IDENTIFICATION OF EFFECTING PROTEINS BY MALDI TOF/TOF MASS SPECTROMETRY OF THE ROOT-KNOT NEMATODE Meloidogyne javanica

José Córdova-Campos, Pedro G Calle-Ulfe, Erick Suarez-Peña, Sandra Mendez-Farroñan, David Enrique Lindo-Seminario, Savina Gutiérrez Calle, Arturo Morales-Pizarro, Virna Cedeño-Escobar, Eric Mialhe-Matonnier, Carlos Condemarín-Montealegre

Abstract


Background: The main problem in grapevine cultivation is root-attachment nematodes cause serious yield problems in most crops worldwide. Through their different infection mechanisms these nematodes synthesize and secrete a mixture of protein-based effectors that they use to penetrate the root, migrate and develop into giant root feeding cells in host plants. The use of new molecular tools such as MALDI TOF/TOF (Matrix-Assisted Laser Desorption/Ionization - Time-Of-Flight) mass spectrometry and PCR (Polymerase Chain Reaction) technique have allowed us to know these proteins and genes in different microorganisms. Objective: To characterize the root-knot nematode Meloidogyne javanica by sequencing the 18S rRNA gene from infected root samples and the effector proteins of juvenile (J2) and adult (J4) stage of M. javanica by MALDI TOF/TOF shotgun proteomics dual mass spectrometry. Methodology: Infected roots of grapevine crop were collected to extract fresh galls and J2 of M. javanica, then inoculated on tomato plants. J4 of M. javanica were used for genomic DNA extraction and sequencing at the 18S rRNA gene level. The J2 and J4 stages of M. javanica were disinfected with sodium hypochlorite (0.5%) and sterile distilled water for protein extraction and analysis with MALDI-TOF/TOF. Finally, the sequences obtained were processed with ProteinPilot™ and Protein BLAST software for the identification of effector proteins of M. javanica. Results: The nematode M. javanica was molecularly identified by PCR amplification of the 18S rDNA gene M. javanica with an identity percentage of 98% from infected root samples and by MALDI TOF/TOF mass spectrometry, effector protein sequences were identified such as: Beta-1,4-endoglucan and polygalaturonase, identified from J2, and expansin B2, CLAVATA3/ESR, Pectate lyase and Chorismato mutase from J4, involved in the different infection processes. In addition, we were able to identify 49 nematode non-effector proteins in both stages related to conserved biological development. Implications: The results indicate the existence of effector proteins related to root gill formation. Conclusions: This study confirms that dual mass spectrometry methodology provides in a rapid and reproducible way a proteomic profile that the galls nematode synthesizes to infect root cells and that can be used in other types of pathogens.

Keywords


proteomics; nematodes; peptide profiling.

Full Text:

PDF

References


Abad, P., and Williamson, V. M., 2010. Plant nematode interaction: a sophisticated dialogue. Advances in Botanical Research, 53, pp. 147-192. http://doi.org/10.1016/S0065-2296(10)53005-2.

Abad, P., Gouzy, J., Aury, J. M., Castagnone-Sereno, P., Danchin, E. G., Deleury, E., and Caillaud, M. C., 2008. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. NatureBbiotechnology, 26(8), pp. 909-915. http://doi.org/10.1038/nbt.1482.

Agrios, G. N., 2005. Plant pathology. New York: Elsevier Academic Press. http://doi.org/10.2307/4442935.

Baum, T. J., Hussey, R. S., and Davis, E. L., 2007. Root-knot and cyst nematode parasitism genes: the molecular basis of plant parasitism. In Genetic engineering (pp. 17-43). Springer US. http://doi.org/10.1007/978-0-387-34504-8_2.

Bellafiore, S., Shen, Z., Rosso, M. N., Abad, P., Shih, P., and Briggs, S. P., 2008. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential. PLoS Pathogens, 4(10), pp. e1000192. http://doi.org/10.1371/journal.ppat.1000192.

Bellincampi, D., Cervone, F., and Lionetti, V., 2014. Plant cell wall dynamics and wall-related susceptibility in plant–pathogen interactions. Frontiers in Plant Science, 5, pp. 228.

Bendezú, M. V., and Alvarez, L. A., 2012. The perfect stage of powdery mildew of grapevine caused by Erysiphe necator found in Peru. Plant Disease, 96(5), pp. 768-768. http://doi.org/10.1094/PDIS-11-11-0985.

Blaxter, M. L., De Ley, P., Garey, J. R., Liu, L. X., Scheldeman, P., Vierstraete, A., and Vida, J. T., 1998. A molecular evolutionary framework for the phylum Nematoda. Nature, 392(6671), pp. 71-75. http://doi.org/10.1038/32160.

Cañuelo, A., and Peragón, J., 2013. Proteomics analysis in Caenorhabditis elegans to elucidate the response induced by tyrosol, an olive phenol that stimulates longevity and stress resistance. Proteomics, 13(20), pp. 3064-3075. http://doi.org/10.1002/pmic.201200579.

Chen, J., Li, Z., Lin, B., Liao, J., and Zhuo, K., 2021. A Meloidogyne graminicola pectate lyase is involved in virulence and activation of host defense responses. Frontiers in Plant Science, 12, 401.

Chitwood, D. J., 2003. Research on plant?parasitic nematode biology conducted by the United States Department of Agriculture–Agricultural Research Service. Pest Management Science, 59(6?7), pp. 748-753. http://doi.org/10.1002/ps.684.

Cotton, J. A., Lilley, C. J., Jones, L. M., Kikuchi, T., Reid, A. J., Thorpe, P., and Eves-van den Akker, S., 2014. The genome and life-stage specific transcriptomes of Globodera pallida elucidate key aspects of plant parasitism by a cyst nematode. Genome biology, 15(3), pp. R43. http://doi.org/10.1186/gb-2014-15-3-r43.

Condemarín Montealegre, C., Oyola Medina, M., Mialhe, E., Quimi Mujica, J., Astudillo Urbina, S., Gutierrez Calle, S., and LeónTemple, G., 2018. Efecto de bacterias nativas del suelo cultivado y prístino sobre el control del nematodo agallador radicular, Meloidogyne javanica. en condiciones in vitro y producción de biomasa. Arnaldoa, 25(2), 515-528.

Davis, E. L., Hussey, R. S., Baum, T. J., Bakker, J., Schots, A., Rosso, M. N., and Abad, P., 2000. Nematode parasitism genes. Annual Review of Phytopathology, 38(1), pp. 365-396. http://doi.org/10.1146/annurev.phyto.38.1.365.

Davis, E. L., Hussey, R. S., Mitchum, M. G., and Baum, T. J., 2008. Parasitism proteins in nematode–plant interactions. Current opinion in plant biology, 11(4), 360-366. http://doi.org/10.1016/j.pbi.2008.04.003.

Doyle, E. A., and Lambert, K. N., 2002. Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica. Molecular Plant-Microbe Interactions, 15(6), pp. 549-556. http://doi.org/10.1094/MPMI.2002.15.6.549.

Doyle, E. A., and Lambert, K. N., 2003. Meloidogyne javanica chorismate mutase 1 alters plant cell development. Molecular Plant-Microbe Interactions, 16(2), pp. 123-131. http://doi.org/10.1094/MPMI.2003.16.2.123.

Drayman, N., Glick, Y., Ben-nun-Shaul, O., Zer, H., Zlotnick, A., Gerber, D., and Oppenheim, A., 2013. Pathogens use structural mimicry of native host ligands as a mechanism for host receptor engagement. Cell Host & Microbe, 14(1), pp. 63-73.

Floyd, R., Abebe, E., Papert, A., and Blaxter, M., 2002. Molecular barcodes for soil nematode identification. Molecular Ecology, 11(4), pp. 839-850. http://doi.org/10.1046/j.1365-294X.2002.01485.x.

Gheysen, G., and Mitchum, M. G., 2011. How nematodes manipulate plant development pathways for infection. Current Opinion in Plant Biology, 14(4), pp. 415-421. http://doi.org/10.1016/j.pbi.2011.03.012.

Goad, D. M., Zhu, C., and Kellogg, E. A., 2017. Comprehensive identification and clustering of CLV3/ESR?related (CLE) genes in plants finds groups with potentially shared function. New Phytologist, 216(2), pp. 605-616.

Guo, X., Chronis, D., De La Torre, C. M., Smeda, J., Wang, X., and Mitchum, M. G., 2015. Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors. Plant Biotechnology Journal, 13(6), pp. 801-810. http://doi.org/10.1111/pbi.12313.

Hewezi, T., 2015. Cellular signaling pathways and posttranslational modifications mediated by nematode effector proteins. Plant Physiology, 169(2), pp. 1018-1026. http://doi.org/10.1104/pp.15.00923.

Hu, L., Cui, R., Sun, L., Lin, B., Zhuo, K., and Liao, J., 2013. Molecular and biochemical characterization of the ?-1, 4-endoglucanase gene Mj-eng-3 in the root-knot nematode Meloidogyne javanica. Experimental Parasitology, 135(1), 15-23. http://doi.org/10.1016/j.exppara.2013.05.012.

Huang, G., Dong, R., Allen, R., Davis, E. L., Baum, T. J., and Hussey, R. S., 2005. Developmental expression and molecular analysis of two Meloidogyne incognita pectate lyase genes. International Journal for Parasitology, 35(6), pp. 685-692. http://doi.org/10.1016/j.ijpara.2005.01.006.

Hussey, R. S. and Barker, K. R., 1973. Histopathology of nodular tissues of legumes infected with certain nematodes. Phytopathology, 66(7), pp. 851-855. http://doi.org/10.1094/Phyto-66-851.

Jones, A. M., Thomas, V., Bennett, M. H., Mansfield, J., and Grant, M., 2006. Modifications to the Arabidopsis defense proteome occur prior to significant transcriptional change in response to inoculation with Pseudomonas syringae. Plant Physiology, 142(4), 1603-1620. http://doi.org/10.1104/pp.106.086231.

Kudla, U., Qin, L., Milac, A., Kielak, A., Maissen, C., Overmars, H., and Helder, J., 2005. Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis. FEBS Letters, 579(11), pp. 2451-2457.

Lee, C., Chronis, D., Kenning, C., Peret, B., Hewezi, T., Davis, E. L., and Mitchum, M. G., 2011. The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development. Plant Physiology, 155(2), pp. 866-880. http://doi.org/10.1104/pp.110.167197.

Lilley, C. J., Bakhetia, M., Charlton, W. L., and Urwin, P. E., 2007. Recent progress in the development of RNA interference for plant parasitic nematodes. Molecular Plant Pathology, 8(5), 701-711. https://doi.org/10.1111/j.1364-3703.2007.00422.x.

Lin, B., Zhuo, K., Wu, P., Cui, R., Zhang, L. H., and Liao, J., 2013. A novel effector protein, MJ-NULG1a, targeted to giant cell nuclei plays a role in Meloidogyne javanica parasitism. Molecular Plant-Microbe Interactions, 26(1), pp. 55-66.

Long, H., Wang, X., Xu, J. H., and Hu, Y. J., 2006. Isolation and characterization of another cDNA encoding a chorismate mutase from the phytoparasitic nematode Meloidogyne arenaria. Experimental parasitology, 113(2), 106-111.

Lu, S. W., Chen, S., Wang, J., Yu, H., Chronis, D., Mitchum, M. G., and Wang, X., 2009. Structural and functional diversity of CLAVATA3/ESR (CLE)-like genes from the potato cyst nematode Globodera rostochiensis. Molecular Plant-Microbe Interactions, 22(9), pp. 1128-1142. http://doi.org/10.1094/MPMI-22-9-1128.

Lindo-Seminario, D., Mendez-Farroñan, S., Canta-Ventura, J., Córdova-Campos, J., Condemarín-Montealegre, C., Gutiérrez-Calle, S., Morales-Pizarro, A., and Mialhe-Matonnier, E., 2022. Endosymbiont microbiota of Meloidogyne javanica adult female infected by Pasteuria penetrans. Tropical and Subtropical Agroecosystems, 25, pp. 132. http://dx.doi.org/10.56369/tsaes.4341

Mann, M., Hendrickson, R. C., and Pandey, A., 2001. Analysis of proteins and proteomes by mass spectrometry. Annual Review of Biochemistry, 70(1), 437-473.

Mitchum, M. G., Hussey, R. S., Baum, T. J., Wang, X., Elling, A. A., Wubben, M., and Davis, E. L., 2013. Nematode effector proteins: an emerging paradigm of parasitism. New Phytologist, 199(4), pp. 879-894. http://doi.org/10.1111/nph.12323.

Mitchum, M. G., Wang, X., Wang, J., and Davis, E. L., 2012. Role of nematode peptides and other small molecules in plant parasitism. Annual Review of Phytopathology, 50, pp. 175-195. http://doi.org/10.1146/annurev-phyto-081211-173008.

Morales Pizarro, A., Javier Alva, J., Álvarez, L. A., Peña Castillo, R., Chanduvi García, R., Granda Wong, C. A., Mayta-Obos, R., Lindo-Seminario, D., and Condori Pacsi, S., 2022. Control in vivo de Phaeoacremonium parasiticum con antagonistas nativos Bacillus, Trichoderma y actinomicetos y su efecto promotor de crecimiento en Vid. Tropical and Subtropical Agroecosystems, 25(3), pp. 116. http://dx.doi.org/10.56369/tsaes.4232.

Nakaewa, R., Kiggundu, A., Talwana, H., Namaganda, J., Lilley, C., Tushemereirwe, W., and Atkinson, H., 2013. Nematode 18S rRNA gene is a reliable tool for environmental biosafety assessment of transgenic banana in confined field trials. Transgenic Research, 22(5), pp. 1003-1010. http://doi.org/10.1007/s11248-013-9712-9.

Opperman, C. H., Bird, D. M., Williamson, V. M., Rokhsar, D. S., Burke, M., Cohn, J., and Houfek, T. D., 2008. Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism. Proceedings of the National Academy of Sciences, 105(39), pp. 14802-14807. http://doi.org/10.1073/pnas.0805946105.

Painter, J. E., and Lambert, K. N., 2003. Meloidogyne javanica chorismate mutase transcript expression profile using real-time quantitative RT-PCR. Journal of Nematology, 35(1), pp. 82. https://pubmed.ncbi.nlm.nih.gov/19265979/

Perry, R. N., and Moens, M., 2011. Introduction to plant-parasitic nematodes; modes of parasitism. In Genomics and molecular genetics of plant-nematode interactions (pp. 3-20). Springer Netherlands. http://doi.org/10.1007/978-94-007-0434-3

Pizarro, D. A. M., Alva, J. J., Álvarez, L. A., Obos, R. M., Anccota, R. A., Castillo, R. P., Chanduvi, G. R., and Seminario, D. L., 2022. Isolation, identification and in vitro evaluation of native isolates of Bacillus, Trichoderma and Streptomyces with potential for the biocontrol of grapevine trunk fungi. Tropical and Subtropical Agroecosystems, 25(2), pp. 086. http://dx.doi.org/10.56369/tsaes.4206

Popeijus, H., Overmars, H., Jones, J., Blok, V., Goverse, A., Helder, J., and Smant, G., 2000. Enzymology: degradation of plant cell walls by a nematode. Nature, 406(6791), pp. 36-37. http://doi.org/10.1038/35017641.

Qin, L., Kudla, U., Roze, E. H., Goverse, A., Popeijus, H., Nieuwland, J., and Bakker, J., 2004. Plant degradation: a nematode expansin acting on plants. Nature, 427(6969), pp. 30-30. http://doi.org/10.1038/427030a.

Rodríguez-Gálvez, E., E. Maldonado., and A. Alves., 2015. Identification and pathogenicity of Lasiodiplodia theobromae causing dieback of table grapes in Peru. European Journal of Plant Pathology, 141(3), pp. 477-489.

Roze, E., Hanse, B., Mitreva, M., Vanholme, B., Bakker, J., and Smant, G., 2008. Mining the secretome of the root?knot nematode Meloidogyne chitwoodi for candidate parasitism genes. Molecular Plant Pathology, 9(1), pp. 1-10. http://doi.org/10.1111/j.1364-3703.2007.00435.x.

Sebastiana, M., Figueiredo, A., Monteiro, F., Martins, J., Franco, C., Coelho, A. V., and Ferreira, S., 2013. A possible approach for gel-based proteomic studies in recalcitrant woody plants. SpringerPlus, 2(1), pp. 210. http://doi.org/10.1186/2193-1801-2-210.

Stare, B. G., Lamovšek, J., Širca, S., and Urek, G., 2012. Assessment of sequence variability in putative parasitism factor, expansin (expB2) from diverse populations of potato cyst nematode Globodera rostochiensis. Physiological and Molecular Plant Pathology, 79, pp. 49-54.

Smant, G., Stokkermans, J. P., Yan, Y., De Boer, J. M., Baum, T. J., Wang, X., and Helder, J., 1998. Endogenous cellulases in animals: isolation of ?-1, 4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proceedings of the National Academy of Sciences, 95(9), pp. 4906-4911. http://doi.org/10.1073/pnas.95.9.4906

Tabb, J. S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A., and Langford, G. M., 1998. Transport of ER vesicles on actin filaments in neurons by myosin V. Journal of Cell Science, 111(21), pp. 3221-3234. http://doi.org/10.1242/jcs.111.21.3221.

Underwood, W., 2012. The plant cell wall: a dynamic barrier against pathogen invasion. Frontiers in plant science, 3, pp. 85.

Vieira, P., and Nemchinov, L. G., 2020. An Expansin-like candidate effector protein from Pratylenchus penetrans modulates immune responses in Nicotiana benthamiana. Phytopathology, 110(3), pp. 684-693.

Vieira, P., Danchin, E. G., Neveu, C., Crozat, C., Jaubert, S., Hussey, R. S., and Rosso, M. N. 2011. The plant apoplasm is an important recipient compartment for nematode secreted proteins. Journal of Experimental Botany, 62(3), pp.1241-1253.

Wang, X., Allen, R., Ding, X., Goellner, M., Maier, T., de Boer, J. M., and Davis, E. L., 2001. Signal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Molecular Plant-Microbe Interactions, 14(4), pp. 536-544. http://doi.org/10.1094/MPMI.2001.14.4.536




URN: http://www.revista.ccba.uady.mx/urn:ISSN:1870-0462-tsaes.v26i1.45186

DOI: http://dx.doi.org/10.56369/tsaes.4518

Bookmark and Share


Copyright (c) 2022 David Enrique Lindo Seminario, José Córdova-Campos1, Pedro G. Calle-Ulfe, Erick Antonio Suarez-Peña1, Carlos Condemarín-Montealegre, Savina Gutiérrez Calle, Arturo Morales, Virna Cedeño-Escobar, Eric Mialhe-Matonnier

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.