The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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A LysM Domain-Containing Protein LtLysM1 Is Important for Vegetative Growth and Pathogenesis in Woody Plant Pathogen Lasiodiplodia theobromae

  • Harishchandra, Dulanjalee Lakmali (Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences) ;
  • Zhang, Wei (Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences) ;
  • Li, Xinghong (Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences) ;
  • Chethana, Kandawatte Wedaralalage Thilini (Centre of Excellence in Fungal Research, School of Science, Mae Fah Luang University) ;
  • Hyde, Kevin David (Centre of Excellence in Fungal Research, School of Science, Mae Fah Luang University) ;
  • Brooks, Siraprapa (Centre of Excellence in Fungal Research, School of Science, Mae Fah Luang University) ;
  • Yan, Jiye (Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences) ;
  • Peng, Junbo (Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences)
  • Received : 2020.05.20
  • Accepted : 2020.07.14
  • Published : 2020.08.01
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Abstract

Lysin motif (LysM) proteins are reported to be necessary for the virulence and immune response suppression in many herbaceous plant pathogens, while far less is documented in woody plant pathogens. In this study, we preliminarily characterized the molecular function of a LysM protein LtLysM1 in woody plant pathogen Lasiodiplodia theobromae. Transcriptional profiles revealed that LtLysM1 is highly expressed at infectious stages, especially at 36 and 48 hours post inoculation. Amino acid sequence analyses revealed that LtLysM1 was a putative glycoprotein with 10 predicted N-glycosylation sites and one LysM domain. Pathogenicity tests showed that overexpressed transformants of LtLysM1 displayed increased virulence on grapevine shoots in comparison with that of wild type CSS-01s, and RNAi transformants of LtLysM1 exhibited significantly decreased lesion length when compared with that of wild type CSS-01s. Moreover, LtLysM1 was confirmed to be a secreted protein by a yeast signal peptide trap assay. Transient expression in Nicotiana benthamiana together with protein immunoblotting confirmed that LtLysM1 was an N-glycosylated protein. In contrast to previously reported LysM protein Slp1 and OsCEBiP, LtLysM1 molecule did not interact with itself based on yeast two hybrid and co-immunoprecipitation assays. These results indicate that LtLysM1 is a secreted protein and functions as a critical virulence factor during the disease symptom development in woody plants.

Keywords

References

  1. Akcapinar, G. B., Kappel, L., Sezerman, O. U. and Seidl-Seiboth, V. 2015. Molecular diversity of LysM carbohydrate-binding motifs in fungi. Curr. Genet. 61:103-113. https://doi.org/10.1007/s00294-014-0471-9
  2. Alcântara, A., Bosch, J., Nazari, F., Hoffmann, G., Gallei, M., Uhse, S., Darino, M. A., Olukayode, T., Reumann, D., Baggaley, L. and Djamei, A. 2019. Systematic Y2H screening reveals extensive effector-complex formation. Front. Plant Sci. 10:1437. https://doi.org/10.3389/fpls.2019.01437
  3. Buist, G., Steen, A., Kok, J. and Kuipers, O. P. 2008. LysM, a widely distributed protein motif for binding to (peptido)glycans. Mol. Microbiol. 68:838-847. https://doi.org/10.1111/j.1365-2958.2008.06211.x
  4. Cao, H., Wang, C., Liu, H., Jia, W. and Sun, H. 2020. Enzyme activities during Benzo[a]pyrene degradation by the fungus Lasiodiplodia theobromae isolated from a polluted soil. Sci Rep. 10:865. https://doi.org/10.1038/s41598-020-57692-6
  5. Chen, X.-L., Shi, T., Yang, J., Shi, W., Gao, X., Chen, D., Xu, X., Xu, J.-R., Talbot, N. J. and Peng, Y.-L. 2014. N-glycosylation of effector proteins by an ${\alpha}$-1,3-mannosyltransferase is required for the rice blast fungus to evade host innate immunity. Plant Cell. 26:1360-1376. https://doi.org/10.1105/tpc.114.123588
  6. Chethana, K. W. T., Li, X., Zhang, W., Hyde, K. D. and Yan, J. 2016. Trail of decryption of molecular research on Botryosphaeriaceae in woody plants. Phytopathol. Mediterr. 55:147-171.
  7. Correia, K. C., Silva, M. A., de Morais, M. A. Jr., Armengol, J., Phillips, A. J. L., Câmara, M. P. S. and Michereff, S. J. 2016. Phylogeny, distribution and pathogenicity of Lasiodiplodia species associated with dieback of table grape in the main Brazilian exporting region. Plant Pathol. 65:92-103. https://doi.org/10.1111/ppa.12388
  8. de Jonge, R., van Esse, H. P., Kombrink, A., Shinya, T., Desaki, Y., Bours, R., van der Krol, S., Shibuya, N., Joosten, M. H. A. J. and Thomma, B. P. H. J. 2010. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329:953-955. https://doi.org/10.1126/science.1190859
  9. El-Gebali, S., Mistry, J., Bateman, A., Eddy, S. R., Luciani, A., Potter, S. C., Qureshi, M., Richardson, L. J., Salazar, G. A., Smart, A., Sonnhammer, E. L. L., Hirsh, L., Paladin, L., Piovesan, D., Tosatto, S. C. E. and Finn, R. D. 2019. The Pfam protein families database in 2019. Nucleic Acids Res. 47:D427-D432. https://doi.org/10.1093/nar/gky995
  10. Fang, A., Han, Y., Zhang, N., Zhang, M., Liu, L., Li, S., Lu, F. and Sun, W. 2016. Identification and characterization of plant cell death-inducing secreted proteins from Ustilaginoidea virens. Mol Plant-Microbe Interact. 29:405-416. https://doi.org/10.1094/MPMI-09-15-0200-R
  11. Felix, C., Meneses, R., Goncalves, M. F. M., Tilleman, L., Duarte, A. S., Jorrin-Novo, J. V., Van de Peer, Y., Deforce, D., Van Nieuwerburgh, F., Esteves, A. C. and Alves, A. 2019. A multiomics analysis of the grapevine pathogen Lasiodiplodia theobromae reveals that temperature affects the expression of virulence- and pathogenicity-related genes. Sci. Rep. 9:13144. https://doi.org/10.1038/s41598-019-49551-w
  12. Goncalves, M. F. M., Nunes, R. B., Tilleman, L.,Van de Peer, Y., Deforce, D., Van Nieuwerburgh, F., Esteves, A. C. and Alves, A. 2019. Dual RNA sequencing of Vitis vinifera during Lasiodiplodia theobromae infection unveils host-pathogen interactions. Int. J. Mol. Sci. 20:6083. https://doi.org/10.3390/ijms20236083
  13. Gu, B., Kale, S. D., Wang, Q., Wang, D., Pan, Q., Cao, H., Meng, Y., Kang, Z., Tyler, B. M. and Shan, W. 2011. Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells. PLoS ONE 6:e27217. https://doi.org/10.1371/journal.pone.0027217
  14. Han, X. and Kahmann, R. 2019. Manipulation of phytohormone pathways by effectors of filamentous plant pathogens. Front. Plant Sci. 10:822. https://doi.org/10.3389/fpls.2019.00822
  15. Jacobs, K. A., Collins-Racie, L. A., Colbert, M., Duckett, M., Golden-Fleet, M., Kelleher, K., Kriz, R., La Vallie, E. R., Merberg, D., Spaulding, V., Stover, J., Williamson, M. J. and McCoy, J. M. 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198:289-296. https://doi.org/10.1016/S0378-1119(97)00330-2
  16. Jones, J. D. G. and Dangl, J. L. 2006. The plant immune system. Nature 444:323-329. https://doi.org/10.1038/nature05286
  17. Klein, R. D., Gu, Q., Goddard, A. and Rosenthal, A. 1996. Selection for genes encoding secreted proteins and receptors. Proc. Natl. Acad. Sci. U. S. A. 93:7108-7113. https://doi.org/10.1073/pnas.93.14.7108
  18. Kombrink, A., Rovenich, H., Shi-Kunne, X., Rojas-Padilla, E., van den Berg, G. C. M., Domazakis, E., de Jonge, R., Valkenburg, D.-J., Sanchez-Vallet, A., Seidl, M. F. and Thomma, B. P. H. J. 2017. Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts. Mol. Plant Pathol. 18:596-608. https://doi.org/10.1111/mpp.12520
  19. Kombrink, A., Sanchez-Vallet, A. and Thomma, B. P. H. J. 2011. The role of chitin detection in plant-pathogen interactions. Microbes Infect. 13:1168-1176. https://doi.org/10.1016/j.micinf.2011.07.010
  20. Kombrink, A. and Thomma, B. P. H. J. 2013. LysM effectors: secreted proteins supporting fungal life. PLoS Pathog. 9:e1003769. https://doi.org/10.1371/journal.ppat.1003769
  21. Lee. S.-J. and Rose, J. K. C. 2012. A yeast secretion trap assay for identification of secreted proteins from eukaryotic phytopathogens and their plant hosts. Methods Mol. Biol. 835:519-530. https://doi.org/10.1007/978-1-61779-501-5_32
  22. Li, Q., Zhang, M., Shen, D., Liu, T., Chen, Y., Zhou, J.-M. and Dou, D. 2016. A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner. Sci. Rep. 6:26951. https://doi.org/10.1038/srep26951
  23. Liu, L., Xu, L., Jia, Q., Pan, R., Oelmuller, R., Zhang, W. and Wu, C. 2019. Arms race: diverse effector proteins with conserved motifs. Plant Signal. Behav. 14:1557008. https://doi.org/10.1080/15592324.2018.1557008
  24. Liu, T., Liu, Z., Song, C., Hu, Y., Han, Z., She, J., Fan, F., Wang, J., Jin, C., Chang, J., Zhou, J.-M. and Chai, J. 2012. Chitininduced dimerization activates a plant immune receptor. Science 336:1160-1164. https://doi.org/10.1126/science.1218867
  25. Livak, K. J. and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-{\Delta}{\Delta}CT}$ method. Methods 25:402-408. https://doi.org/10.1006/meth.2001.1262
  26. Macho, A. P. and Zipfel, C. 2014. Plant PRRs and the activation of innate immune signaling. Mol. Cell 54:263-272. https://doi.org/10.1016/j.molcel.2014.03.028
  27. Marshall, R., Kombrink, A., Motteram, J., Loza-Reyes, E., Lucas, J., Hammond-Kosack, K. E., Thomma, B. P. H. J. and Rudd, J. J. 2011. Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on Wheat. Plant Physiol. 156:756-769. https://doi.org/10.1104/pp.111.176347
  28. Mentlak, T. A., Kombrink, A., Shinya, T., Ryder, L. S., Otomo, I., Saitoh, H., Terauchi, R., Nishizawa, Y., Shibuya, N., Thomma, B. P. H. J. and Talbot, N. J. 2012. Effector-mediated suppression of chitin-triggered immunity by Magnaporthe oryzae is necessary for rice blast disease. Plant Cell 24:322-335. https://doi.org/10.1105/tpc.111.092957
  29. Newman, M.-A., Sundelin, T., Nielsen, J. T. and Erbs, G. 2013. MAMP (microbe-associated molecular pattern) triggered immunity in plants. Front. Plant Sci. 4:139. https://doi.org/10.3389/fpls.2013.00139
  30. Oh, S.-K., Young, C., Lee, M., Oliva, R., Bozkurt, T. O., Cano, L. M., Win, J., Bos, J. I. B., Liu, H.-Y., van Damme, M., Morgan, W., Choi, D., Van der Vossen, E. A. G., Vleeshouwers, V. G. A. A. and Kamoun, S. 2009. In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2. Plant Cell 21:2928-2947. https://doi.org/10.1105/tpc.109.068247
  31. Paolinelli-Alfonso, M., Villalobos-Escobedo, J. M., Rolshausen, P., Herrera-Estrella, A., Galindo-Sanchez, C., Lopez-Hernandez, J. F. and Hernandez-Martinez, R. 2016. Global transcriptional analysis suggests Lasiodiplodia theobromae pathogenicity factors involved in modulation of grapevine defensive response. BMC Genomics 17:615. https://doi.org/10.1186/s12864-016-2952-3
  32. Rodriguez-Galvez, E., Maldonado, E. and Alves, A. 2015. Identification and pathogenicity of Lasiodiplodia theobromae causing dieback of table grapes in Peru. Eur. J. Plant Pathol. 141:477-489. https://doi.org/10.1007/s10658-014-0557-8
  33. Romero-Contreras, Y. J., Ramirez-Valdespino, C. A., Guzman-Guzman, P., Macias-Segoviano, J. I., Villagomez-Castro, J. C. and Olmedo-Monfil, V. 2019. Tal6 from Trichoderma atroviride is a LysM effector involved in mycoparasitism and plant association. Front. Microbiol. 10:2231. https://doi.org/10.3389/fmicb.2019.02231
  34. Rovenich, H., Zuccaro, A. and Thomma, B. P. H. J. 2016. Convergent evolution of filamentous microbes towards evasion of glycan-triggered immunity. New Phytol. 212:896-901. https://doi.org/10.1111/nph.14064
  35. Sanchez-Vallet, A., Saleem-Batcha, R., Kombrink, A., Hansen, G., Valkenburg, D.-J., Thomma, B. P. H. J. and Mesters, J. R. 2013. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. eLife 2:e00790. https://doi.org/10.7554/eLife.00790
  36. Schmitz, A. M., Pawlowska, T. E. and Harrison, M. J. 2019. A short LysM protein with high molecular diversity from an arbuscular mycorrhizal fungus, Rhizophagus irregularis. Mycoscience 60:63-70. https://doi.org/10.1016/j.myc.2018.09.002
  37. Selin, C., de Kievit, T. R., Belmonte, M. F. and Dilantha Fernando, W. G. 2016. Elucidating the role of effectors in plantfungal interactions: progress and challenges. Front. Microbiol. 7:600.
  38. Shimizu, T., Nakano, T., Takamizawa, D., Desaki, Y., Ishii-Minami, N., Nishizawa, Y., Minami, E., Okada, K., Yamane, H., Kaku, H. and Shibuya, N. 2010. Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J. 64:204-214. https://doi.org/10.1111/j.1365-313X.2010.04324.x
  39. Stergiopoulos, I. and de Wit, P. J. G. M. 2009. Fungal effector proteins. Annu. Rev. Phytopathol. 47:233-263. https://doi.org/10.1146/annurev.phyto.112408.132637
  40. Takahara, H., Hacquard, S., Kombrink, A., Hughes, H. B., Halder, V., Robin, G. P., Hiruma, K., Neumann, U., Shinya, T., Kombrink, E., Shibuya, N., Thomma, B. P. H. J. and O'Connell, R. J. 2016. Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity. New Phytol. 211:1323-1337. https://doi.org/10.1111/nph.13994
  41. Thomma, B. P. H. J., Nurnberger, T. and Joosten, M. H. A. J. 2011. Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant Cell 23:4-15. https://doi.org/10.1105/tpc.110.082602
  42. Urbez-Torres, J. R., Leavitt, G. M., Guerrero, J. C., Guevara, J. and Gubler, W. D. 2008. Identification and pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the causal agents of bot canker disease of grapevines in Mexico. Plant Dis. 92:519-529. https://doi.org/10.1094/PDIS-92-4-0519
  43. Urbez-Torres, J. 2011. The status of Botryosphaeriaceae species infecting grapevines. Phytopathol. Mediterr. 50:5-45.
  44. Win, J., Chaparro-Garcia, A., Belhaj, K., Saunders, D. G. O., Yoshida, K., Dong, S., Schornack, S., Zipfel, C., Robatzek, S., Hogenhout, S. A. and Kamoun. S. 2012. Effector biology of plant-associated organisms: concepts and perspectives. Cold Spring Harb. Symp. Quant. Biol. 77:235-247.
  45. Yan, J.-Y., Xie, Y., Zhang, W., Wang, Y., Liu, J.-K., Hyde, K. D., Seem, R. C., Zhang, G.-Z., Wang, Z.-Y., Yao, S.-W., Bai, X.-J., Dissanayake, A. J., Peng, Y.-L. and Li, X.-H. 2013. Species of Botryosphaeriaceae involved in grapevine dieback in China. Fungal Divers. 61:221-236. https://doi.org/10.1007/s13225-013-0251-8
  46. Yan, J. Y., Zhao, W. S., Chen, Z., Xing, Q. K., Zhang, W., Chethana, K. W. T., Xue, M. F., Xu, J. P., Phillips, A. J. L., Wang, Y., Liu, J. H., Liu, M., Zhou, Y., Jayawardena, R. S., Manawasinghe, I. S., Huang, J. B., Qiao, G. H., Fu, C. Y., Guo, F. F., Dissanayake, A. J., Peng, Y. L., Hyde, K. D. and Li, X. H. 2018. Comparative genome and transcriptome analyses reveal adaptations to opportunistic infections in woody plant degrading pathogens of Botryosphaeriaceae. DNA Res. 25:87-102. https://doi.org/10.1093/dnares/dsx040
  47. Zeng, T., Rodriguez-Moreno, L., Mansurkhodzaev, A., Wang, P., van den Berg, W., Gasciolli, V., Cottaz, S., Fort, S., Thomma, B. P. H. J., Bono, J.-J., Bisseling, T. and Limpens, E. 2020. A lysin motif effector subverts chitin-triggered immunity to facilitate arbuscular mycorrhizal symbiosis. New Phytol. 225:448-460. https://doi.org/10.1111/nph.16245
  48. Zipfel, C. 2014. Plant pattern-recognition receptors. Trends Immunol. 35:345-351. https://doi.org/10.1016/j.it.2014.05.004