Two Candidate Meloidogyne javanica Effector Genes, MjShKT and MjPUT3: A Functional Investigation of Their Roles in Regulating Nematode Parasitism.

During parasitism, root-knot nematode Meloidogyne spp. inject molecules termed effectors that have multifunctional roles in construction and maintenance of nematode feeding sites. As an outcome of transcriptomic analysis of Meloidogyne javanica, we identified and characterized two differentially expressed genes encoding the predicted proteins MjShKT, carrying a Stichodactyla toxin (ShKT) domain, and MjPUT3, carrying a ground-like domain, both expressed during nematode parasitism of the tomato plant. Fluorescence in-situ hybridization revealed expression of MjShKT and MjPUT3 in the dorsal esophageal glands, suggesting their injection into host cells. MjShKT expression was upregulated during the parasitic life stages, to a maximum at the mature female stage, whereas MjPUT3 expression increased in third- to fourth-stage juveniles. Subcellular in-planta localization of MjShKT and MjPUT3 using a fused fluorescence marker indicated MjShKT co-occurrence with the endoplasmic reticulum, the perinuclear endoplasmatic reticulum, and the Golgi organelle markers, while MjPUT3 localized, to some extent, within the endoplasmatic reticulum and was clearly observed within the nucleoplasm. MjShKT inhibited programmed cell death induced by overexpression of MAPKKKα and Gpa2/RBP-1. Overexpression of MjShKT in tomato hairy roots allowed an increase in nematode reproduction, as indicated by the high number of eggs produced on roots overexpressing MjShKT. Roots overexpressing MjPUT3 were characterized by enhanced root growth, with no effect on nematode development on those roots. Investigation of the two candidate effectors suggested that MjShKT is mainly involved in manipulating the plant effector-triggered immune response toward establishment and maintenance of active feeding sites, whereas MjPUT3 might modulate roots morphology in favor of nematode fitness in the host roots. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Minichromosome Maintenance Complex Component 2 (MjMCM2) of Meloidogyne javanica is a potential effector regulating the cell cycle in nematode-induced galls.

Root-knot nematodes Meloidogyne spp. induce enlarged multinucleate feeding cells-galls-in host plant roots. Although core cell-cycle components in galls follow a conserved track, they can also be usurped and manipulated by nematodes. We identified a candidate effector in Meloidogyne javanica that is directly involved in cell-cycle manipulation-Minichromosome Maintenance Complex Component 2 (MCM2), part of MCM complex licensing factor involved in DNA replication. MjMCM2, which is induced by plant oxilipin 9-HOT, was expressed in nematode esophageal glands, upregulated during parasitic stages, and was localized to plant cell nucleus and plasma membrane. Infected tomato hairy roots overexpressing MjMCM2 showed significantly more galls and egg-mass-producing females than wild-type roots, and feeding cells showed more nuclei. Phylogenetic analysis suggested seven homologues of MjMCM2 with unknown association to parasitism. Sequence mining revealed two RxLR-like motifs followed by SEED domains in all Meloidogyne spp. MCM2 protein sequences. The unique second RxLR-like motif was absent in other Tylenchida species. Molecular homology modeling of MjMCM2 suggested that second RxLR2-like domain is positioned on a surface loop structure, supporting its function in polar interactions. Our findings reveal a first candidate cell-cycle gene effector in M. javanica-MjMCM2-that is likely secreted into plant host to mimic function of endogenous MCM2.