Insights into Novel Non-Fumigant Nematicides: Physiological and Cellular Responses of Meloidogyne Incognita and Other Plant-Parasitic Nematodes

Plant-parasitic nematodes (PPN) pose a severe threat to crop production with the economic losses due to nematode parasitism in excessive of US$ 80 billion each year. Meloidogyne incognita, a globally distributed pest with a diverse host range, contributes significantly to this economic loss. The most reliable way to manage PPN is often through chemical controls, however, many fumigant and non-fumigant chemical controls are being phased out due to their harmful effects to both applicators and the environment. As new nematicide enter the market, there is a need for basic information on the toxicity of these new nematicides to PPN including M. incognita. The aim of this dissertation research is to determine foundational knowledge of how novel nematicides, fluopyram, fluensulfone, and fluazaindolizine affect M. incognita movement and motility, reproduction, and transcription in comparison with the traditional nematicide oxamyl. In Chapter 2, 24hr dose-response curves were established for M. incognita second-stage juveniles (J2) exposed to fluopyram, fluensulfone, fluazaindolizine, and oxamyl. Using the dose-response curves, sublethal doses were obtained to examine their effect on M. incognita J2 motility and fecundity on a susceptible host. Fluopyram was determined to be the most toxic nematicide to J2, but did not reduce nematode reproduction at sublethal doses. Fluensulfone and oxamyl both had similar levels of toxicity at 24-hrs and fluazaindolizine was the least toxic. Oxamyl, fluensulfone, and fluazaindolizine all were able to suppress nematode reproduction even at sublethal doses. Motility was unaffected by any compound, however, the level of activity of motile nematodes varied amongst compounds. The novel nematicide fluazaindolizine has been shown to be PPN specific in its toxicity, necessitating the exploration of what PPN genera are susceptible to this compound and how might populations within a species vary in their susceptibility to this compound. Therefore, in Chapter 3 dose-response curves were generated for populations of 3 different Meloidogyne species along with Globodera ellingtonae, Xiphinema americanum, and Pratylenchus neglectus, and P. penetrans. In addition to dose-response curves, the effects of a pre-exposure to fluazaindolizine on reproduction of 3 different Meloidogyne species was also examined. Sensitivity to fluazaindolizine varied >10-fold within species of Meloidogyne, but this did not translate to a change in the ability of fluazaindolizine to suppress reproduction. Pratylenchus species were found not to be susceptible to fluazaindolizine, but Globodera ellingtonae, Xiphinema americanum were; expanding the range of PPN known to be sensitive to this compound. Chapters 4 and 5 examined the changes in M. incognita transcription in response to 24-hr exposure to fluopyram, fluensulfone, fluazaindolizine, and oxamyl. The aim of these two chapters was to provide context for the physiological responses seen in M. incognita to nematicides, determine potential modes-of-action for fluensulfone and fluazaindolizine, and gain greater understanding how nematicides are toxic to nematodes. Transcriptional changes were examined of various components of cellular function including cellular detoxification, fatty-acid retinoid-binding proteins, transcriptional regulators of oxidative stress, beta-fatty acid oxidation, acetylcholine neuron potential, the citric acid cycle, and oxidative phosphorylation. Although the most toxic nematicide, fluopyram had limited impacts on expression of cellular detoxification or other stress-responses, but expression patterns in the citric acid cycle and oxidative phosphorylation pathways support the mode-of-action, succinate dehydrogenase inhibitor, described in fungi. Fluensulfone and fluazaindolizine both resulted in robust transcriptional responses with 1,208 and 2,611 DE genes, respectively. These compounds had strong impacts on cellular detoxification, causing differential regulation of transcription factors and mixed regulation of genes in the detox pathway. Expression data indicated two potential pathways of interest for mode-of-actions: β-fatty acid oxidation pathway and 2-Oxoglutarate dehydrogenase of the TCA cycle for fluensulfone and fluazaindolizine, respectively. Oxamyl negatively affected expression of cellular detoxification, but expression changes in acetylcholine neuron components also provided strong evidence to support its functionality as an acetylcholinesterase inhibitor. Overall, the research in this dissertation aimed at understanding nematicide toxicity to PPN from the organismal to the cellular level to support more well-informed use of these nematicides in managing PPN

The plant‐parasitic cyst nematode effector GLAND4 is a DNA‐binding protein

Cyst nematodes are plant pathogens that infect a wide range of economically important crops. One parasitic mechanism employed by cyst nematodes is the production and in planta delivery of effector proteins to modify plant cells and suppress defenses to favor parasitism. This study focused on GLAND4, an effector of Heterodera glycines and H. schachtii, the soybean and sugar beet cyst nematodes, respectively. We showed that GLAND4 is recognized by the plant cellular machinery and is transported to the plant nucleus, an organelle where little is known about plant nematode effector functions. We showed that GLAND4 has DNA-binding ability and repressed reporter gene expression in a plant transcriptional assay. One DNA-fragment that bound to GLAND4 was localized in an Arabidopsis chromosomal region associated with the promoters of two lipid transfer protein (LTP) genes. These LTPs have known defense functions and are downregulated in the nematode feeding site. When expressed in Arabidopsis, the presence of GLAND4 caused downregulation of the two LTP genes in question, which was associated also with increased susceptibility to the plant-pathogenic bacterium Pseudomonas syringae. Furthermore, overexpression of one of the LTP genes reduced plant susceptibility to H. schachtii and P. syringae, confirming that LTP repression likely suppresses plant defenses. This study made GLAND4 one of a small subset of characterized plant nematode nuclear effectors and identified GLAND4 as the first DNA-binding plantparasitic nematode effector

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis.

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode

The plant‐parasitic cyst nematode effector GLAND4 is a DNA‐binding protein

Cyst nematodes are plant pathogens that infect a wide range of economically important crops. One parasitic mechanism employed by cyst nematodes is the production and in planta delivery of effector proteins to modify plant cells and suppress defenses to favor parasitism. This study focused on GLAND4, an effector of Heterodera glycines and H. schachtii, the soybean and sugar beet cyst nematodes, respectively. We showed that GLAND4 is recognized by the plant cellular machinery and is transported to the plant nucleus, an organelle where little is known about plant nematode effector functions. We showed that GLAND4 has DNA-binding ability and repressed reporter gene expression in a plant transcriptional assay. One DNA-fragment that bound to GLAND4 was localized in an Arabidopsis chromosomal region associated with the promoters of two lipid transfer protein (LTP) genes. These LTPs have known defense functions and are downregulated in the nematode feeding site. When expressed in Arabidopsis, the presence of GLAND4 caused downregulation of the two LTP genes in question, which was associated also with increased susceptibility to the plant-pathogenic bacterium Pseudomonas syringae. Furthermore, overexpression of one of the LTP genes reduced plant susceptibility to H. schachtii and P. syringae, confirming that LTP repression likely suppresses plant defenses. This study made GLAND4 one of a small subset of characterized plant nematode nuclear effectors and identified GLAND4 as the first DNA-binding plantparasitic nematode effector.This is the peer reviewed version of the following article: Barnes, Stacey N., Catherine L. Wram, Melissa G. Mitchum, and Thomas J. Baum. "The plant‐parasitic cyst nematode effector GLAND4 is a DNA‐binding protein." Molecular plant pathology 19, no. 10 (2018): 2263-2276, which has been published in final form at doi: 10.1111/mpp.12697. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.</p

Unraveling Microbial Endosymbiosis Dynamics in Plant-Parasitic Nematodes with a Genome Skimming Strategy

Bacterial endosymbionts, in genera Wolbachia and Cardinium, infect various arthropods and some nematode groups. Manipulating these microbial symbionts presents a promising biocontrol strategy for managing disease-causing parasites. However, the diversity of Wolbachia and Cardinium in nematodes remains unclear. This study employed a genome skimming strategy to uncover their occurrence in plant-parasitic nematodes, analyzing 52 populations of 12 species. A metagenome analysis revealed varying endosymbiont genome content, leading to the categorization of strong, weak, and no evidence for endosymbiont genomes. Strong evidence for Wolbachia was found in five populations, and for Cardinium in one population, suggesting a limited occurrence. Strong Wolbachia evidence was noted in Pratylenchus penetrans and Radopholus similis from North/South America and Africa. Heterodera glycines from North America showed strong Cardinium evidence. Weak genomic evidence for Wolbachia was observed in Globodera pallida, Meloidogyne incognita, Rotylenchus reniformis, Pratylechus coffeae, Pratylenchus neglectus, and Pratylenchus thornei; for Cardinium was found in G. pallida, R. reniformis and P. neglectus; 27/52 populations exhibited no endosymbiont evidence. Wolbachia and Cardinium presence varied within nematode species, suggesting non-obligate mutualism. Wolbachia and Cardinium genomes differed among nematode species, indicating potential species-specific functionality. This study advances knowledge of plant-parasitic nematode–bacteria symbiosis, providing insights for downstream eco-friendly biocontrol strategies

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode.This article is published as Vieira P, Myers RY, Pellegrin C, Wram C, Hesse C, Maier TR, et al. (2021) Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis. PLoS Pathog 17(11): e1010036. https://doi.org/10.1371/journal.ppat.1010036. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication