Silencing the conserved small nuclear ribonucleoprotein SmD1 target gene alters susceptibility to root-knot nematodes in plants.

Root-knot nematodes (RKNs) are among the most damaging pests of agricultural crops. Meloidogyne is an extremely polyphagous genus of nematodes that can infect thousands of plant species. A few genes for resistance (R-genes) to RKN suitable for use in crop breeding have been identified, but virulent strains and species of RKN have emerged that render these R-genes ineffective. Secretion of RKN effectors targeting plant functions mediates the reprogramming of root cells into specialized feeding cells, the giant cells, essential for RKN development and reproduction. Conserved targets among plant species define the more relevant strategies for controlling nematode infection. The EFFECTOR18 (EFF18) protein from M. incognita interacts with the spliceosomal small nuclear ribonucleoprotein D1 (SmD1) in Arabidopsis (Arabidopsis thaliana), disrupting its function in alternative splicing regulation and modulating the giant cell transcriptome. We show here that EFF18 is a conserved RKN-specific effector that targets this conserved spliceosomal SmD1 protein in Solanaceae. This interaction modulates alternative splicing events produced by tomato (Solanum lycopersicum) in response to M. incognita infection. The alteration of SmD1 expression by virus-induced gene silencing in Solanaceae affects giant cell formation and nematode development. Thus, our work defines a promising conserved SmD1 target gene to develop broad resistance for the control of Meloidogyne spp. in plants.

AUXIN RESPONSIVE FACTOR8 regulates development of the feeding site induced by root-knot nematodes in tomato.

Root-knot nematodes (RKN) from the genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression, and auxin is known to be a key player in their development. However, little is known about how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their miRNA167 regulators, were identified as robust gene-miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-ß-glucuronidase (GUS) fusions showed the up-regulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR (clustered regularly interspaced palindromic repeats) mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.

Gene copy number variations as signatures of adaptive evolution in the parthenogenetic, plant-parasitic nematode Meloidogyne incognita.

Adaptation to changing environmental conditions represents a challenge to parthenogenetic organisms, and until now, how phenotypic variants are generated in clones in response to the selection pressure of their environment remains poorly known. The obligatory parthenogenetic root-knot nematode species Meloidogyne incognita has a worldwide distribution and is the most devastating plant-parasitic nematode. Despite its asexual reproduction, this species exhibits an unexpected capacity of adaptation to environmental constraints, for example, resistant hosts. Here, we used a genomewide comparative hybridization strategy to evaluate variations in gene copy numbers between genotypes of M. incognita resulting from two parallel experimental evolution assays on a susceptible vs. resistant host plant. We detected gene copy number variations (CNVs) associated with the ability of the nematodes to overcome resistance of the host plant, and this genetic variation may reflect an adaptive response to host resistance in this parthenogenetic species. The CNV distribution throughout the nematode genome is not random and suggests the occurrence of genomic regions more prone to undergo duplications and losses in response to the selection pressure of the host resistance. Furthermore, our analysis revealed an outstanding level of gene loss events in nematode genotypes that have overcome the resistance. Overall, our results support the view that gene loss could be a common class of adaptive genetic mechanism in response to a challenging new biotic environment in clonal animals.

Development of Diagnostic SCAR Markers for Meloidogyne graminicola, M. oryzae, and M. salasi Associated with Irrigated Rice Fields in Americas.

Root-knot nematodes (RKN) cause important production losses of rice (Oryza sativa L.) in the world. Together with Meloidogyne graminicola Golden and Birchfield 1965, M. oryzae Maas, Sanders and Dede, 1978 and M. salasi López, 1984 have been causing damages in irrigated rice fields in Central and South America. In addition, six other RKN species may occur in rice fields in other regions of the world. Correct identification of Meloidogyne spp. is difficult but essential for the management of rice RKNs. The objective of this study was to develop some species-specific molecular markers for the diagnosis of South American RKN rice-related species. Isozyme phenotypes indicated the occurrence of some RKN species in the Brazilian samples, namely M. graminicola, M. oryzae, M. javanica, and two cryptic species designated as Meloidogyne sp. 2 and Meloidogyne sp. 3. Random amplified polymorphic DNA (RAPD) analysis of 16 isolates revealed interspecific genetic polymorphism between Meloidogyne spp., but isolates belonging to the same species (i.e., sharing the same esterase phenotype) always clustered together, whatever the species considered. Specific SCAR markers of 230, 120, and 160 bp were developed for M. graminicola, M. oryzae, and M. salasi, respectively. These SCAR markers may be potential molecular tools for application in routine diagnostic procedures subject to their validation with other rice RKN field populations in the world.

The root-knot nematode effector MiEFF12 targets the host ER quality control system to suppress immune responses and allow parasitism.

Root-knot nematodes (RKNs) are microscopic parasitic worms able to infest the roots of thousands of plant species, causing massive crop yield losses worldwide. They evade the plant's immune system and manipulate plant cell physiology and metabolism to transform a few root cells into giant cells, which serve as feeding sites for the nematode. RKN parasitism is facilitated by the secretion in planta of effector molecules, mostly proteins that hijack host cellular processes. We describe here a conserved RKN-specific effector, effector 12 (EFF12), that is synthesized exclusively in the oesophageal glands of the nematode, and we demonstrate its function in parasitism. In the plant, MiEFF12 localizes to the endoplasmic reticulum (ER). A combination of RNA-sequencing analysis and immunity-suppression bioassays revealed the contribution of MiEFF12 to the modulation of host immunity. Yeast two-hybrid, split luciferase and co-immunoprecipitation approaches identified an essential component of the ER quality control system, the Solanum lycopersicum plant bap-like (PBL), and basic leucine zipper 60 (BZIP60) proteins as host targets of MiEFF12. Finally, silencing the PBL genes in Nicotiana benthamiana decreased susceptibility to Meloidogyne incognita infection. Our results suggest that EFF12 manipulates PBL function to modify plant immune responses to allow parasitism.

The Meloidogyne incognita Nuclear Effector MiEFF1 Interacts With Arabidopsis Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases to Promote Parasitism.

Root-knot nematodes are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks. They induce the differentiation of root cells into specialized multinucleate hypertrophied feeding cells known as giant cells. Nematode effectors synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet play a key role in giant cell ontogenesis. The Meloidogyne incognita MiEFF1 is one of the rare effectors of phytopathogenic nematodes to have been located in vivo in feeding cells. This effector specifically targets the giant cell nuclei. We investigated the Arabidopsis functions modulated by this effector, by using a yeast two-hybrid approach to identify its host targets. We characterized a universal stress protein (USP) and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) as the targets of MiEFF1. We validated the interaction of MiEFF1 with these host targets in the plant cell nucleus, by bimolecular fluorescence complementation (BiFC). A functional analysis with Arabidopsis GUS reporter lines and knockout mutant lines showed that GAPCs were induced in giant cells and that their non-metabolic functions were required for root-knot nematode infection. These susceptibility factors are potentially interesting targets for the development of new root-knot nematode control strategies.

Tracking changes in life-history traits related to unnecessary virulence in a plant-parasitic nematode.

Evaluating trade-offs in life-history traits of plant pathogens is essential to understand the evolution and epidemiology of diseases. In particular, virulence costs when the corresponding host resistance gene is lacking play a major role in the adaptive biology of pathogens and contribute to the maintenance of their genetic diversity. Here, we investigated whether life-history traits directly linked to the establishment of plant-nematode interactions, that is, ability to locate and move toward the roots of the host plant, and to invade roots and develop into mature females, are affected in Meloidogyne incognita lines virulent against the tomato Mi-1.2 resistance gene. Virulent and avirulent near-isogenic lines only differing in their capacity to reproduce or not on resistant tomatoes were compared in single inoculation or pairwise competition experiments. Data highlighted (1) a global lack of trade-off in traits associated with unnecessary virulence with respect to the nematode ability to successfully infest plant roots and (2) variability in these traits when the genetic background of the nematode is considered irrespective of its (a)virulence status. These data suggest that the variation detected here is independent from the adaptation of M. incognita to host resistance, but rather reflects some genetic polymorphism in this asexual organism.

The map-1 gene family in root-knot nematodes, Meloidogyne spp.: a set of taxonomically restricted genes specific to clonal species.

Taxonomically restricted genes (TRGs), i.e., genes that are restricted to a limited subset of phylogenetically related organisms, may be important in adaptation. In parasitic organisms, TRG-encoded proteins are possible determinants of the specificity of host-parasite interactions. In the root-knot nematode (RKN) Meloidogyne incognita, the map-1 gene family encodes expansin-like proteins that are secreted into plant tissues during parasitism, thought to act as effectors to promote successful root infection. MAP-1 proteins exhibit a modular architecture, with variable number and arrangement of 58 and 13-aa domains in their central part. Here, we address the evolutionary origins of this gene family using a combination of bioinformatics and molecular biology approaches. Map-1 genes were solely identified in one single member of the phylum Nematoda, i.e., the genus Meloidogyne, and not detected in any other nematode, thus indicating that the map-1 gene family is indeed a TRG family. A phylogenetic analysis of the distribution of map-1 genes in RKNs further showed that these genes are specifically present in species that reproduce by mitotic parthenogenesis, with the exception of M. floridensis, and could not be detected in RKNs reproducing by either meiotic parthenogenesis or amphimixis. These results highlight the divergence between mitotic and meiotic RKN species as a critical transition in the evolutionary history of these parasites. Analysis of the sequence conservation and organization of repeated domains in map-1 genes suggests that gene duplication(s) together with domain loss/duplication have contributed to the evolution of the map-1 family, and that some strong selection mechanism may be acting upon these genes to maintain their functional role(s) in the specificity of the plant-RKN interactions.