Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis

Abstract

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode.

The origin, deployment, and evolution of a plant-parasitic nematode effectorome

“…Therefore, the lowest expression bin enriched in either -but in most cases both -effector-annotated genes and genes encoding putatively secreted proteins was used to define a cutoff for gland cell expression in each library. Above this cutoff, gland-expressed genes encoding putatively secreted proteins were considered putative effectors (Vieira et al 2021(Vieira et al , 2020Espada et al 2018). A majority (64%) of previously effector-annotated genes were recaptured above expression thresholds in gland cell libraries using this approach.…”

Functional Characterization of ShK Domain-Containing Protein in the Plant-Parasitic Nematode Bursaphelenchus xylophilus

“…In this study, we have identified 96 transcripts encoding for ShK domain-containing proteins from one of the most damaging PPN, B. xylophilus [9,33], and showed that the putative secreted proteins were enriched in these sequences-64% of these sequences encode a predicted signal peptide-which indicates that it might belong to secretory pathways. These proteins have also been found in the secretome of several animal parasitic nematodes (e.g., Teladorsagia circumcincta) [36] and other plant-parasitic nematodes, including B. xylophilus, P. penetrans, R. similis, and M. javanica [23][24][25]37]. The number of predicted ShK sequences constitutes one of the largest known protein families in Nematoda (e.g., T. circumcincta) [16,36], which is also observed in our data from B. xylophilus.…”

“…The nervous system is composed of major dorsal and ventral nerves along the body of the nematode, nearly associated with the epidermis, containing cell bodies that make and receive synapses with other neurons [39]. Some parasitic-related genes have been localized in nervous system tissues (e.g., R. similis) [23]. Curiously, a PWN catalase, which was used as a positive control for our ROS experiment, expressed in a transgenic C. elegans (for B. xylophilus catalase, Bxy-ctl-2, and BXY_1745800), was also localized in the nervous system [35].…”

“…Several studies showed the relevance of pharyngeal gland cells as a producer of several proteins that are secreted and potentially related to parasitism. The ShK domain-containing proteins that have also been identified in other plant-parasitic nematodes, such as P. penetrans [24], R. similis [23], M. javanica [25], and H. schachtii [26], are also expressed in the pharyngeal gland cells. M. javanica secreted ShK protein, MjShKT, which is expressed in the dorsal gland cell and upregulated in parasitic stages and manipulates the plant effector-triggered immune response [25].…”

“…The ShKT domain-containing proteins in the phylum Nematoda are present in the free-living Caenorhabditis elegans, with ca. 66 secreted ShKT proteins containing multiple copies of the ShK toxin domain [20], in the entomopathogenic nematode Steinernema carpocapsae [21,22], in the plant-parasitic nematodes Radopholus similis [23], Pratylenchus penetrans [24], Meloidogyne javanica [25], Heterodera schachtii [26], and in the animal-parasitic species Toxocara canis [15] and Trichostrongylus colubriformis [27]. In the parasitic and venomous species, the peptides with the ShKT domain have been shown to function as K + channel blockers [15,[28][29][30].…”