Plant-parasitic nematodes produce at least one structurally unique class of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant hosts. Herein we describe a protein of the plant-parasitic root-knot nematode Meloidogyne javanica, which is a member of the nematode-specific fatty-acid- and retinol-binding (Mj-FAR-1) family of proteins. The mj-far-1 mRNA was detected through M. javanica pre-parasitic J2s, migratory and sedentary parasitic stages by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Immunolocalization assays demonstrate that the FAR protein of Meloidogyne is secreted during sedentary stages, as evidenced by the accumulation of FAR at the nematode cuticle surface and along the adjacent host root tissues. Tomato roots constitutively expressing mj-far-1 demonstrated an increased susceptibility to root-knot nematodes infection as observed by accelerated gall induction and expansion, accompanied by a higher percentage of nematodes developing into mature females compared to control roots. RNA interference assays that expressed double-stranded RNA complementary to mj-far-1 in transgenic tomato lines specifically reduced nematode infection levels. Histological analysis of nematode-infested roots indicated that in roots overexpressing mj-far-1, galls contained larger feeding cells and might support a faster nematode development and maturation. Roots overexpressing mj-far-1 suppressed jasmonic acid responsive genes such as the proteinase inhibitor (Pin2) and γ-thionin, illustrating the possible role of Mj-FAR-1 in manipulating the lipid based signaling in planta. This data, suggests that Meloidogyne FAR might have a strategic function during the interaction of the nematode with its plant host. Our study present the first demonstration of an in planta functional characterization and localization of FAR proteins secreted by plant-parasitic nematodes. It provides evidence that Mj-FAR-1 facilitates infection most likely via the manipulation of host lipid-based defenses, as critical components for a successful parasitism by plant-parasitic nematodes.

BackgroundThe secreted Meloidogyne javanica fatty acid- and retinol-binding (FAR) protein Mj-FAR-1 is involved in nematode development and reproduction in host tomato roots. To gain further insight into the role of Mj-FAR-1 in regulating disease development, local transcriptional changes were monitored in tomato hairy root lines with constitutive mj-far-1 expression compared with control roots without inoculation, and 2, 5 and 15 days after inoculation (DAI), using mRNA sequencing analysis.ResultsGene-expression profiling revealed a total of 3970 differentially expressed genes (DEGs) between the two lines. Among the DEGs, 1093, 1039, 1959, and 1328 genes were up- or downregulated 2-fold with false discovery rate < 0.001 in noninoculated roots, and roots 2, 5, and 15 DAI compared with control roots, respectively. Four main groups of genes that might be associated with Mj-FAR-1-mediated susceptibility were identified: 1) genes involved in biotic stress responses such as pathogen-defense mechanisms and hormone metabolism; 2) genes involved in phenylalanine and phenylpropanoid metabolism; 3) genes associated with cell wall synthesis, modification or degradation; and 4) genes associated with lipid metabolism. All of these genes were overrepresented among the DEGs. Studying the distances between the treatments, samples from noninoculated roots and roots at 2 DAI clustered predominantly according to the temporal dynamics related to nematode infection. However, at the later time points (5 and 15 DAI), samples clustered predominantly according to mj-far-1 overexpression, indicating that at these time points Mj-FAR-1 is more important in defining a common transcriptome.ConclusionsThe presence of four groups of DEGs demonstrates a network of molecular events is mediated by Mj-FAR-1 that leads to highly complex manipulation of plant defense responses against nematode invasion. The results shed light on the in vivo role of secreted FAR proteins in parasitism, and add to the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1426-3) contains supplementary material, which is available to authorized users.

The responses of two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, to infection by the sedentary nematodes root-knot nematode (Meloidogyne javanica) and cyst nematode (Heterodera schachtii) were analysed in transgenic Arabidopsis seedlings. The tissue localization of LOX3 and LOX4 gene expression using β-glucuronidase (GUS) reporter gene constructs showed local induction of LOX3 expression when second-stage juveniles reached the vascular bundle and during the early stages of plant-nematode interaction through gall and syncytia formation. Thin sections of nematode-infested knots indicated LOX3 expression in mature giant cells, and high expression in neighbouring cells and those surrounding the female body. LOX4 promoter was also activated by nematode infection, although the GUS signal weakened as infection and disease progressed. Homozygous insertion mutants lacking LOX3 were less susceptible than wild-type plants to root-knot nematode infection, as reflected by a decrease in female counts. Conversely, deficiency in LOX4 function led to a marked increase in females and egg mass number and in the female to male ratio of M. javanica and H. schachtii, respectively. The susceptibility of lox4 mutants was accompanied by increased expression of allene oxide synthase, allene oxide cyclase and ethylene-responsive transcription factor 4, and the accumulation of jasmonic acid, measured in the roots of lox4 mutants. This response was not found in lox3 mutants. Taken together, our results reveal that LOX4 and LOX3 interfere differentially with distinct metabolic and signalling pathways, and that LOX4 plays a major role in controlling plant defence against nematode infection.

The behavior of naturally virulent Meloidogyne isolates toward the tomato resistance gene Mi in major tomato-growing areas in Israel was studied for the first time. Virulence of seven selected isolates was confirmed over three successive generations on resistant (Mi-carrying) and susceptible (non-Mi-carrying) tomato cultivars. Diagnostic markers verified the predominance of Meloidogyne javanica among virulent isolates selected on resistant tomato cultivars or rootstocks. To better understand the determinants of nematode selection on Mi-carrying plants, reproduction of Mi-avirulent and virulent isolates Mjav1 and Mjv2, respectively, measured as eggs per gram of root, on non-Mi-carrying, heterozygous (Mi/mi) and homozygous (Mi/Mi) genotypes was evaluated. Although no reproduction of Mjav1 was observed on Mi/Mi genotypes, some reproduction was consistently observed on Mi/mi plants; reproduction of Mjv2 on the homozygous and heterozygous genotypes was similar to that on susceptible cultivars, suggesting a limited quantitative effect of the Mi gene. Histological examination of giant cells induced by Mi-virulent versus avirulent isolates confirmed the high virulence of Mjv2 on Mi/mi and Mi/Mi genotypes, allowing the formation of well-developed giant-cell systems despite the Mi gene. Analysis of the plant defense response in tomato Mi/Mi, Mi/mi, and mi/mi genotypes to both avirulent and virulent isolates was investigated by quantitative real-time polymerase chain reaction. Although the jasmonate (JA)-signaling pathway was clearly upregulated by avirulent and virulent isolates on the susceptible (not carrying Mi) and heterozygous (Mi/mi) plants, no change in signaling was observed in the homozygous (Mi/Mi) resistant line following incompatible interaction with the avirulent isolate. Thus, similar to infection promoted by the avirulent isolate on the susceptible genotype, the Mi-virulent isolate induced the JA-dependent pathway, which might promote tomato susceptibility during the compatible interaction with the homozygous (Mi/Mi) resistant line. These results have important consequences for the management of Mi resistance genes for ensuring sustainable tomato farming.

Natural variation in the root-knot nematode Meloidogyne incognita is problematic for breeding programs: populations possessing similar morphological characteristics can produce different reactions on the same host. We collected 30 widely dispersed M. incognita populations from protected pepper production systems in major pepper-growing regions of Israel and accurately identified their virulence characteristics by modified differential host test in a growth chamber on tomato, tobacco, cotton, melon, pepper, and peanut. Galling indices and reproduction were determined on the different hosts. All populations fit the published scheme for M. incognita race 2, except for reproduction on cotton plants by five out of 25 tested M. incognita populations, indicating host-range variations. Reaction of three genes that confer resistance to M. incognita—Me1, Me3 and N—to the collected populations was evaluated. Several M. incognita populations induced galling and reproduced successfully on pepper genotypes carrying Me3 and N, whereas plant resistance conferred by Me1 was more robust for all examined populations. Moreover, the effect of genetic background on Me1 resistance demonstrated a relative advantage of several genotypes in nematode infestations. Efficiency of Me3 under local nematode infestation was further studied with a homozygous line carrying two Me3 alleles. Reproduction of virulent populations on the homozygotes (Me3/Me3) and heterozygotes (Me3/Me3+) was similar, suggesting a limited quantitative effect of Me3. These results present the first characterization of host range, reproduction, and molecular aspects of M. incognita from Israel and highlight the importance of taking a multidimensional approach in pepper-breeding programs for resistance to M. incognita.

Plant-parasitic nematodes are extremely destructive pathogens with a cosmopolitan distribution and a host range that affects most crops. They are characterized by distinct parasitic lifestyles, e.g., as sedentary or migratory endo-or ectoparasites, resulting in high losses in yield and revenue. Possessing limited lipid metabolism, they produce one or two structurally unique classes of small α-helix-rich fatty acid and retinol binding (FAR) proteins that have no counterpart in other organisms. We investigated the sequence and structural characteristics of the FAR protein of the root-knot nematode Meloidogyne javanica (Mj-FAR-1) in comparison to other studied FAR proteins. Protein sequence analyses enabled phylogenetic clustering according to trophic groups and lifestyles. Bioinformatics analysis of the FAR protein sequences revealed ten likely core amino acids representing the trophic-group clustering. Clear modifications of four of these amino acids from less reactive (nonpolar, with aliphatic R group) to more reactive (positively or negatively charged R groups, or uncharged polar R groups) might distinguish free-living from parasitic nematode species. Structural predictions of the mature Mj-FAR-1 protein and its ligand-binding pockets, suggest that adaptation toward parasitism is associated with increased reactivity of the second pocket residues, as well as those on the protein surface. Subcellular localization of Mj-FAR-1 with or without its signal peptide was determined by Agrobacterium infiltration of N-terminal mCherrytagged protein into Nicotiana benthamiana leaves. Intact Mj-FAR-1 with its signal peptide was predominantly localized along the plasma membrane surrounding plant cells while removing the signal peptide resulted in additional localization within the cell nucleus. The nuclear localization agreed with in-silico analysis of the Mj-FAR-1 sequence and sheds new light on its function in manipulating the plant response. Our study provides the first basic structural information and subcellular localization of the plant-parasitic Mj-FAR-1 protein.Keywords Fatty acid and retinol binding (FAR) protein .Plant-parasitic nematode . Root-knot nematode . Meloiodgyne javanica . Parasitism . Effector protein Abbreviations MjMeloidogyne javanica FAR Fatty acid and retinol binding (protein) Eur J Plant Pathol

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Regenerative processes depend on the interpretation of signals to coordinate cell behaviors. The role of ubiquitin-mediated signaling in regeneration is not well understood. To investigate how ubiquitylation might specifically impact tissue regeneration, we are studying planarians that are capable of regenerating from nearly any injury using a population of stem cells. Here we used RNAi to screen RING/U-box E3 ubiquitin ligases that are highly expressed in planarian stem cells and stem cell progeny. RNAi screening identified nine genes with functions in regeneration, including the spliceosomal factor prpf19 and epigenetic regulator rnf2; based on roles in developmental processes, we further explored these two genes. We found that prpf19 was required for survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation. Because RNF2 is the catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), we also examined other cofactors of rnf2 and observed a striking phenotype of regional tissue misspecification in cbx and phc RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down rnf2 or phc and found that the set of genes differentially expressed were largely non-overlapping despite being predicted to function in the same complex. Using in situ hybridization, we showed that rnf2 regulates gene expression levels within a tissue type, whereas phc is necessary for the spatial restriction of gene expression. This work uncovered roles for RING/U-box E3 ligases in stem cell regulation and regeneration and identified differential gene targets for PRC1 factors required for maintaining cell-type-specific gene expression in planarians.