ZmMPK14’ün Bitki Savunmasında Görev Aldığının Mukayeseli Ekspresyon Analiziyle Belirlenmesi

Abstract

Mitojenle-Aktive olan Protein Kinazlar (MAPKs) stimulusla aktive olan sensörlerle yanıt mekanizmaları arasında sinyal iletiminde kaskadlar halinde fonksiyonel olan moleküllerdir. Bu kaskadların son basamağında bir grup C MAPK olan ZmMAPK14 duyarlı ve dirençli genotipi cDNA-AFLP çalışmalarında diferansiyel ekspresyon gösteren bir mesaj olarak tanımlanmıştır: Puccinia sorghi (Ps) inokülasyonuyla dirençli genotipte göreceli bir indüksiyon görülürken, duyarlı genotipte belirgin bir represyon tespit edilmiştir. RT-qPCR çalışmaları diğer taraftan Ps inokülasyonuyla her iki genotipte farklı düzeylerde indüksiyon olduğunu göstermiştir: Duyarlı genotipte kontrole göre 1.5- 2.5 kat arasında değişim gösteren bir ekspresyon artışı görülürken dirençli genotipte 12. h’de 149 kat ile başlayan ve 72. h’de 477 kat tepe değerine ulaşılan dramatik göreceli bir artış gözlenmiştir. Söz konusu MAP Kinazın bitki savunmasında rolüyle ilgili daha somut bulgular elde etmek için, kitin, Salisilik Asit (SA) ve H2O2 uygulamalarıyla ZmMapk14 ekspresyonunda değişim her iki genotipte 0-24 aralığını kapsayan altı örnekleme noktasında çalışılmıştır. Tüm uygulamalar dirençli genotipte istatistiki önemli indüksiyonlar ortaya çıkarırken, duyarlı genoptipte ekspresyon değişimleri Ps uygulamasındakine benzer düzede kompleks ve sınırlı olarak tespit edilmiştir. Sonuçlar, ZmMAPK14 ekspresyonunun gerçekleştirilen uygulamalarla değişim gösterdiğini ve Ps’e karşı bitki savunmasında fonksiyonel olduğunu göstermektedir. 

Keywords

• ZmMAPK14
• Bitki Savunması
• SA
• Kitin
• H2O2

Project Number

209T002

Involvement of ZmMPK14 in Plant Defense Revealed by Comparative Expression Analysis

Abstract

Mitogen-Activated Protein Kinases (MAPKs) function in signaling pathways as modules cascading between stimulus activated sensors and response mechanisms.ZmMAPK14, a group C final MAPK of this cascade was identified as a differentially expressed message in cDNA-AFLP studies of both susceptible and resistant genotypes, where a gradual induction was displayed in the resistant genotype while a clear repression occurred in the susceptible genotype in response to Puccinia sorghi (Ps) inoculation. RT-qPCR verification studies, however, did not reveal the same pattern of expression in that both displayed inductions at different levels. Ps inoculation induced a limited expression increase fluctuating between 1.5 and 2.5-fold in the susceptible genotype while a dramatic upregulation starting at 12 h with a 149-fold and gradually increasing to a maximum level of 477 folds at 72 h in resistant genotype was observed. To obtain further evidence about its role in plant defense, ZmMapk14 expression in response to applications of chitin, Salicylic Acid (SA) and H2O2 at six time points covering a 0-24 h interval were studied in both genotypes. All treatments induced ZmMapk14 expression in the resistant genotype significantly at different levels while the expressional changes in the susceptible were more complex and limited similar to the Ps inoculation levels in the susceptible. Overall, the results show that ZmMAPK14 display differential expression in resistant and susceptible genotypes in response to Ps inoculation and applied defense compounds, and it presumably functions in plant defense to Ps.

Keywords

• ZmMAPK14
• Plant Defense
• SA
• Chitin
• H2O2

Supporting Institution

The Council of Turkish Scientific and Technological Research.

Project Number

209T002

References

1. Tena, G., Boudsocq, M., & Sheen, J. (2011). Protein kinase signaling networks in plant innate immunity. Current Opinion in Plant Biology, 14(5), 519–529.
2. Zhang, M., Su, J., Zhang, Y., Xu. J., & Zhang, S. (2018). Conveying internal and external signals via MAPKs. Current Opinion in Plant Biology, 45, 1–10.
3. Taj, G., Agarwal, P., Grant, M., & Kumar, A. (2010). MAPK machinery in plants Recognition and response to different stresses through multiple signal transduction pathways. Plant Signaling & Behavior, 5(11), 1370-1378.
4. Meng, X., & Zhang, S. (2013). MAPK cascades in plant disease resistance signaling. Annual Review of Phytopathology, 51, 245–266.
5. Kong, X., Lv, W., Zhang, S., Jiang, S., & Li, D. (2013). Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase, PLoS ONE, 8 e57714.
6. Kong, X., Pan, J., Zhang, D., Jiang, S., Cai, G., Wang, L., & Li, D. (2013). Identification of mitogen-activated protein kinase kinase gene family and MKK–MAPK interaction network in maize. Biochemical and Biophysical Research Communications, 441, 964–969.
7. Wei, K., Wang, Y., Zhong, X., & Pan, S. (2014). Protein kinase structure, expression and regulation in maize drought signalling. Molecular Breeding 34, 583–602.
8. Liu, Y., Zhang, D., Wang, L., & Li, D. (2013). Genome-Wide Analysis of Mitogen-Activated Protein Kinase Gene Family in Maize. Plant Molecular Biology Reporter, 31, 1446–1460.

9. Mohanta, T. K., Arora, P. K., Mohanta, N., Parida, P., & Bae, H. (2015). Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants. BMC Genomics, 16:58, 1-20.
10. Sun, W., Chen, H., Wang, J., Sun, H. W., Yang, S. K., Sang, Y. L., Lu, X. B., & Xu, X. H. (2015). Expression analysis of genes encoding mitogen-activated protein kinases in maize provides a key link between abiotic stress signaling and plant reproduction” Functional and Integrative Genomics, 15, 107–120.
11. Adachi, H., Nakano, T., Miyagawa, N., Ishihama, N., Yoshioka, M., Katou, Y., Yaeno, T., Shirasu, K., & Yoshioka, H. (2015). WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in. Nicotiana benthamiana. The Plant Cell, 27, 2645–2663.
12. Cheng, Z., Li, J. F., Niu, Y., Zhang, X. C., Woody, O. Z., Xiong, Y., Djonovic, S., Miller, Y., Bush, J., McConkey, J. B., Sheen, J., & Ausubel, F. M. (2015). Pathogen-secreted proteases activate a novel plant immune pathway. Nature, 521, 213-216.
13. Devendrakumar, K. H., Li, X., & Zhang, Y. (2018). MAP Kinase signaling: interplays between plant and effector-triggered immunity. Cellular and Molecular Life Sciences, 75, 2981–2989.
14. Tsuda, K., Mine, A., Bethke, G., Igarashi, D., Botanga, C. J., Tsuda, Y., Glazebrook, J., Sato, M., & Katagiri, F. (2013). Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana. PLoS Genetics 9, e1004015.
15. Bigeard, J., Colcombet, J., & Hirt, H. (2015). Signaling Mechanisms in Pattern-Triggered Immunity (PTI). Molecular Plant, 8, 521–539.
16. Bi, G., & Zhou, J. (2017). MAP Kinase signaling pathways: A hub of plant-microbe interactions. Cell Host &Microbe, 27, 270-273.
17. Xiang, T., Zong, N., Zou, Y., Wu, Y., Zhang, J., Xing, W., Li, Y., Tang, X., Zhu, L., Chai, J., & Zhou, J. M. (2008). Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Current Biology, 18, 74-80.
18. Yamaguchi, K., Yamada, K., Kazuya, I. K., Yoshimura, S., Hayashi, N., Uchihashi, K., Ishihama, N., Kishi-Kaboshi, M., Takahashi, A., & Tsuge, S.. et. al. (2013). A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity. Cell Host & Microbe, 13, 347–357.
19. Shin, H. Y., You, M. K., Jeung, J. U., & Shin, J. S. (2014).OsMPK3 is a TEY-type rice MAPK in group C and phosphorylates OsbHLH65, a transcription factor binding to E-box. Plant Cell Rep. 33, 1343–1353.
20. Wang, M., Zhang, Y., Wang, J., Wu, X., & Guo, X. (2007). A novel MAP kinase gene in cotton (Gossypium hirsutum L.), GhMAPK, is involved in response to diverse environmental stresses. Journal of Biochemistry and Molecular Biology, 40, 325-332.
21. Zhang, L., Dongmei, X., Luo, L., Meng, F., Li, Y., Wu, C., & Guo, X. (2011). Cotton GhMPK2 is involved in multiple signaling pathways and mediates defense responses to pathogen infection and oxidative stress. FEBS Journal, 278, 1367–1378.
22. Sun, T., Nitta, Y., Zhang, Q., Wu, D., Tian, H., Lee, J. S., & Zhang, Y. (2018). Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling. EMBO reports, 19, e45324.
23. Gao, M., Liu, J., Bi, D., Zhang, Z., Cheng, F., Chen, S., & Zhang, Y. (2008). MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen- activated protein kinase cascade to regulate innate immunity in plants. Cell Res, 18(12), 1190–1198.
24. Hirt, H. (1999). Transcriptional upregulation of signaling pathways: more complex than anticipated? Trends Plant Science, 4, 7–8.
25. Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H., & Ohashi, Y. (1995). Tobacco MAP kinase: a possible mediator in wound signal transduction pathways. Science, 270, 1988–1992.
26. Wu, J., Hettenhausen, C., Meldau, S., & Baldwin, I. T. (2007). Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. Plant Cell, 19, 1096-1122.
27. Zong, X., Li, D., Gu, L., Li, D., Liu, L., & Hu, X. (2009). Abscisic acid and hydrogen peroxide induce a novel maize group C MAP kinase gene, ZmMPK7, which is responsible for the removal of reactive oxygen species. Planta, 229, 485-495.
28. Pan, J., Zhang, M., Kong, X., Xing, X., Liu, Y., Zhou, Y., Liu, Y., Sun, L., & Li, D. (2012) ZmMPK17, a novel maize group D MAP kinase, is involved in multiple stress responses. Planta, 235, 661-676.
29. Zhang, B., Ramonell, K., Someville, S., & Stacey, G. (2002). Characterization of early, chitin-induced gene expression in Arabidopsis. Molecular Plant Microbe Interactions, 15, 963-970.
30. Südüpak, M. A. (2014). A cDNA-AFLP protocol with reciprocally arranged 2-enzyme sequential digestion and silver staining detection. Turkish Journal of Biology, 38, 260-270.
31. Livak, K. J.& Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25, 402-408.
32. Reyna, N. S. & Yang, Y. (2006). Molecular analysis of the rice MAP Kinase gene family in relation to Magnaporthe grisea infection. Molecular Plant Microbe Interactions, 19, 530-540.
33. Mur, L. A,, Kenton, P., Loyd, A. J., Ougham, H., & Prats, E. (2008). The hypersensitive response; the centenary is upon us but how much do we know? J. Experimental Botany, 59, 501–520.
34. Shetty, N. P., Jørgensen, H. J. L., Jensen, J. D., Collinge, D. B., & Shetty, H. S. (2008). Roles of reactive oxygen species in interactions between plants and pathogens. European Journal Plant Pathology, 121, 267-280.
35. Liu, Y. & He, C. (2017). A review of redox signaling and the control of MAP kinase pathway in plants. Plant Redox Biology, 11, 192–204.
36. Zhang, S., & Klessig, D. F.(2001). MAPK cascades in plant defense signaling. Trends in Plant Science 6(11), 520-527.
37. [37] Zhang, J., Shao, F., Li, Y., Cui, H., Chen, L., Li, H., Zou, Y., Long, C.,Lan, L., Chai, J., Chen, S., Tang, X., & Zhou, J. M. (2007). A Pseudomonas syringe Effector inactivates MAPKs to supress PAMP-induced immunity in plants. Cell Host &Microbe, 1, 175–185.
38. Wang, J., Ding, H., Zhang, A., Ma, F., Cao, J., & Jiang, M. (2010). A novel MAPK, MAPK3, is involved in response to diverse environmental cues. Journal of Integrative. Plant Biology, 52, 442-452.
39. Jalmi, S. K., & Sinha, A. K. (2015). ROS mediated MAPK signaling in abiotic and biotic stress- striking similarities and differences. Frontiers in Plant Science 6, 769.
40. Sewelam, N., Kazan, K., Thomas-Hall, S. R., Kidd, B. N., Manners, J. M., & Schenk, P. M. (2013). Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen Species Signaling in Arabidopsis. PLoS ONE 8(8), e70289.
41. Mine, A., Seyfferth, C., Kracher, B., Berens, M. L., Becker, D., & Tsuda, K. (2018). The defense phytohormone signaling network enables rapid, high-amplitude transcriptional reprogramming during effector-triggered immunity. The Plant Cell, 30, 1199–1219.
42. Gangappa, S. N., & Botto, J. F. (2016). The Multifaceted Roles of HY5 in plant growth and development. Mol. Plant, 9, 1353-1365.
43. Kumar, D. (2014). Salicylic acid signaling in disease resistance. Plant Science, 228, 127-134.
44. [Wan, J., Zhang, X. C., Neece, D., Ramonell, K. M., Clough, S., Kim, S. Y., Stacey, M. G., & Stacey, G. (2008). A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis. Plant Cell, 20, 471-481.
45. Wan, J., Zhang, S., & Stacey, G. (2004). Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin. Molecular Plant. Pathology, 5, 125–135.
46. Rushton, P. J., & Somssich, I. E. (1998). Transcriptional control of plant genes responsive to pathogens. Current Opinion in Plant Biology, 1, 311-315.
47. Eulgem, T, Rushton, P. J., Robatzek, S., & Somssich, I. E. (2000). The WRKY superfamily of plant transcription factors. Trends Plant Science, 5, 199-206.
48. Ülker, B., & Somssich, I.E.(2004). WRKY transcription factors: from DNA binding towards biological function. Current Opinion in Plant Biology, 7, 491–498.
49. Zhang, L., Dongmei, X., Li, S., Gao, Z., Zhao, S., Shi, J., Wu, C., & Guo, X. (2011). A cotton group C MAP kinase gene, GhMPK2, positively regulates salt and drought tolerance in tobacco. Plant Molecular Biology, 77, 17–31.