bentgrass

Site-specific recombination systems originating from bacterium and yeast have been widely used for targeted DNA rearrangement in various heterologous species, yet the genome-wide off-target potential and biological effects of recombinases in the host remain poorly understood. Here, we investigated how sustained expression of two widely used bacteriophage recombinases, Cre and φC31 influences plant growth, abiotic stress responses, and transcriptome reprogramming, and examined the presence and activity of the putative cryptic recombinase target sites in a representative perennial grass species, creeping bentgrass (Agrostis stolonifera). Independent transgenic lines constitutively expressing Cre or φC31 all exhibited enhanced plant growth with the φC31 lines displaying the strongest and most coordinated increase in shoot and root biomass. The φC31 lines also exhibited improved drought and salt tolerance associated with sustained relative water content, cell membrane integrity, and proline accumulation, whereas the Cre lines did not show a significant difference from wild type in salt stress response, but exhibited increased drought susceptibility. Genome-wide in silico analysis identified numerous loxP-like and att-like putative Cre/φC31 targets in the creeping bentgrass genome. However, transient reporter assays only detected robust Cre-mediated DNA recombination on canonical loxP sites, not on the highest-scoring loxP-like putative Cre target candidates. No φC31-mediated DNA recombination was detected under the conditions tested. Transcriptome analysis revealed extensive transcriptional reprogramming in the φC31 lines, including enrichment of defense- and hormone-related processes, whereas the Cre lines showed enrichment mainly in growth-, photosynthesis-, and energy metabolism-associated processes without comparable immune-response activation.