Background

Plant-parasitic nematodes (PPNs) form a diverse group of microscopic roundworms and pose a serious threat to many crop plants, significantly impacting agricultural productivity [1,2]. Through their parasitic interactions, PPNs disrupt normal plant functions, leading to reduced nutrient and water uptake, stunted growth, and, in severe infestations, plant death [3].

PPNs secrete various enzymes and effectors that modify the host plant's cellular processes to support nematode development while suppressing the plant's immune responses [4]. Among the most damaging PPNs are sedentary root-knot (Meloidogyne spp.) and cyst nematodes (Heterodera spp. and Globodera spp.), as well as migratory lesion nematodes (Pratylenchus spp.) [5,6]. The endoparasitic nematodes induce sophisticated feeding sites in host roots, which serve as continuous nutrient sources for their development. While root-knot nematodes induce giant cells embedded in gall tissue, cyst nematodes establish so-called syncytia in plant roots.

The analyses of transcriptomes of syncytia and giant cells have shown that nematodes can suppress plant defense during the establishment of their feeding sites [7,8]. For instance, functional analysis of genes involved in pathways such as callose deposition, phytoalexin biosynthesis, reactive oxygen species (ROS) production, and antimicrobial peptide synthesis has revealed an overexpression of these genes in nematode-infected roots [9–12]. Similarly, genes related to lignin biosynthesis in the phenylpropanoid pathway have been studied in response to nematode infection, showing comparable results (Ali and Wieczorek, unpublished data). The phenylpropanoid pathway in plants leads to the biosynthesis of diverse secondary metabolites, including lignin, lignans, flavonoids, and phenolic compounds [13]. These compounds play essential roles in plant defense, pigmentation, structural integrity, rigidity, and the mechanical strength of cell walls [14]. Lignin, a key component of plant cell walls, is crucial for reinforcing structural integrity and serves as a protective barrier against invading pathogens [15]. Interestingly, DIR proteins, which are involved in lignin biosynthesis, do not function as enzymes. Instead, they guide the process by positioning monolignols in specific orientations, ensuring their proper coupling during polymerization [16]. This unique ability to control the spatial arrangement of lignin precursors facilitates the formation of a robust and intricate lignin network. Such a network not only strengthens the cell wall but also enhances plant defense by making it more difficult for pathogens to penetrate and infect the plant [17].

Disruption of lignin biosynthesis pathways has been shown to increase susceptibility to pathogens in Arabidopsis and other plants, highlighting the crucial role of these pathways in plant resistance [18]. Therefore, the present protocol has been developed to detect lignin in roots infected by the cyst nematode H. schachtii and the root-knot nematode M. incognita, which induce syncytia and galls, respectively. The staining method uses phloroglucinol-HCl, which reacts with the cinnamaldehyde end groups of lignin, producing a red-violet color. This method has not been widely used for staining lignin in nematode-induced feeding sites. Instead, many scientific publications analyze other cell wall polymers, such as cellulose and callose, using histochemical approaches or specific antibodies (e.g., [19,20]). However, Nakagami et al. (2020) used a 2% phloroglucinol solution to stain 3- and 5-day-old galls induced by M. incognita in Arabidopsis wild type and the mutant del1-1 [21]. The description of the method in this report is not very detailed. Additionally, the authors stained only whole root fragments containing the feeding sites and did not perform tissue sectioning to obtain a more detailed picture of lignin localization. Similarly, Sato et al. [22] used this method to stain entire 3-day-old galls induced by M. arenaria in Solanum torvum, demonstrating its applicability to Solanum species. However, the description of the staining protocol in this study is not detailed, and the authors refer to a histochemistry book by Jensen (1962) [23], which is not easily accessible. Hence, this protocol provides, for the first time, a detailed description of lignin staining in both whole nematode-feeding sites and sectioned feeding sites induced in Arabidopsis, allowing for a more precise localization of lignin deposition. Its comprehensive methodology enhances reproducibility and enables a clearer understanding of cell wall modifications in response to nematode infection.