PFLP提升PAMP-triggered immunity於抗細菌性軟腐病之研究

Study on PFLP-intensified PAMP-triggered immunity to bacterial soft rot disease

洪銓佑(Chuan-Yu Hong)

指導教授 ： 林宜賢

國立屏東科技大學/農學院/植物醫學系所/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00266

摘要

細菌性軟腐病是世界上重要的細菌性病害之一。運用源自甜椒可表現plant ferredoxin-like protein之pflp基因所獲得的轉殖作物已證明可防治此病害的發生。PFLP強化植物抗病也證實與其提升植物的PAMP-triggered immunity (PTI)有關，但其對細胞內訊號傳遞的影響至今仍不清楚。因此，本研究將探討PFLP提升植物對細菌性軟腐病抗病的過程中，PFLP對PTI訊號傳遞路徑之影響。首先利用Pectobacterium carotovorum subsp. carotovorum Ecc17的鞭毛蛋白(FliC)作為誘導物，於胞外表現PFLP之阿拉伯芥上確認PFLP能提升植物H2O2快速產生及癒傷葡聚醣累積等PTI指標。隨後，以細菌鞭毛蛋白上具保守性的胺基酸flg22Pst作為誘導物，亦發現PFLP可增加flg22Pst所誘導的H2O2快速產生、癒傷葡聚醣的累積以及與防禦相關的H2O2累積、過敏性反應與AtrbohD的基因表現。為了解PFLP在強化PTI的過程中是否與MAPK與CDPK之訊號傳遞路徑有關，分別以FRK1與PHI1之基因表現進行分析，結果顯示PFLP可以強化由flg22Pst誘導的MAPK路徑，但僅能延長flg22Pst誘導的CDPK路徑之訊號。在MPK6與CDPK6之基因表現上，PFLP卻無法增加flg22Pst所誘導MPK6與CDPK6的基因表現，推測PFLP可能強化激酶的活性。為進一步了解PFLP強化癒傷葡聚醣累積是否與MAPK路徑有關，結果發現PFLP在mkk5與mpk6突變株上亦能增加flg22Pst所誘導的癒傷葡聚醣累積，顯示PFLP可同時強化癒傷葡聚醣的累積與MAPK路徑兩條路徑。在以軟腐細菌Ecc17進行接種試驗之結果顯示，PFLP提升阿拉伯芥的抗病能力在mekk1, mkk5, mpk6突變株上均顯著下降。此外，PFLP亦能強化由flg22Pst所誘導的水楊酸路徑之PR1以及茉莉酸/乙烯路徑PDF1.2之基因表現，說明PFLP可能多元地提升植物的防禦訊號。綜合以上結果推測PFLP提升植物對細菌性軟腐病之抗病性與其強化PTI的多重防禦路徑有關，而MAPK路徑扮演關鍵之角色。

關鍵字

Plant ferredoxin-like protein (PFLP) ； PAMP-triggered immunity (PTI) ； 細菌性軟腐病 ； MAPK (mitogen-activated protein kinase) ； CDPK (calcium-dependent protein kinase)

並列摘要

Bacterial soft rot disease was an important disease worldwide. It has been proven that transferring plant ferredoxin-like protein (pflp) to various crops was able to control bacterial soft rot disease. PFLP-mediated resistance against bacterial soft rot pathogen was associated with the intensification of PAMP-triggered immunity (PTI), but its effect on intracellular signaling was still unknown. Thus, this study will discuss what effect on PTI signaling by PFLP in PFLP-intensified resistance to bacterial soft rot disease. Firstly, it was confirmed PFLP could improve the response of PTI, including rapid H2O2 generation and callose deposition in Arabidopsis upon the existence of PFLP and flagellin (FliC). Subsequently, the results also exhibited that PFLP intensified rapid generation of H2O2, callose deposition, accumulation of H2O2, and hypersensitive response (HR) while the existence of the conserved amino acid of bacterial flagellin, flg22Pst. In addition, PFLP increased the expression of AtrbohD transcripts when flg22Pst was used as an elicitor. To gain more insight about whether PFLP-intensified PTI are related to MAPK and CDPK pathway, results indicated PFLP could intensify flg22Pst-induced MAPK pathway, but only prolong flg22Pst-induced CDPK pathway based on mRNA transcripts of marker genes, FRK1 and PHI1 in MAPK pathway and CDPK pathway, respectively. The data also revealed that the increasing expression of these two genes may be due to PFLP-intensified activities of MAPK and CDPK. To understand if PFLP-intensified callose deposition is related to MAPK pathway, results showed that PFLP intensified flg22Pst-induced callose deposition in mkk5, and mpk6 plants, implying PFLP could intensify flg22Pst-induced callose deposition and MAPK pathway, simultaneously. The data indicated PFLP-intensified resistance is reduced in mekk1, mkk5, and mpk6 plants after inoculation with Pectobacterium carotovorum subsp. carotovorum Ecc17. Besides, PFLP also intensified flg22Pst-induced salicylic acid pathway and jasmonic acid/ethylene pathway based on increasing marker gene expression of PR1 and PDF1.2, respectively. These results suggested that PFLP could intensified multiple defense signalings. According to the above results, PFLP-intensified resistance to P. carotovorum subsp. carotovorum Ecc17 may be associated with multiple PTI signaling, and MAPK pathway plays the key role in PFLP-intensified resistanceified resistance.

並列關鍵字

Plant ferredoxin-like protein (PFLP) ； PAMP-triggered immunity (PTI) ； bacterial soft rot disease ； MAPK (mitogen-activated protein kinase) ； CDPK (calcium-dependent protein kinase)

參考文獻

12. Chao, Y. C., Hsu, S. T., and Tzeng, K. C. 2010. Bactericidal efficacy of chlorine dioxide against Pectobacterium chrysanthemi and its control for bacterial soft rot on stalk of Oncidium cut flower. Plant Pathology Bulletin 19: 127-136.

44. Liau, C. H., Lu, J. C., Prasad, V., Hsiao, H. H., You, S. J., Lee, J. T., Yang, N. S., Huang, H. E., Feng, T. Y., Chen, W. H., and Chan, M. T. 2003. The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid. Transgenic Research 12: 329-336.

5. Aslam, S. N., Erbs, G., Morrissey, K. L., Newman, M. A., Chinchilla, D., Boller, T., Molinaro, A., Jackson, R. W., and Cooper, R. M. 2009. Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses. Molecular Plant Pathology 10: 375-387.

6. Beckers, G. J. M., Jaskiewicz, M., Liu, Y., Underwood, W. R., He, S. Y., Zhang, S., and Conrath, U. 2009. Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. The Plant Cell 21: 944-953.

7. Bethke, G., Unthan, T., Uhrig, J. F., Poschl, Y., Gust, A. A., Scheel, D., and Lee, J. 2009. Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proceedings of the National Academy of Sciences 19: 8067-8072.