RESUMEN
Understanding the transcriptional regulatory characteristics throughout the embryogenesis of plant-parasitic nematodes is crucial for elucidating their developmental processes' uniqueness. However, a challenge arises due to the lack of suitable technical methods for synchronizing the age of plant-parasitic nematodes embryo, it is difficult to collect detailed transcriptome data at each stage of embryonic development. Here, we recorded the 11 embryonic developmental time-points of endophytic nematode Meloidogyne incognita (isolated from Wuhan, China), Heterodera glycines (isolated from Wuhan, China), and Ditylenchus destructor (isolated from Jinan, China) species, and constructed transcriptome datasets of single embryos of these three species utilizing low-input smart-seq2 technology. The datasets encompassed 11 complete embryonic development stages, including Zygote, 2-cell, 4-cell, 8-cell, 24-44 cell, 64-78 cell, Comma, 1.5-fold, 2-fold, Moving, and L1, each stage generated four to five replicates, resulting in a total of 162 high-resolution transcriptome libraries. This high-resolution cross-species dataset serves as a crucial resource for comprehending the embryonic developmental properties of plant-parasitic nematodes and for identifying functional regulatory genes during embryogenesis.
Asunto(s)
Plantas , Transcriptoma , Tylenchoidea , Animales , Desarrollo Embrionario/genética , Tylenchoidea/embriología , Tylenchoidea/genética , Plantas/parasitologíaRESUMEN
BACKGROUND: Egg hatching in Meloidogyne incognita is a highly regulated developmental event and is strictly correlated with temperature. It has been demonstrated that exposure of M. incognita eggs to low temperature seriously affects their embryonic development. On the other hand, clear evidence has shown that M. incognita is able to overwinter at subzero soil temperatures in certain open fields. Therefore, subtle physiological and genetic adaptations may occur in M. incognita to minimize freezing injuries. OBJECTIVE: A growing body of evidence indicates that cold acclimation plays a large role in an individual organism's ability to cope with freezing-induced cellular damage. Given the decreasing temperatures in late autumn or early winter, we hypothesize that natural cold acclimation occurring during these periods may assist M. incognita in overwintering. METHODS: Transcriptomic analysis and functional enrichment analyses were used to identify and annotate differentially expressed genes (DEGs) in acclimated eggs. The expression of DEGs involved in signal transduction and protein assembly was subsequently validated by reverse transcription quantitative PCR (RT-qPCR). RESULTS: Relatively long-term preacclimation at 4 °C significantly accelerated the hatching of M. incognita eggs that were subjected to freezing at - 1 °C. Using a transcriptomic approach, we further identified 686 and 460 up- and downregulated transcripts, respectively, in pre-cold-acclimated eggs. Additionally, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology annotations for functional enrichment analyses of the differentially expressed genes (DEGs). CONCLUSION: The phenomenon in which M. incognita safely overwinters at subzero soil temperatures in certain areas may be attributed to the natural cold acclimation occurring in late autumn. Here, the identification of DEGs between acclimated and nonacclimated eggs will provide us with promising directions for future studies on the mechanisms of M. incognita freezing tolerance.
Asunto(s)
Aclimatación , Respuesta al Choque por Frío , Transcriptoma , Tylenchoidea/genética , Animales , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Óvulo/metabolismo , Tylenchoidea/embriología , Tylenchoidea/metabolismoRESUMEN
BACKGROUND: Many parasites regulate their development to synchronize their life cycle with a compatible host. The parasitic nematode Heterodera glycines displays incomplete host-mediated hatching behavior wherein some H. glycines individuals hatch only in the presence of a host-derived cue while others hatch in water alone. Furthermore, H. glycines shows variable hatching behavior based on oviposition location. The mechanisms regulating this hatching variability are unknown. In this study, we established a detailed timeline of the H. glycines pre-hatch development from early embryogenesis to the pre-hatched J2. These descriptive data were then used to test hypotheses regarding the effect of host stimulus and oviposition location on pre-hatch development. RESULTS: We found that H. glycines develops from a single-cell egg to a fully formed J2 in approximately 172 hours. The stylet-based mouthpart, which is used to pierce the eggshell during hatching, is not completely formed until late in pre-hatch J2 development and is preceded by the formation of stylet protractor muscles. We also found that the primary motor nervous system of H. glycines did not complete development until late in pre-hatch J2 development. These data suggest possible structural requirements for H. glycines hatching. As expected, exposure of H. glycines eggs to host-derived cues increased the percentage of nematodes that hatched. However, exposure to hatching cues did not affect pre-hatch development. Similarly, we found no obvious differences in the pre-hatch developmental timeline between eggs laid in an egg sac or retained within the mother. CONCLUSIONS: The pattern of early embryonic development in H. glycines was very similar to that recently described in the related parasitic nematode Meloidogyne incognita. However, the speed of H. glycines pre-hatch development was approximately three times faster than reported for M. incognita. Our results suggest that hatching stimulants do not affect embryogenesis itself but only influence the hatching decision once J2 development is complete. Similarly, the oviposition location does not alter the rate of embryogenesis. These results provide insight into the primary survival mechanism for this important parasite.
Asunto(s)
Glycine max/parasitología , Tylenchoidea/embriología , Animales , Interacciones Huésped-Parásitos , Nódulos de las Raíces de las Plantas/parasitologíaRESUMEN
BACKGROUND: Detailed descriptions of the early development of parasitic nematodes are seldom available. The embryonic development of the plant-parasitic nematode Meloidogyne incognita was studied, focusing on the early events. RESULTS: A fixed pattern of repeated cell cleavages was observed, resulting in the appearance of the six founder cells 3 days after the first cell division. Gastrulation, characterized by the translocation of cells from the ventral side to the center of the embryo, was seen 1 day later. Approximately 10 days after the first cell division a rapidly elongating two-fold stage was reached. The fully developed second stage juvenile hatched approximately 21 days after the first cell division. CONCLUSIONS: When compared to the development of the free-living nematode Caenorhabditis elegans, the development of M. incognita occurs approximately 35 times more slowly. Furthermore, M. incognita differs from C. elegans in the order of cell divisions, and the early cleavage patterns of the germ line cells. However, cytoplasmic ruffling and nuclear migration prior to the first cell division as well as the localization of microtubules are similar between C. elegans and M. incognita.
Asunto(s)
Desarrollo Embrionario , Raíces de Plantas/parasitología , Tylenchoidea/embriología , Animales , División Celular , Linaje de la Célula , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , ADN/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Femenino , Gastrulación , Óvulo/citología , Filogenia , Tylenchoidea/citologíaRESUMEN
Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control. On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (si)RNA-soaked M. incognita eggs. Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH(2))-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal. Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality. To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s, which recovered within 24h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M. incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-eri-1 transcript which encodes an RNAi-inhibiting siRNA exonuclease. We observe a marked up-regulation of Mi-eri-1 transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RNAi-based reverse genetic screens in plant parasitic nematodes.
Asunto(s)
Diferenciación Celular , Óvulo/citología , Interferencia de ARN , ARN Interferente Pequeño/genética , Tylenchoidea/embriología , Tylenchoidea/genética , Animales , Femenino , Técnicas de Silenciamiento del Gen , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Masculino , Óvulo/enzimología , Óvulo/crecimiento & desarrollo , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Tylenchoidea/enzimología , Tylenchoidea/fisiologíaRESUMEN
Anhydrobiotic (i.e., life without water) organisms are known to produce group 3 late embryogenesis abundant (G3LEA) proteins during adaptation to severely water-deficient conditions. Their primary amino acid sequences are composed largely of loosely conserved 11-mer repeat units. However, little information has been obtained for the structural and functional roles of these repeat units. In this study, we first explore the consensus sequences of the 11-mer repeat units for several native G3LEA proteins originating from anhydrobiotic organisms among insects (Polypedilum vanderplanki), nematodes, and plants. Next, we synthesize four kinds of model peptides (LEA models), each of which consists of four or two repeats of the 11-mer consensus sequences for each of the three organisms. The structural and thermodynamic properties of the LEA models were examined in solution, in dehydrated and rehydrated states, and furthermore in the presence of trehalose, since a great quantity of this sugar is known to be produced in the dried cells of most anhydrobiotic organisms. The results of Fourier transform infrared (FTIR) spectroscopic measurements indicate that all of the LEA models transform from random coils to alpha-helical coiled coils on dehydration and return to random coils again on rehydration, both with and without trehalose. In contrast, such structural changes were never observed for a control peptide with a randomized amino acid sequence. Furthermore, our differential scanning calorimetry (DSC) measurements provide the first evidence that the above 11-mer motif-containing peptides themselves vitrify with a high glass transition temperature (>100 degrees C) and a low enthalpy relaxation rate. In addition, they play a role in reinforcing the glassy matrix of the coexisting trehalose. On the basis of these results, we discuss the underlying mechanism of G3LEA proteins as desiccation stress protectants.
Asunto(s)
Adaptación Fisiológica , Deshidratación , Vidrio/química , Proteínas del Helminto/síntesis química , Proteínas de Insectos/síntesis química , Biosíntesis de Péptidos/fisiología , Proteínas de Plantas/síntesis química , Secuencias de Aminoácidos , Animales , Brassica napus/embriología , Proteínas de Caenorhabditis elegans/síntesis química , Secuencia de Consenso , Dípteros/embriología , Gossypium/embriología , Estructura Secundaria de Proteína , Secuencias Repetitivas de Aminoácido , Trehalosa/síntesis química , Tylenchoidea/embriologíaRESUMEN
An efficient technique was developed for separating early and late stages of embryonic development in eggs of Heterodera glycines. This technique takes advantage of density changes that occur during embryogenesis in the developing embryo and egg to partition the egg within a sucrose step gradient. Sorted samples of eggs separated with 82% enrichment for pre-gastrula early embryos and 93% enrichment for first and second stage unhatched juveniles as late embryos. Subpopulations enriched for either developmental stage are available for use in generating stage-specific cDNA libraries, normalization of subpopulations to synchronize development, biochemical characterization, and many other uses.