ABSTRACT
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.
Subject(s)
Parasites , Tylenchoidea , Animals , Tylenchoidea/physiology , Parasites/genetics , Apoptosis , Gene Expression Profiling , Cell Nucleus/metabolism , Plant Roots/parasitology , Plant DiseasesABSTRACT
CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15-30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.
ABSTRACT
Potato virus Y (PVY) is the most common virus infecting potato worldwide. We analysed potato tuber PVY infections from the major Israeli growing region in 2014-2017. Isolates were characterized by multiplex PCR according to Chikh-Ali et al. (Plant Disease 97, 1370, 2013), whose primers were not fully compatible with the Israeli isolates. New primers were designed for a multiplex PCR assay to differentiate the Israeli isolates. Three recombinant strains were observed: PVYNTNa (72% of the isolates), PVYNWi (24%) and PVYSyr-III (found only in 2015). The archetypal PVYO strain was found only once. The classical PVY strains have recently been displaced by recombinant forms, with PVYNTNa dominating. The Israeli isolates appear very similar to those of Europe (the seed tuber source), except for PVYSyr-III.
