GFP labeling of a Bradyrhizobium strain and an attempt to track the crack entry process during symbiosis with peanuts

Compared to the well-studied model legumes, where symbiosis is established via root hair entry, the peanut is infected by Bradyrhizobium through the crack entry, which is less common and not fully understood. Crack entry is, however, considered a primitive symbiotic infection pathway, which could be potentially utilized for engineering non-legume species with nitrogen fixation ability. We utilized a fluorescence-labeled Bradyrhizobium strain to help in understanding the crack entry process at the cellular level. A modified plasmid pRJPaph-bjGFP, harboring the codon-optimized GFP gene and tetracycline resistance gene, was created and conjugated into Bradyrhizobium strain Lb8, an isolate from peanut nodules, through tri-parental mating. Microscopic observation and peanut inoculation assays confirmed the successful GFP tagging of Lb8, which is capable of generating root nodules. A marking system for peanut root potential infection sites and an optimized sample preparation protocol for cryostat sectioning was developed. The feasibility of using the GFP-tagged Lb8 for observing crack entry was examined. GFP signal was detected at the nodule primordial stage and the following nodule developmental stages with robust GFP signals observed in infected cells in the mature nodules. Spherical bacteroids in the root tissue were visualized at the nodules' inner cortex under higher magnification, reflecting the trace along the rhizobial infection path. The GFP labeled Lb8 can serve as an essential tool for plant-microbe studies between the cultivated peanut and Bradyrhizobium, which could facilitate further study of the crack entry process during the legume-rhizobia symbiosis.