RNA Isolation from Nematode-Induced Feeding Sites in Arabidopsis Roots Using Laser Capture Microdissection.

ABSTRACT

Nematodes are diverse multicellular organisms that are most abundantly found in the soil. Most nematodes are free-living and feed on a range of organisms. Based on their feeding habits, soil nematodes can be classified into four groups bacterial, omnivorous, fungal, and plant-feeding. Plant-parasitic nematodes (PPNs) are a serious threat to global food security, causing substantial losses to the agricultural sector. Root-knot and cyst nematodes are the most important of PPNs, significantly limiting the yield of commercial crops such as sugar beet, mustard, and cauliflower. The life cycle of these nematodes consists of four molting stages (J1-J4) that precede adulthood. Nonetheless, only second-stage juveniles (J2), which hatch from eggs, are infective worms that can parasitize the host's roots. The freshly hatched juveniles (J2) of beet cyst nematode, Heterodera schachtii, establish a permanent feeding site inside the roots of the host plant. A cocktail of proteinaceous secretions is injected into a selected cell which later develops into a syncytium via local cell wall dissolution of several hundred neighboring cells. The formation of syncytium is accompanied by massive transcriptional, metabolic, and proteomic changes inside the host tissues. It creates a metabolic sink in which solutes are translocated to feed the nematodes throughout their life cycle. Deciphering the molecular signaling cascades during syncytium establishment is thus essential in studying the plant-nematode interactions and ensuring sustainability in agricultural practices. However, isolating RNA, protein, and metabolites from syncytial cells remains challenging. Extensive use of laser capture microdissection (LCM) in animal and human tissues has shown this approach to be a powerful technique for isolating a single cell from complex tissues. Here, we describe a simplified protocol for Arabidopsis-Heterodera schachtii infection assays, which is routinely applied in several plant-nematode laboratories. Next, we provide a detailed protocol for isolating high-quality RNA from syncytial cells induced by Heterodera schachtii in the roots of Arabidopsis thaliana plants.