The transcriptome of Nacobbus aberrans reveals insights into the evolution of sedentary endoparasitism in plant-parasitic nematodes

Abstract

Within the phylum Nematoda, plant-parasitism is hypothesized to have arisen independently on at least four occasions. The most economically damaging plant-parasitic nematode species, and consequently the most widely studied, are those that feed as they migrate destructively through host roots causing necrotic lesions (migratory endoparasites) and those that modify host root tissue to create a nutrient sink from which they feed (sedentary endoparasites). The false root-knot nematode Nacobbus aberrans is the only known species to have both migratory endoparasitic and sedentary endoparasitic stages within its life cycle. Moreover, its sedentary stage appears to have characteristics of both the root-knot and the cyst nematodes. We present the first large-scale genetic resource of any false-root knot nematode species. We use RNAseq to describe relative abundance changes in all expressed genes across the life cycle to provide interesting insights into the biology of this nematode as it transitions between modes of parasitism. A multigene phylogenetic analysis of N. aberrans with respect to plant-parasitic nematodes of all groups confirms its proximity to both cyst and root-knot nematodes. We present a transcriptome-wide analysis of both lateral gene transfer events and the effector complement. Comparing parasitism genes of typical root-knot and cyst nematodes to those of N. aberrans has revealed interesting similarities. Importantly, genes that were believed to be either cyst nematode, or root-knot nematode, "specific" have both been identified in N. aberrans. Our results provide insights into the characteristics of a common ancestor and the evolution of sedentary endoparasitism of plants by nematodes.

Keywords: RNAseq; effectors; host mimics; lateral gene transfer; phylogenetics; plant-parasitic nematode.

Fig. 1.—

The life cycle of Nacobbus species on potato. Dashed arrows represent transitions through the life cycle, whereas solid arrows represent movement of the nematode. J2s hatch from eggs in the soil and locate plant roots. J2s migrate destructively (i.e., intracellularly) through roots. J2s moult without feeding, either in roots or in the soil, into the J3. This stage, and the subsequent J4, can enter or leave roots and also migrate destructively and in addition cause lesions and necrosis. The J3 and J4 are the dauer (survival) stages and are able to tolerate adverse environmental conditions such as low humidity. After the final moult the vermiform females migrate to healthy root tissue, where they establish a permanent plant-derived, multinucleate feeding site known as a syncytium that is formed inside a root gall. Females feed from the syncytium for several weeks as their body swells and eggs are produced. Eggs are deposited in a gelatinous matrix to form egg masses in the soil.

Fig. 2.—

Transcripts clustered by differential expression. For all expression clusters, mean centred log fold change of expression is plotted for each of the three biological replicates for each life stage in the following order: J2, mixed J3/J4/female migratory (Mig), and sedentary female (Sed). A total of 7,731 differentially expressed transcripts clustered into 26 clusters based on expression profile. Clusters are manually grouped into ten super clusters with summarized expression.

Fig. 3.—

Local phylogenetic history of sedentary PPN: Nuclear protein-coding multigene phylogeny. Concatenated multigene protein alignment of 65 CEGs present in all 15 species. Using Longidorus elongatus as a root: Nacobbus aberrans lies at the base of the root-knot nematodes. Node numbers represent bootstrap support values for 100 iterations, and species are highlighted by their different life strategies.

Fig. 4.—

Comparison of nematode-derived plant peptide mimics between genera. (a) A comparison between the CLE and CEP-like peptides in Arabidopsis thaliana and PPN. No CEP-like sequences were identifiable in either N. aberrans or Globodera pallida. (b) Two of the three sequences containing CLE-like motifs in the Nacobbus aberrans transcriptome contain putative signal peptides (2 and 3) and have expression peaks during the sedentary life stage (2 and 3) compared with juvenile or migratory stages. Data for A. thaliana and M. hapla are modified from Bird et al. (Bird et al. 2014).

Fig. 5.—

Expression comparison of 4D06-like gene families of Globodera pallida and Nacobbus aberrans. Each line contains expression data from a single gene across the life cycle. G. pallida 4D06-like genes are characterized by highest expression in the earliest stages of sedentary endoparasitism. Nacobbus aberrans 4D06-like sequences follow a similar expression pattern with peaks of expression during sedentary parasitic stages.

Fig. 6.—

Putative effector 4D06: Multispecies protein alignment. Midpoint rooted protein phylogeny of 4D06-like sequences of Nacobbus aberrans (gray), Rotylenchulus reniformis (red), Globodera pallida (green), and G. rostochiensis (blue) is divided into two groups. Group 1 is dominated by cyst nematode sequences. Group 2 contains almost all of the N. aberrans sequences, 75% of R. reniformis sequences, 40% of G. rostochiensis sequences, and 3.4% of G. pallida sequences and is therefore presumably ancestral. MEME analysis has identified a highly conserved motif, termed “PCCP” that is present with a conserved location in all sequences in group 1 and absent in group 2.