Genome-Guided Reanalysis of Root-Knot Nematode Meloidogyne Incognita Esophageal Gland Cell-Enriched Sequence Tag Libraries: A Resource for the Discovery of Novel Effectors

HomePhytoFrontiers™Ahead of PrintGenome-Guided Reanalysis of Root-Knot Nematode Meloidogyne incognita Esophageal Gland Cell-Enriched Sequence Tag Libraries: A Resource for the Discovery of Novel Effectors Previous RESOURCE ANNOUNCEMENT OPENOpen Access licenseGenome-Guided Reanalysis of Root-Knot Nematode Meloidogyne incognita Esophageal Gland Cell-Enriched Sequence Tag Libraries: A Resource for the Discovery of Novel EffectorsMelissa G. Mitchum, Raquel O. Rocha, Guozhong Huang, Tom R. Maier, Thomas J. Baum, and Richard S. HusseyMelissa G. Mitchum†Corresponding author: M. G. Mitchum; E-mail Address: melissa.mitchum@uga.eduhttps://orcid.org/0000-0002-9086-6312Department of Plant Pathology and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, GA 30602Search for more papers by this author, Raquel O. Rochahttps://orcid.org/0000-0003-3264-2385Department of Plant Pathology and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, GA 30602Search for more papers by this author, Guozhong HuangDepartment of Plant Pathology and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, GA 30602Search for more papers by this author, Tom R. MaierDepartment of Plant Pathology, Entomology and Microbiology, Iowa State University, Ames, IA 50011Search for more papers by this author, Thomas J. Baumhttps://orcid.org/0000-0001-9241-3141Department of Plant Pathology, Entomology and Microbiology, Iowa State University, Ames, IA 50011Search for more papers by this author, and Richard S. HusseyDepartment of Plant Pathology and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, GA 30602Search for more papers by this authorAffiliationsAuthors and Affiliations Melissa G. Mitchum1 † Raquel O. Rocha1 Guozhong Huang1 Tom R. Maier2 Thomas J. Baum2 Richard S. Hussey1 1Department of Plant Pathology and Institute of Plant Breeding, Genetics, and Genomics, University of Georgia, Athens, GA 30602 2Department of Plant Pathology, Entomology and Microbiology, Iowa State University, Ames, IA 50011 Published Online:9 Jan 2023https://doi.org/10.1094/PHYTOFR-09-22-0099-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleMost plant-parasitic nematode species (PPNs) use a hollow, protrusible stylet to secrete effector proteins originating from three large, secretory esophageal gland cells (two subventral and one dorsal) to parasitize host plants (Hussey 1989). These stylet-secreted effector proteins are packaged into secretory granules within the gland cells, marking them for secretion from the nematode stylet (Mitchum et al. 2013). Sedentary endoparasitic nematodes such as root-knot nematodes (RKNs) (Meloidogyne spp.) secrete effector proteins to aid penetration and migration through root tissues and facilitate the establishment and maintenance of a permanent feeding site within the vascular cylinder of the root (Caillaud et al. 2008). The identification and functional characterization of stylet-secreted effectors, therefore, is not only critical for understanding the complex molecular interactions between the nematode and its host but also opens the door for devising novel strategies for nematode control by interfering with key steps in the infection process. Thus, identifying genes encoding stylet-secreted effector proteins has been an intense focus for researchers.Microaspiration was the first approach employed to isolate gland-cell-enriched RNA for molecular analyses, specifically the construction and sequencing of gland-enriched complementary DNA (cDNA) libraries (Gao et al. 2001; Wang et al. 2001). The coupling of expressed sequence tag (EST) sequencing and in situ hybridization led to the discovery of dozens of candidate stylet-secreted effectors, opening the door to a new era of research in molecular plant–nematode interactions (Gao et al. 2003; Huang et al. 2003, 2004). Now, decades later, the ‘omics’ era has ushered in the release of annotated plant-parasitic nematode genomes (Abad et al. 2008; Eves-van den Akker et al. 2016; Masonbrink et al. 2021; Opperman et al. 2008), life-stage-specific transcriptomes (Blanc-Mathieu et al. 2017), genome-wide effector prediction algorithms (Da Rocha et al. 2021; Grynberg et al. 2020), and the identification of cis-regulatory motifs associated with effector gene expression (Eves-van den Akker et al. 2016). Hundreds of candidate effectors preceded by a signal peptide (SP) for secretion have been identified from multiple genera; however, determining whether these genes are expressed exclusively within the esophageal gland cells requires further evidence of in situ localization within the gland cells.The southern RKN Meloidogyne incognita is one of the most damaging pathogens to crop production worldwide (Jones et al. 2013). Comprehensive mining of the available annotated M. incognita genome for putative secreted proteins (PSPs) has identified 2,811 proteins (Grynberg et al. 2020). Of these, 2,146 PSPs contained a predicted effector motif (Vens et al. 2011). However, it is impossible to know if these proteins are being produced in the nematode's esophageal gland cells without cell-type-specific analyses. More recently, conserved cis-regulatory elements enriched in the promoters of gland-expressed effectors have been identified and offer an additional filtering step for computational effector prediction pipelines (Da Rocha et al. 2021; Espada et al. 2018; Eves-van den Akker et al. 2016). In situ hybridization (De Boer et al. 1998) and immunolocalization studies (Hussey et al. 1990; Smant et al. 1998) are the gold standard for confirming esophageal gland expression; however, these procedures are labor intensive and impractical to apply to thousands of potential candidates. Consequently, researchers have developed techniques to microaspirate gland cell contents (Gao et al. 2003; Huang et al. 2003; Rutter et al. 2014) or isolate intact gland cells (Maier et al. 2013, 2021; Vieira et al. 2020) for molecular analyses. Thus, we reasoned that there might be value in carrying out a reanalysis of ESTs generated from sequencing M. incognita gland-enriched cDNA libraries (Huang et al. 2003, 2004). At the time the gland-enriched cDNA libraries were sequenced in the early 2000s, no nematode genomes were available. The sequences identified in these cDNA libraries have a higher likelihood of being expressed in the glands and, therefore, could help researchers to prioritize candidate effector gene sequences mined from the genome.Two sets of M. incognita ESTs previously generated from sequencing microaspirated gland-cell-enriched cDNA libraries were queried using Blast+ or BlastN against the version 3.0 M. incognita genome-annotated messenger RNA (mRNA) sequences to obtain the corresponding M. incognita gene identities (Minc gene IDs). Library 1 (migfha1_35) represented 1,964 sequences generated by Huang et al. (2003). Library 2 (migfhb1_16) represented 1,321 sequences generated by Huang et al. (2004). The BlastN output was parsed with grep and saved to an Excel file. Of 3,285 ESTs queried, 2,201 (67%) had high homology to the predicted mRNA sequences, which ranged from 77 to 100% identity (Fig. 1A). Many of the Minc mRNA sequences were homologous to two or more of the ESTs. Further analysis revealed that the ESTs in question were derived from the same mRNA, albeit having slightly different sequence lengths. When the ESTs with homology to a single Minc mRNA sequence were accounted for, 1,478 unique Minc sequence IDs, in total, were identified. We then proceeded with a filtering pipeline to identify potentially novel M. incognita stylet-secreted effector proteins (Fig. 1A). We first cross-referenced this list with the list of 2,811 PSPs identified by Grynberg et al. (2020) from the M. incognita protein set using SignalP v4 (available at https://doi.org/10.15454/JCYZDI). This analysis reduced the list of putative esophageal gland effector gene candidates to 127 (Fig. 1A; Supplementary Table S1), of which 57 were included in the list of 1,331 secreted proteins specific to PPNs. All but three of these were among the predicted 1,040 PPN-specific M. incognita candidate effector proteins predicted by Grynberg et al. (2020) based on the presence of a N-terminal SP, lack of a transmembrane domain (TMD), and a MERCI effector motif (i.e., harbors at least one motif known to be enriched in effector proteins in the 100 first amino acids) (Vens et al. 2011) (available at https://doi.org/10.15454/CSTXU2). Of the 127 candidates, 12 were found in the list of the 457 predicted dorsal gland (DG) effector proteins based on the presence of a predicted DOG box (available at https://doi.org/10.15454/2O77EF) (Da Rocha et al. 2021). Twenty-seven of the identified PPN-specific effectors were either rediscovered effectors or shared high sequence similarity with previously published effectors (Huang et al. 2003, 2004; Nguyen et al. 2018). The remaining 30 candidates identified here represent newly discovered novel M. incognita effectors with potential expression in the esophageal gland cells (Table 1). None of these genes were among the list of M. incognita genes with evidence for gland cell-specific expression via in situ hybridization (https://doi.org/10.15454/P5YIGX) (Da Rocha et al. 2021). The 30 novel effector candidates were grouped based on their assigned developmental expression (Blanc-Mathieu et al. 2017) cluster according to Da Rocha et al. (2021) (https://doi.org/10.15454/YM2DHE). Three were not assigned to an expression cluster. Of the remaining 27 novel effector candidates, 8 were in expression cluster C, 6 in expression cluster D, 4 in expression cluster G, and 9 in expression cluster H. Of the 30 novel effector candidates, 3 had a predicted cis-acting DOG box sequence in their promoters. In all, 25 of the 30 novel effector candidates hit to a similar sequence (>40% amino acid similarity) in M. hapla, and 6 sequences hit to similar sequences (>40% amino acid similarity) in the soybean cyst nematode Heterodera glycines. Of the 30 novel effector candidates, 9 had one or more nuclear localization signals predicted with NLStradamus (Nguyen Ba et al. 2009), and 11 had a protein domain as predicted with Pfam (Mistry et al. 2021).Fig. 1. Identification of Meloidogyne incognita candidate effector genes. A, Diagram of the filtering pipeline used for a genome-guided reanalysis of gland-enriched expressed sequence tag (EST) sequences for the identification of novel effectors from the M. incognita. B to D, In situ hybridization with gene-specific digoxigenin-labeled probes for Minc3s00020g01295 showing dorsal gland (DG)-specific expression in B, early and C, late parasitic second-stage juveniles and D, a young adult female nematode. Bars = 10 µM. ER = endoplasmic reticulum, Mi and Minc = M. incognita, pJ2 = parasitic second-stage juvenile, PPN = plant-parasitic nematode, and TMD = transmembrane domain.Download as PowerPointTable 1. Summary of the 30 novel Meloidogyne incognita candidate effector genes identified in this studyQuery EST IDaMinc IDbDOG boxcNLSdBLASTp M. haplaeBLASTp Heterodera glycineseppJ2fpJ2–pJ4fFfClustergPfam domainhMigfha9D07.ab1Minc3s00339g10525−−−−000−N/AMigfha12E07.ab1Minc3s01030g20014−−+−1.170.810.74−PF11797Migfhab13D10.ab1Minc3s00375g11238−++−0.560.510.56−PF03057Migfha35H01.ab1Minc3s00013g00834−−++1.730.520.35CN/AMigfha28H09.ab1Minc3s00020g01295l−+−−2.940.550.45CPF11398Migfha13H11.ab1Minc3s00100g04543−−−−2.621.340.96CN/AMigfha31D09.ab1Minc3s00126g05384−−++1.671.120.82CPF06024Migfha7B01.ab1Minc3s01616g25127−−+−1.751.150.65CN/AMigfha34H01.ab1Minc3s08040g41830−−−−1.901.251.44CPF07172Migfha11F02.ab1Minc3s03654g34440−−+−1.641.020.80CN/AMigfha13E05.ab1Minc3s00610g15098+−+−1.861.070.54CN/AMigfha16G10.ab1Minc3s00081g03896−−++1.910.470.20DN/AMigfha14A12.ab1Minc3s01578g24852−++−1.140.500.57DPF19960Migfha25H11.ab1Minc3s01573g24822i−++−3.221.860.74DN/AMigfha19H12.ab1Minc3s03360g33617i−++−3.231.890.64DN/AMigfha32E04.ab1Minc3s03978g35225i−++−3.171.850.69DN/AMigfhab16A06.ab1Minc3s06371g39735−−+−1.490.630.48DPF15326Migfha12B11.ab1Minc3s00145g05984−−+−1.622.642.28GN/AMigfhab1B03.ab1Minc3s00324g10230−++−1.071.631.06GPF03176Migfha23A09.ab1Minc3s00351g10779−−+−0.571.280.19GPF00341Migfhab4E01.ab1Minc3s01206g21700+++−0.531.400.96GN/AMigfha19B11.ab1Minc3s00071g03524j−−+−0.203.072.98HN/AMigfha20D11.ab1Minc3s00110g04843j−−+−0.182.532.40HN/AMigfha25B12.ab1Minc3s03303g33468j−−+−0.122.952.76HN/AMigfha6C06.ab1Minc3s00076g03731−−+−0.272.101.98HPF02969Migfha21B06.ab1Minc3s00646g15573k−−+−0.451.311.41HN/AMigfhab9A12.ab1Minc3s02745g31424k−−+−0.531.942.02HN/AMigfha5E09.ab1Minc3s01498g24281−−−−0.471.170.94HPF05814Migfhab1A11.ab1Minc3s08340g42160−+−+1.032.532.45HN/AMigfhab1B04.ab1Minc3s01550g24644l+−++0.441.180.97HN/AaExpressed sequence tag (EST) clone names (Huang et al. 2003, 2004).bMeloidogyne incognita genome ID, version 3.cPredicted DOG box (Da Rocha et al. 2021).dNuclear localization signals (NLS) predicted with NLStradamus (Nguyen Ba et al. 2009).eBlastP amino acid sequence similarity > 40%.fLife-stage expression (Da Rocha et al. 2021). ppJ2 = preparasitic second-stage juvenile; pJ2-pJ4 = mixed stages; F = adult female.gGene expression cluster (Da Rocha et al. 2021).hProtein domains predicted using Pfam (Mistry et al. 2021). N/A = not applicable.iProtein sequences share >80% amino acid similarity.jProtein sequences share >80% amino acid similarity.kProtein sequences share >80% amino acid similarity.lConfirmed dorsal gland specific expression via in situ hybridization (this study and Rocha et al. 2021).Table 1. Summary of the 30 novel Meloidogyne incognita candidate effector genes identified in this studyView as image HTML We selected Minc3s00020g01295 and Minc3s01550g24644 for in situ hybridization analysis on selected life stages using established protocols (De Boer et al. 1998). Minc3s00020g01295 expression was localized specifically within the DG cell of early parasitic juveniles through young adult female life stages (Fig. 1B to D). Expression of Minc3s01550g24644, which we also identified in a parallel study to enrich for the DG cells of adult RKN females, was also specifically expressed in the DG cell of adult RKN females (Rocha et al. 2021). Minc3s01550g24644 but not Minc3s00020g01295 contained a predicted DOG box; however, both genes were expressed in the DG cell, suggesting possible DOG box-independent regulation of a subset of DG effectors.In addition to the 30 new PPN-specific novel candidate effector gene sequences, we identified 70 non-PPN-specific putative M. incognita gland effector candidates, including many with annotations that may have important roles in parasitism (Supplementary Table S1). Among these are several candidates that have since been reported by other groups as bona fide gland-expressed nematode effector proteins with important roles in plant–nematode interactions, including FMRFamide-related (Banakar et al. 2020), calreticulin (Jaouannet et al. 2013), C-type lectin (Zhao et al. 2021), SXP/RAL-2 (Tytgat et al. 2005), fatty acid and retinol binding (Iberkleid et al. 2013), and transthyretin-like effectors (Lin et al. 2016). However, not all of these have been studied in M. incognita. An assortment of secreted metallo, cysteine, serine, and aspartic proteases and proteinase inhibitors were also identified. Nematode proteases shown to be expressed in the nematode intestine are likely to play a role in digestion (Neveu et al. 2003; Shingles et al. 2007); however, there is also increasing evidence of a role in plant parasitism as expression in esophageal gland cells and secretion into plant tissues has been confirmed (Pu et al. 2022; Vieira et al. 2011; Wang et al. 2016). Studies on gland-expressed housekeeping genes also offer promise for garnering new insights into plant parasitism. Recent work by Lilley et al. (2018) demonstrated that neofunctionalization of glutathione synthetase (GST) housekeeping genes has given rise to secreted GST effectors important for establishing plant–nematode interactions. Additionally, we discovered several new M. incognita gland effector candidates sharing similarity with proteins of unknown function in other free-living or parasitic nematodes (Supplementary Table S1) that have yet to be explored but may have evolved a unique function in plant–nematode interactions.Much to our surprise, our genome-guided reanalysis of the gland-enriched EST sequences did not rediscover all of the previously reported in situ confirmed gland-expressed effector candidates. M. incognita effector gene sequences 7E12, 7H08, 10G02, 14E06, 16D10, 17H02, 21E02, 28B04, and 35F03 (Huang et al. 2003) and 1D08B, 2G06B, 5C03B, and 6D09B (Huang et al. 2004) did not result in a Minc ID hit in the genome. Sequence 16D10 is a well characterized Meloidogyne effector (Huang et al. 2006a, b), prompting us to interrogate the genome in more depth. We determined that the 16D10 sequence was indeed present but is not annotated in the current genome. Manual annotation using available ESTs could be undertaken to make improvements to the current version of the genome. Another set of previously reported effector sequences, including 2G10, 4D03, 6G07, 8D05, 19F07, 25B10, 31H06, 34F06, 35A02, and 35E04 (Huang et al. 2003) and 2B02B and 4F05B (Huang et al. 2004) had a corresponding Minc ID hit in the genome but were not rediscovered in our analysis for various reasons that we traced to either errors in the Minc open reading frame prediction that led to a missed SP prediction or detection of a TMD that excluded it from the analysis by Da Rocha et al. (2021). Our results caution reliance on genome mining alone for discovery of effectors.In summary, we have used a novel genome-guided pipeline for the reanalysis of an archived set of gland-enriched ESTs to further expand the current effector repertoire of M. incognita and provide a community resource that will serve to aid in the selection and prioritization of relevant candidates for future functional studies. Our approach may also have utility for other nematodes and archived datasets such as the soybean cyst nematode H. glycines, for which similar gland-enriched cDNA libraries were generated (Gao et al. 2001, 2003; Wang et al. 2001) and a current genome now exists (Masonbrink et al. 2021).Data AvailabilityThe candidate Minc effector gene sequences are available in Supplementary Table S1.AcknowledgmentsWe thank W. Lorenz and M. Alabady of the Georgia Genomics and Bioinformatics Core facility for bioinformatics assistance.The author(s) declare no conflict of interest.Literature CitedAbad, P., Gouzy, J., Aury, J.-M., Castagnone-Sereno, P., Danchin, E. G. J., Deleury, E., Perfus-Barbeoch, L., Anthouard, V., Artiguenave, F., Blok, V. C., Caillaud, M. C., Coutinho, P. M., Dasilva, C., De Luca, F., Deau, F., Esquibet, M., Flutre, T., Goldstone, J. V., Hamamouch, N., Hewezi, T., Jaillon, O., Jubin, C., Leonetti, P., Magliano, M., Maier, T. R., Markov, G. V., McVeigh, P., Pesole, G., Poulain, J., Robinson-Rechavi, M., Sallet, E., Ségurens, B., Steinbach, D., Tytgat, T., Ugarte, E., van Ghelder, C., Veronico, P., Baum, T. J., Blaxter, M., Bleve-Zacheo, T., Davis, E. L., Ewbank, J. J., Favery, B., Grenier, E., Henrissat, B., Jones, J. T., Laudet, V., Maule, A. G., Quesneville, H., Rosso, M. N., Schiex, T., Smant, G., Weissenbach, J., and Wincker, P. 2008. 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This is an open access article distributed under the CC BY-NC-ND 4.0 International license.DetailsFiguresLiterature CitedRelated Just PublishedISSN:2690-5442 Metrics Article History Published: 9 Jan 2023Accepted: 3 Oct 2022 InformationCopyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.Funding University of Georgia Office of the President and Georgia Agricultural Experiment Station Keywordsbioinformaticseffectoresophageal gland cellhost–parasite interactionsMeloidogyne incognitanematodepathogen effectorsThe author(s) declare no conflict of interest.PDF download