Efficient fluorescence-based localization technique for real-time tracking endophytes route in host-plants colonization

Bacterial isolates that enhance plant growth and suppress plant pathogens growth are essential tools for reducing pesticide applications in plant production systems. The objectives of this study were to develop a reliable fluorescence-based technique for labeling bacterial isolates selected as biological control agents (BCAs) to allow their direct tracking in the host-plant interactions, understand the BCA localization within their host plants, and the route of plant colonization. Objectives were achieved by developing competent BCAs transformed with two plasmids, pBSU101 and pANIC-10A, containing reporter genes eGFP and pporRFP, respectively. Our results revealed that the plasmid-mediated transformation efficiencies of antibiotic-resistant competent BCAs identified as PSL, IMC8, and PS were up 84%. Fluorescent BCA-tagged reporter genes were associated with roots and hypocotyls but not with leaves or stems and were confirmed by fluoresence microscopy and PCR analyses in colonized Arabidopsis and sorghum. This fluorescence-based technique's high resolution and reproducibility make it a platform-independent system that allows tracking of BCAs spatially within plant tissues, enabling assessment of the movement and niches of BCAs within colonized plants. Steps for producing and transforming competent fluorescent BCAs, as well as the inoculation of plants with transformed BCAs, localization, and confirmation of fluorescent BCAs through fluorescence imaging and PCR, are provided in this manuscript. This study features host-plant interactions and subsequently biological and physiological mechanisms implicated in these interactions. The maximum time to complete all the steps of this protocol is approximately 3 months.