Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo

Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The crystal structure of C. elegans FAR-7 is the first structure of a FAR protein, and it exhibits a novel fold. It differs radically from the mammalian fatty acid-binding proteins and has two ligand binding pockets joined by a surface groove. The first can accommodate the aliphatic chain of fatty acids, whereas the second can accommodate the bulkier retinoids. In addition to demonstrating lipid binding by fluorescence spectroscopy, we present evidence that retinol binding is positively regulated by casein kinase II phosphorylation at a conserved site near the bottom of the second pocket. far-7::GFP (green fluorescent protein) expression shows that it is localized in the head hypodermal syncytia and the excretory cell but that this localization changes under starvation conditions. In conclusion, our study provides the basic structural and functional information for investigation of inhibitors of lipid binding by FAR proteins.     

Fergusobia/Fergusonina-induced Shoot Bud Gall Development on Melaleuca quinquenervia

Fergusobia nematodes and Fergusonina flies are mutualists that cause a variety of gall types on myrtaceous plant buds and young leaves. The biology of an isolate of the gall complex was studied in its native range in Australia for possible use in southern Florida as a biological control agent against the invasive broad-leaved paperbark tree, Melaleuca quinquenervia. Timed studies with caged Fergusonina flies on young branches of M. quinquenervia revealed that females are synovigenic with lifetime fecundities of 183 ± 42 (standard error; SE) eggs and longevities of 17 ± 2 days. None of the male flies but all dissected female flies contained parasitic female nematodes (range = 3-15), nematode eggs (12-112), and nematode juveniles (78-1,750). Female flies deposited eggs (34 ± 6; 8-77 per bud) and nematode juveniles (114 ± 15; 44-207 per bud) into bud apices within 15 days. Histological sections of shoot buds suggested that nematodes induce the formation of hypertrophied, uninucleate plant cells prior to fly larval eclosion. Enlarged size, granular cytoplasm, and enlarged nucleus and nucleolus characterized these cells, which appeared similar to those of other species galled by nematodes in the Anguinidae. Observations of ovipositional behavior revealed that female Fergusonina sp. create diagnostic oviposition scars. The presence of these scars may facilitate recognition of host use during specificity screening.     

Changes in Morphology of Trichostrongylus colubriformis Eggs and Juveniles Caused by Bacillus thuringiensis israelensis

Eggs and rhabditiform juveniles of the ruminant parasite Trichostrongylus colubriformis developed normally in Caenorhabditis briggsae Maintenance Medium. A toxin from a crystal-enriched preparation of the bacterium Bacillus thuringiensis israelensis was lethal to nematode eggs and juveniles within 24 hours and to eggs and juveniles after 24 hours of development. Treated eggs had refractive granules and development was arrested, whereas nontreated eggs developed normally. Eggs treated after 24 hours of development contained juveniles that were granulated, had esophageal derangements, and were moribund or dead. The ovicidal toxin from B. t. israelensis may facilitate microbial control of parasitic nematodes.     

Observations on the Foliar Nematode,Aphelenchoides besseyi,Infecting Tuberose and Rice in India

The foliar nematode Aphelenchoides besseyi causes white tip disease in rice (Oryza sativa L.) and floral malady in tuberose (Polianthes tuberosa L.). This nematode is widely distributed in the rice fields of many states of India, including West Bengal (WB), Andhra Pradesh (AP), Madhya Pradesh (MP) and Gujarat (GT). In order to generate information on intraspecific variations of A. besseyi as well as to confirm the identity of the nematode species infecting these important crops, morphological observation was undertaken of A. besseyi isolated from tuberose and rice from WB and rice from AP, MP and GT. The molecular study was only done for rice and tuberose populations from AP and WB. The variations were observed among the populations in the tail, esophageal and anterior regions, including the occurrence of four as well as six lateral lines in the lateral fields. The morphometrics of observed populations showed variations and those could be regarded as a consequence of host-induced or geographical variations. PCR amplification of the rDNA ITS 1 and 2 region of rice (AP) and tuberose (WB) populations of A. besseyi generated one fragment of approximately 830 bp, and the size of the ITS region was 788 bp and 791 bp for tuberose and rice population, respectively. Alignment of the two sequences showed almost 100% similarity. Blast analysis revealed a very high level of similarity of both the Indian strains to a Russian population. The Indian and Russian strains could be differentiated using restriction enzyme Bccl. Host tests revealed that rice (cv. IET 4094), oat (cv. OS-6) and teosinte (cv. TL-1) showed a typical distortion due to the infection of A. besseyi. Five germplasm lines of oat showed no infection of the nematode under field conditions. Local cultivars of onion, maize, chrysanthemum, gladiolus, and Sorghum halepense were also not infected by A. besseyi.     

Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.     

Aboveground herbivory induced jasmonates disproportionately reduce plant reproductive potential by facilitating root nematode infestation

Different plant feeders, including insects and parasitic nematodes, can influence each other by triggering systemic changes in their shared host plants. In most cases, however, the underlying mechanisms are unclear, and the consequences for plant fitness are not well understood. We studied the interaction between leaf feeding Manduca sexta caterpillars and root parasitic nematodes in Nicotiana attenuata. Simulated M. sexta attack increased the abundance of root parasitic nematodes in the field and facilitated Meloidogyne incognita reproduction in the glasshouse. Intact jasmonate biosynthesis was found to be required for both effects. Flower counts revealed that the jasmonate‐dependent facilitation of nematode infestation following simulated leaf attack reduces the plant's reproductive potential to a greater degree than would be expected from the additive effects of the individual stresses. This work reveals that jasmonates mediate the interaction between a leaf herbivore and root parasitic nematodes and illustrates how plant‐mediated interactions can alter plant's reproductive potential. The selection pressure resulting from the demonstrated fitness effects is likely to influence the evolution of plant defense traits in nature.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.     

The Cyst Nematode SPRYSEC Protein RBP-1 Elicits Gpa2- and RanGAP2-Dependent Plant Cell Death

Plant NB-LRR proteins confer robust protection against microbes and metazoan parasites by recognizing pathogen-derived avirulence (Avr) proteins that are delivered to the host cytoplasm. Microbial Avr proteins usually function as virulence factors in compatible interactions; however, little is known about the types of metazoan proteins recognized by NB-LRR proteins and their relationship with virulence. In this report, we demonstrate that the secreted protein RBP-1 from the potato cyst nematode Globodera pallida elicits defense responses, including cell death typical of a hypersensitive response (HR), through the NB-LRR protein Gpa2. Gp-Rbp-1 variants from G. pallida populations both virulent and avirulent to Gpa2 demonstrated a high degree of polymorphism, with positive selection detected at numerous sites. All Gp-RBP-1 protein variants from an avirulent population were recognized by Gpa2, whereas virulent populations possessed Gp-RBP-1 protein variants both recognized and non-recognized by Gpa2. Recognition of Gp-RBP-1 by Gpa2 correlated to a single amino acid polymorphism at position 187 in the Gp-RBP-1 SPRY domain. Gp-RBP-1 expressed from Potato virus X elicited Gpa2-mediated defenses that required Ran GTPase-activating protein 2 (RanGAP2), a protein known to interact with the Gpa2 N terminus. Tethering RanGAP2 and Gp-RBP-1 variants via fusion proteins resulted in an enhancement of Gpa2-mediated responses. However, activation of Gpa2 was still dependent on the recognition specificity conferred by amino acid 187 and the Gpa2 LRR domain. These results suggest a two-tiered process wherein RanGAP2 mediates an initial interaction with pathogen-delivered Gp-RBP-1 proteins but where the Gpa2 LRR determines which of these interactions will be productive.     

Identification of neuropeptides,flp-1 and flp-12 targeting neuromuscular system of rice root knot nematode (RRKN) Meloidogyne graminicola

Root-knot nematodes (RKNs), Meloidogyne spp, are found in all temperate and tropical areas, and are among the most damaging plant pathogens worldwide. M. graminincola is an economically important root parasite on upland, lowland and deepwater rice. FMRFamide-like peptides (FLPs) play significant role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time, we have cloned and characterized two neuropeptide genes (flp-1 and flp-12) from the cDNA of preparasitic second stage juveniles of M. graminicola. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting function as an extra-cellular protein. We have found highly conserved motif LFRGR in flp-1. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.     

Plant Parasitic Nematodes Associated with Leatherleaf Fern

Seven species of plant parasitic nematodes were found to be associated with leatherleaf fern (Rumohra adiantiformis) in central Florida. Of these, Pratylenchus penetrans, Tylenchorhynchus claytoni, and Criconemoides curvatum were commonly encountered. Nematode communities generally included two or three species of plant parasitic nematodes, with greatest diversity in nematode species occurring in ferneries shaded by oak trees. Species diversity was not correlated with fernery age. Leatherleaf fern was tolerant of P. penetrans and T. claytoni in microplot tests.     

The Aphelenchoides genomes reveal substantial horizontal gene transfers in the last common ancestor of free-living and major plant-parasitic nematodes

Aphelenchoides besseyi is a plant-parasitic nematode (PPN) in the family Aphelenchoididae capable of infecting more than 200 plant species. A. besseyi is also a species complex with strains exhibiting varying pathogenicity to plants. We present the genome and annotations of six Aphelenchoides species, four of which belonged to the A. besseyi species complex. Most Aphelenchoides genomes have a size of 44.7–47.4 Mb and are among the smallest in clade IV, with the exception of A. fujianensis, which has a size of 143.8 Mb and is one of the largest. Phylogenomic analysis successfully delimited the species complex into A. oryzae and A. pseudobesseyi and revealed a reduction of transposon elements in the last common ancestor of Aphelenchoides. Synteny analyses between reference genomes indicated that three chromosomes in A. besseyi were derived from fission and fusion events. A systematic identification of horizontal gene transfer (HGT) genes across 27 representative nematodes allowed us to identify two major episodes of acquisition corresponding to the last common ancestor of clade IV or major PPNs, respectively. These genes were mostly lost and differentially retained between clades or strains. Most HGT events were acquired from bacteria, followed by fungi, and also from plants; plant HGT was especially prevalent in Bursaphelenchus mucronatus. Our results comprehensively improve the understanding of HGT in nematodes.     

Structural and binding studies of phosphopantetheine adenylyl transferase from Acinetobacter baumannii

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4′-phosphopantetheine (pPant) yielding 3′- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway_. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri‑sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10⁻⁸ M and 10⁻⁵ M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Å resolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.     

Reproduction and Development of Meloidogyne incognita and M. javanica on Guardian Peach Rootstock

Guardian peach rootstock was evaluated for susceptibility to Meloidogyne incognita race 3 (Georgia-peach isolate) and M. javanica in the greenhouse. Both commercial Guardian seed sources produced plants that were poor hosts of M. incognita and M. javanica. Reproduction as measured by number of egg masses and eggs per plant, eggs per egg mass, and eggs per gram of root were a better measure of host resistance than number of root galls per plant. Penetration, development, and reproduction of M. incognita in Guardian (resistant) and Lovell (susceptible) peach were also studied in the greenhouse. Differences in susceptibility were not attributed to differential penetration by the infectivestage juveniles (J2) or the number of root galls per plant. Results indicated that M. incognita J2 penetrated Guardian roots and formed galls, but that the majority of the nematodes failed to mature and reproduce.     

Top 10 plant‐parasitic nematodes in molecular plant pathology

The aim of this review was to undertake a survey of researchers working with plant‐parasitic nematodes in order to determine a ‘top 10’ list of these pathogens based on scientific and economic importance. Any such list will not be definitive as economic importance will vary depending on the region of the world in which a researcher is based. However, care was taken to include researchers from as many parts of the world as possible when carrying out the survey. The top 10 list emerging from the survey is composed of: (1) root‐knot nematodes (Meloidogyne spp.); (2) cyst nematodes (Heterodera and Globodera spp.); (3) root lesion nematodes (Pratylenchus spp.); (4) the burrowing nematode Radopholus similis; (5) Ditylenchus dipsaci; (6) the pine wilt nematode Bursaphelenchus xylophilus; (7) the reniform nematode Rotylenchulus reniformis; (8) Xiphinema index (the only virus vector nematode to make the list); (9) Nacobbus aberrans; and (10) Aphelenchoides besseyi. The biology of each nematode (or nematode group) is reviewed briefly.     

A Nematode Calreticulin,Rs-CRT,Is a Key Effector in Reproduction and Pathogenicity of Radopholus similis

Radopholus similis is a migratory plant-parasitic nematode that causes severe damage to many agricultural and horticultural crops. Calreticulin (CRT) is a Ca²⁺-binding multifunctional protein that plays key roles in the parasitism, immune evasion, reproduction and pathogenesis of many animal parasites and plant nematodes. Therefore, CRT is a promising target for controlling R. similis. In this study, we obtained the full-length sequence of the CRT gene from R. similis (Rs-crt), which is 1,527-bp long and includes a 1,206-bp ORF that encodes 401 amino acids. Rs-CRT and Mi-CRT from Meloidogyne incognita showed the highest similarity and were grouped on the same branch of the phylogenetic tree. Rs-crt is a multi-copy gene that is expressed in the oesophageal glands and gonads of females, the gonads of males, the intestines of juveniles and the eggs of R. similis. The highest Rs-crt expression was detected in females, followed by juveniles, eggs and males. The reproductive capability and pathogenicity of R. similis were significantly reduced after treatment with Rs-crt dsRNA for 36 h. Using plant-mediated RNAi, we confirmed that Rs-crt expression was significantly inhibited in the nematodes, and resistance to R. similis was significantly improved in transgenic tomato plants. Plant-mediated RNAi-induced silencing of Rs-crt could be effectively transmitted to the F2 generation of R. similis; however, the silencing effect of Rs-crt induced by in vitro RNAi was no longer detectable in F1 and F2 nematodes. Thus, Rs-crt is essential for the reproduction and pathogenicity of R. similis.