Electropermeabilization-based fluorescence in situ hybridization of whole-mount plant parasitic nematode specimens

A fluorescence in situ hybridization (FISH) protocol was developed for nematodes in which nucleic acid probes are introduced within the organism via electroporation. This modification of existing FISH protocols removes numerous chemical wash steps, and thus, reduces protocol time and specimen loss while improving hybridization sensitivity. The presented work is optimized for juveniles of soybean cyst nematode (SCN; Heterodera glycines) and has been used to identify both host and associated-microbial (viral) targets. Moreover, through the use of two different long wavelength fluorophores, two probes can be colocalized within one individual. This protocol may be adapted to identify targets-of-interest within other life stages and nematode species. This protocol improves: • Hands-on protocol time (by approximately 1.5 h). • Specimen loss (fewer aspiration steps). • Hybridization (allows colocalization with two nucleic acid probes and increases sensitivity).