Isolation and Characterization of Avirulence Genes in Magnaporthe oryzae

Authors

  • Mui Sie Jee
  • Leonard Whye Kit Lim
  • Martina Azelin Dirum
  • Sara Ilia Che Hashim
  • Muhammad Shafiq Masri
  • Hui Ying Tan
  • Lee San Lai
  • Freddy Kuok San Yeo
  • Hung Hui Chung

DOI:

https://doi.org/10.33736/bjrst.389.2017

Keywords:

Plant disease, rice blast, Sarawak, Magnaporthe oryzae

Abstract

Magnaporthe oryzae is a fungal pathogen contributing to rice blast diseases globally via their Avr (avirulence) gene. Although the occurrence of M. oryzae has been reported in Sarawak since several decades ago, however, none has focused specifically on Avr genes, which confer resistance against pathogen associated molecular pattern-triggered immunity (PTI) in host. The objective of this study is to isolate Avr genes from M. oryzae 7’ (a Sarawak isolate) that may contribute to susceptibility of rice towards diseases. In this study, AvrPiz-t, AVR-Pik, Avr-Pi54, and AVR-Pita1 genes were isolated via PCR and cloning approaches. The genes were then compared with set of similar genes from related isolates derived from NCBI. Results revealed that all eight Avr genes (including four other global isolates) shared similar N-myristoylation site and a novel motif. 3D modeling revealed similar β-sandwich structure in AvrPiz-t and AVR-Pik despite sequence dissimilarities. In conclusion, it is confirmed of the presence of these genes in the Sarawak (M. oryzae) isolate. This study implies that Sarawak isolate may confer similar avirulence properties as their counterparts worldwide. Further R/Avr gene-for-gene relationship studies may aid in strategic control of rice blast diseases in future.

References

Agrios, G.N. (2005). Plant pathology. Fifth Edition. Burlington, MA: Elsevier Academic Press.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Research, 25(17): 3389-3402.

https://doi.org/10.1093/nar/25.17.3389

Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel, V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti, R., Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I., & Stockinger, H. (2012). ExPASy: SIB bioinformatics resource portal. Nucleic Acids Research, 40 (W1): W597-W603.

https://doi.org/10.1093/nar/gks400

Bailey, T.L. & Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. In Altman, R., Brutlag, D., Karp, P., Lathrop, R. & Searls, D. (Eds.), Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology. Menlo Park, CA: AAAI Press. Pp 28-36.

Boyle, P.C., Schwizer, S., Hind, S.R., Kraus, C. M., De la Torre Diaz, S., He, B. & Martin, G.B. (2016). Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry. Plant Methods, 12 (1): 38.

https://doi.org/10.1186/s13007-016-0138-2

Cesari, S., Thilliez, G., Ribot, C., Chalvon, V., Michel, C., Jauneau, A., Rivas, S., Alaux, L., Kanzaki, H., Okuyama, Y., Morel, J.B., Fournier, E., Tharreau, D., Terauchi, R. & Kroj, T. (2013). The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. The Plant Cell, 25(4): 1463-1481.

https://doi.org/10.1105/tpc.112.107201

Chen, X.L., Shi, T., Yang, J., Shi, W., Gao, X., Chen, D., Xu, X., Xu, J.R., Talbot, N.J. & Peng, Y. -L. (2014). N-glycosylation of effector proteins by an ɑ-1, 3-mannosyltransferase is required for the rice blast fungus to evade host innate immunity. The Plant Cell, 26(3): 1360-1376.

https://doi.org/10.1105/tpc.114.123588

de Castro, E., Sigrist, C.J.A., Gattiker, A., Bulliard, V., Langendijk-Genevaux, P.S., Gasteiger, E., Bairoch, A. & Hulo, N. (2006). ScanProsite: Detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Research, 34: W362-W365.

https://doi.org/10.1093/nar/gkl124

de Guillen, K., Ortiz-Vallejo, D., Gracy, J., Fournier, E., Kroj, T. & Padilla, A. (2015). Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi. PLoS Pathogens, 11 (10): e1005228.

https://doi.org/10.1371/journal.ppat.1005228

Devanna, N.B., Vijayan, J. & Sharma, T.R. (2014). The blast resistance gene Pi54 of cloned from Oryza officinalis interacts with Avr-Pi54 through Its novel non-LRR domains. PLoS One, 9(8): e104840.

https://doi.org/10.1371/journal.pone.0104840

Fatah, T., Rafii, M.Y., Rahim, H.A., Meon, S., Azhar, M. & Latif, M. A. (2014). Cloning and analysis of QTL linked to blast disease resistance in Malaysian rice variety Pongsu Seribu 2. International Journal of Agriculture & Biology, 16(2): 395-400.

Food and Agriculture Organization of the United Nations (2002). FAO Rice Information, Volume 3. Rome, Italy: FAO.

Freeley, M., Kelleher, D. & Long, A. (2011). Regulation of protein kinase C function by phosphorylation on conserved and nonconserved sites. Cellular Signaling, 23(5): 753-762.

https://doi.org/10.1016/j.cellsig.2010.10.013

Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D. & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In Walker, J.M. (Ed.), The Proteomics Protocols Handbook, 1st edition. Totowa, NJ: Humana Press. Pp. 571-607.

https://doi.org/10.1385/1-59259-890-0:571

Harman, G.E., Howell, C.R., Viterbo, A., Chet, I. & Lorito, M. (2004). Trichoderma species--opportunistic, avirulent plant symbionts. Nature Reviews Microbiology, 2(1): 43-56.

https://doi.org/10.1038/nrmicro797

Holm, L. & Laakso, L. M. (2016). Dali server update. Nucleic Acids Research, 44 (W1): 351-W355.

https://doi.org/10.1093/nar/gkw357

Horbach, R., Navarro-Quesada, A.R., Knoggec, W. & Deising, H.B. (2011). When and how

to kill a plant cell: Infection strategies of plant pathogenic fungi. Journal of Plant Physiology, 168(1): 51-62.

https://doi.org/10.1016/j.jplph.2010.06.014

Jones, J.D.G. & Dangl, J.L. (2006). The plant immune system. Nature, 444: 323-329.

https://doi.org/10.1038/nature05286

Jones, P., Binns, D., Chang, H.Y., Fraser, M., Li, W., McAnulla, C., McWilliam, H., Maslen, J., Mitchell, A., Nuka, G., Pesseat, S., Quinn, A.F., Sangrador-Vegas, A., Scheremetjew, M., Yong, S.Y., Lopez, R. & Hunter, S. (2014). InterProScan 5: Genomescale protein function classification. Bioinformatics, 30(9): 1236-1240.

https://doi.org/10.1093/bioinformatics/btu031

Joosten, M.H.A.J., Honée, G., van Kan, J.A. L. & de Wit, P.J.G.M. (1997). The gene-forgene

concept in plant-pathogen interactions: Tomato-Cladosporium fulvum. In Carrroll, G.C. & Tudzynski, P. (Eds.), The mycota V. Plant relationships Part b. Berlin, Germany: Springer-Verlag. Pp 3-16.

Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. & Sternberg, M.J. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10(6): 845-858.

https://doi.org/10.1038/nprot.2015.053

Kibbe, W.A. (2007). OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Research, 35: W43-W46.

https://doi.org/10.1093/nar/gkm234

Kotz, A., Wagener, J., Engel, J., Routier, F.H., Echtenacher, B., Jacobsen, I., Heesemann, J.

& Ebel, F. (2010). Approaching the secrets of N-glycosylation in Aspergillus fumigatus: Characterization of the AfOch1 protein. PLoS One, 5(12): e15729.

https://doi.org/10.1371/journal.pone.0015729

Lamb, C.J. (1994). Plant disease resistance genes in signal perception and transduction. Cell, 76: 419-422.

https://doi.org/10.1016/0092-8674(94)90106-6

Laskowski, R.A., Watson, J.D. & Thornton, J.M. (2005). ProFunc: A server for predicting protein function from 3D structure. Nucleic Acids Research, 33 (suppl_2): W89-W93.

https://doi.org/10.1093/nar/gki414

Li, W., Wang, B., Wu, J., Lu, G., Hu, Y., Zhang, X., Zhang, Z., Zhao, Q., Feng, Q., Zhang, H., Wang, Z., Wang, G-L., Han, B., Wang, Z. & Zhou, B. (2009). The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t. Molecular Plant-Microbe Interactions Journal, 22(4): 411-420.

https://doi.org/10.1094/MPMI-22-4-0411

Lovell, S.C., Davis, I.W., Arendall III, W.B., de Bakker, P.I.W., Michael Word, J., Prisant, M.G., Richardson, J.S. & Richardson, D.C. (2002). Structure validation by Calpha geometry: phi, psi and Cbeta deviation. Proteins: Structure, Function & Genetics, 50(3): 437-450.

https://doi.org/10.1002/prot.10286

Marchler-Bauer, A., Derbyshire, M.K., Gonzales, N.R., Lu, S., Chitsaz, F., Geer, L. Y., Geer, R.C., He, J., Gwadz, M., Hurwitz, D.I., Lanczycki, C.J., Lu, F., Marchler, G.H., Song, J.S., Thanki, N., Wang, Z., Yamashita, R.A., Zhang, D., Zheng, C. & Bryant, S.H. (2015). CDD:

NCBI's conserved domain database. Nucleic Acids Research, 43(D1): D222-D226.

https://doi.org/10.1093/nar/gku1221

Markaryan, A., Morozova, I., Yu, H. & Kolattukudy, P.E. (1994). Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung. Infection and Immunity, 62(6): 2149-2157.

https://doi.org/10.1128/iai.62.6.2149-2157.1994

Maqbool, A., Saitoh, H., Franceschetti, M., Stevenson, C.E.M., Uemura, A., Kanzaki, H., Kamoun, S., Terauchi, R. & Banfield, M.J. (2015). Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor. eLife, 4: e08709.

https://doi.org/10.7554/eLife.08709

Martin, K.J. & Rygiewicz, P.T. (2005). Fungalspecific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology, 5:28.

https://doi.org/10.1186/1471-2180-5-28

Miki, S., Matsui, K., Kito, H., Otsuka, K., Ashizawa, T., Yasuda, N., Fukiya, S., Sato, J., Hirayae, K., Fujita, Y., Nakajima, T., Tomita, F. & Sone, T. (2009). Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae. Molecular Plan Pathology, 10(3): 361-374.

https://doi.org/10.1111/j.1364-3703.2009.00534.x

Moriya, K., Nagatoshi, K., Noriyasu, Y., Okamura, T., Takamitsu, E., Suzuki, T. & Utsumi, T. (2013). Protein N-myristoylation plays a critical role in the endoplasmic reticulum morphological change induced by overexpression of protein Lunapark, an integral membrane protein of the endoplasmic reticulum. PLoS One, 8(11): e78235.

https://doi.org/10.1371/journal.pone.0078235

Nimchuk, Z., Marois, E., Kjemtrup, S., Leister, R.T., Katagiri, F. & Dangl, J.L. (2000). Eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type III effector proteins from Pseudomonas syringae. Cell, 101(4): 353-363.

https://doi.org/10.1016/S0092-8674(00)80846-6

Nyarko, A., Singarapu, K.K., Figueroa, M., Manning, V.A., Pandelova, I., Wolpert, T.J., Ciuffetti, L.M. & Barbar, E. (2014). Solution NMR structures of Pyrenophora tritici-repentis ToxB and its inactive homolog reveal potential determinants of toxin activity. The Journal of Biological Chemistry, 289: 25946-25956.

https://doi.org/10.1074/jbc.M114.569103

Orbach, M.J., Farrall, L., Sweigard, J.A., Chumley, F.G. & Valent, B. (2000). A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pita. The Plant Cell, 12(11): 2019-2032.

https://doi.org/10.1105/tpc.12.11.2019

Park, C.H., Shirsekar, G., Bellizzi, M., Chen, S., Songkumarn, P., Xie, X., Shi, X., Ning, Y., Zhou, B., Suttiviriya, P., Wang, M., Umemura, K. & Wang, G. -L. (2016). The E3 ligase APIP10 connects the effector AvrPiz-t to the NLR receptor Piz-t in rice. PLoS Pathogens, 12(3): e1005529.

https://doi.org/10.1371/journal.ppat.1005529

Petersen, T.N., Brunak, S., von Heijne, G. & Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmemberane regions. Nature Methods, 8: 785-786.

https://doi.org/10.1038/nmeth.1701

Rafiqi, M., Gan, P.H.P., Ravensdale, M., Lawrence, G.J., Ellis, J.G., Jones, D.A., Hardham, A.R. & Dodds, P.N. (2010). Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen. The Plant Cell, 22(6): 2017-2032.

https://doi.org/10.1105/tpc.109.072983

Ray, S., Singh, P. K., Gupta, D. K., Mahato, A. K., Sarkar, C., Rathour, R., Singh, N.K. &

Sharma, T.R. (2016). Analysis of Magnaporthe oryzae genome reveals a fungal effector, which is able to induce resistance response in transgenic rice line containing resistance gene, Pi54. Frontiers in Plant Science, 7: 1140.

https://doi.org/10.3389/fpls.2016.01140

Ribot, C., Césari, S., Abidi, I., Chalvon, V., Bournaud, C., Vallet, J., Lebrun, M.H., Morel, J.B. & Kroj, T. (2013). The Magnaporthe oryzae effector AVR1-CO39 is translocated into rice cells independently of a fungal-derived machinery. The Plant Journal, 74(1): 1-12.

https://doi.org/10.1111/tpj.12099

Salamon, J.A., Acuña, R. & Dawe, A. L. (2010). Phosphorylation of phosducin-like protein BDM-1 by protein kinase 2 (CK2) is required for virulence and Gβ subunit stability in the fungal plant pathogen Cryphonectria parasitica. Molecular Microbiology, 76(4): 848-860.

https://doi.org/10.1111/j.1365-2958.2010.07053.x

Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D. & Higgins, D.G. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7: 539.

https://doi.org/10.1038/msb.2011.75

Sweigard, J.A., Carroll, A.M., Kang, S., Farrall, L., Chumley, F.G. & Valent, B. (1995). Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. The Plant Cell, 7(8): 1221-1233.

https://doi.org/10.1105/tpc.7.8.1221

Talbot, N.J. & Wilson, R.A. (2009). Under pressure: Investigating the biology of plant infection by Magnaporthe oryza. Nature Reviews Microbiology, 7(3): 185-195.

https://doi.org/10.1038/nrmicro2032

Tampakaki, A.P., Bastaki, M., Mansfield, J.W. & Panopoulos, N.J. (2002). Molecular

determinants required for the avirulence function of AvrPphB in bean and other plants. Molecular Plant-Microbe Interactions, 15(3): 292-300.

https://doi.org/10.1094/MPMI.2002.15.3.292

Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6: Molecular

Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30(12): 2725-2729.

https://doi.org/10.1093/molbev/mst197

Tanweer, F.A., Rafii, M.Y., Sijam, K., Rahim, H.A., Ahmed, F., Latif, M.A. & Ashkani, S. (2015). Cloning and characterization of two major blast resistance genes Pi-b and Pi-kh from Malaysian rice variety Pongsu Seribu 2. Plant Omics Journal, 8(3): 257-263.

Thines, E., Eilbert, F., Sterner, O. & Anke, H. (1998). Inhibitors of appressorium formation in Magnaporthe grisea: A new approach to control rice blast disease. Pest Management Science, 54(3): 314-316.

https://doi.org/10.1002/(SICI)1096-9063(1998110)54:3<314::AID-PS837>3.0.CO;2-H

Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Geurts, R. & Leunissen, J.A. M. (2007). Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Research, 35(suppl_2): W71-W74.

https://doi.org/10.1093/nar/gkm306

Valera, H.G.A. & Lee, J. (2016). Do rice prices follow a random walk? Evidence from Markov switching unit root tests for Asian markets. Agricultural Economics, 47(6):683-695.

https://doi.org/10.1111/agec.12265

Wu, J., Kou, Y., Bao, J., Li, Y., Tang, M., Zhu, X., Ponaya, A., Xiao, G., Li, J., Li, C., Song,

M.Y., Cumagun, C.J.R., Deng, Q., Lu, G., Jeon, J.S., Naqvi, N. & Zhou, B. (2015).

Comparative genomics identifies the Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9-mediated blast resistance in rice. New Phytologist, 206(4): 1463-1475.

Xue, C., Park, G., Choi, W., Zheng, L., Dean, R.A. & Xua, J. (2002). Two novel fungal virulence genes specifically expressed in appressoria of the rice blast fungus. The Plant Cell, 14(9): 1-13.

https://doi.org/10.1105/tpc.003426

Yoshida, K., Saitoh, H., Fujisawa, S., Kanzaki, H., Matsumura, H., Yoshida, K., Tosa, Y.,

Chuma, I., Tokano, Y., Win, J., Kamoun, S. & Terauchi, R. (2009). Association genetics

reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae. The Plant Cell, 21(5): 1573-1591.

https://doi.org/10.1105/tpc.109.066324

Yu, C-S., Chen, Y.-C., Lu, C.-H. & Hwang, JK. (2006). Prediction of protein subcellular

https://doi.org/10.1002/prot.21018

localization. Proteins: Structure, Function and Bioinformatics, 64(3): 643-651.

Zhang, J., Bayram Akcapinar, G., Atanasova, L., Rahimi, M.J., Przylucka, A., Yang, D., Kubicek, C. P., Zhang, R., Shen, Q. & Druzhinina, I.S. (2016). The neutral metallopeptidase NMP1 of Trichoderma guizhouense is required for mycotrophy and self-defence. Environmental Microbiology, 18(2): 580-597.

https://doi.org/10.1111/1462-2920.12966

Zhang, S., Wang, L., Wu, W., He, L., Yang, X. & Pan, Q. (2015). Function and evolution of

Magnaporthe oryzae avirulence gene AvrPib responding to the rice blast resistance gene

Pib. Scientific Reports, 5: 11642.

Zhang, Z. M., Zhang, X., Zhou, Z.R., Hu, H. Y., Liu, M., Zhou, B. & Zhou, J. (2013). Solution structure of the Magnaporthe oryzae avirulence protein AvrPiz-t. Journal of Biomolecular NMR, 55: 219-223.

https://doi.org/10.1007/s10858-012-9695-5

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2017-07-03

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Jee, M. S., Whye Kit Lim, L., Azelin Dirum, M., Che Hashim, S. I., Masri, M. S., Ying Tan, H., San Lai, L., San Yeo, F. K., & Hui Chung, H. (2017). Isolation and Characterization of Avirulence Genes in Magnaporthe oryzae. Borneo Journal of Resource Science and Technology, 7(1), 31–42. https://doi.org/10.33736/bjrst.389.2017

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