Multiplexed Fluorescent In Situ Hybridization in Plant-Parasitic Nematodes.

Non-model organisms often lack the methodological and technological tools that are available for model systems. This is particularly true for plant-parasitic nematodes. Despite causing significant agricultural losses worldwide, their mechanisms of parasitism remain incompletely understood. One major limitation is the difficulty in validating the spatial expression of genes involved in pathogenesis, known as effectors. In situ hybridization (ISH) remains one of the most accessible and cost-effective approaches for gene expression analysis. However, traditional chromogenic ISH on fragmented nematodes offers limited spatial resolution and cannot support multiplexed detection, making it difficult to definitively localize effector expression to the specialized secretory cells that produce them. In order to address these limitations, I developed a protocol for whole-mount plant-parasitic nematode preparation, called the "Sperling Prep," for use with stains, antibodies, and ISH using third-generation hybridization chain reaction (HCR™). The HCR method enables single-transcript detection through fluorescent probe amplification. The probe amplification allows multiplexed detection of multiple transcripts since it works with adapters that can be coupled to different fluorophores. Combining the Sperling Prep with HCR enables spatially resolved single-molecule detection of transcripts within intact nematodes. This makes it possible to co-localize putative effectors with markers of secretory cells, thereby providing evidence of effector identity. Furthermore, the protocol enables quantification of gene expression across the entire organism, facilitating comparisons of expression differences under different biological conditions, such as during plant infection. Here, I present a detailed protocol of the published method for whole-mount HCR in plant-parasitic nematodes, offering a powerful and scalable method for identifying and validating novel effectors of parasitism.