植物菌質體 PHYL1 蛋白干擾 miR396 調控 SVP 基因 之表現並引發畸形花之形成

Phytoplasma PHYL1 effector induces abnormal flower phenotypes by interfering miR396/SVP regulation

楊喬茵(Chiao-Yin Yang)

指導教授 ： 林長平

共同指導教授 ： 林詩舜(Shih-Shun Lin)

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10765

摘要

花生簇葉病之菌質體 (peanut witches’ broom phytoplasma, PnWB) 感染日日春後花器形態會產生綠化及葉化病徵。在PnWB 菌質體中發現和Aster yellows witches’ broom 菌質體造成花器葉化相似之植物菌質體效應蛋白，依其功能將之命名為 PHYL1。當PHYL1 蛋白於阿拉伯芥大量表現時花器會有葉化現象，根據其他團隊研究顯示此現象的形成是因為PHYL1會降解開花相關的MADS-domain transcription factor (MTFs)。然而本實驗室發現在 PHYL1植物中，除葉化花外，尚有類似 SVP (AtSVP) 於阿拉伯芥大量表現時會產生的畸型花。目前對於PHYL1如何調控抑制開花之相關蛋白仍待進一步了解。本實驗室首次發現 SVP帶有 miR396的 target site，且不論於日日春或阿拉伯芥葉化花中miR396表現量皆較低。因此本實驗室假設PHYL1 蛋白會抑制miR396表現量，以及 miR396會調控 SVP基因的表現量，最終導致花器葉化。首先為了證明miR396會調控SVP，因此於菸草上暫時性的表現miR396和SVP，以確定SVP表現量會受miR396影響。此外，亦進一步以螢光報導分析 (reporter assay)確立miR396調控SVP 是藉由所發現的target site。同時，於阿拉伯芥atmiR396a 突變株進行分析時，發現其AtSVP 的表現量較對照組高，而過量表現 Cr_MIR396之轉殖株其 AtSVP表現量下降。由以上實驗皆可觀察到 miR396 確實會調控SVP 的表現。本實驗室探討 PHYL1 蛋白發現其具不穩定性以及進核之特性。另外，從 ChIP 試驗中我們發現 PHYL1蛋白會附著於 genomic DNA 上。未來希望探討PHYL1如何抑制miR396表現，最終串聯PHYL1、miR396以及SVP三者關係以了解植物菌質體造成花變葉之機制。

關鍵字

花生簇葉病菌質體 ； PHYL1 ； SVP ； miRNA396 ； 蛋白轉譯抑制

並列摘要

Phytoplasma cause virescence and phyllody symptoms in infected-plants. PHYL1 effector of Peanut witches’ broom (PnWB) phytoplasma triggers leafy flower phenotype in transgenic Arabidopsis. Recent studies show that SAP54, a PHYL1 ortholog, triggers flowering related MADS-domain transcription factor (MTFs) proteasomal degrade. Here, we provide a new evidence to demonstrate that how PHYL1 mediate leafy flower in plants. Transgenic Arabidopsis expressing PHYL1 gene (PHYL1 plants) exhibits not only leafy flower phenotype, but also showed abnormal flower phenotype, which is identical with the phenotype in transgenic Arabidopsis overexpressing SVP gene (AtSVP plant). In addition, our previous data indicated that a microRNA 396 (miR396) target site was found on MADS-domain of SVP gene. Moreover, the expressions of miR396 were low in both leafy flowers of PnWB-infected plants and PHYL1 plants. According to these observations, we hypothesized that PHYL1 represses miR396 expression, resulting in abnormal expression of SVP. To evaluate this hypothesis, we performed transient expression analysis, reporter assay and genetic approach to confirm the relationship between miR396 and SVP gene. The results of various analyses indicated one direction is that miR396 triggers SVP down-regulation. Moreover, degradome profiles indicated that miR396-mediate SVP down-regulation via mRNA decay that might be caused by translation inhibition. Furthermore, we observed some properties of PHYL1, including protein instability, nucleus localization, and the ability to bind to genomic DNA. This study demonstrated a new possible mechanism for PHYL1 effector to trigger leafy flower, and also uncovers a miRNA-mediated mRNA decay in plants.

並列關鍵字

PnWB phytoplasma ； PHYL1 ； SVP ； miRNA396 ； translation repression

參考文獻

References

Alonso, J. M. 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana Science 301:1849-1849.

Bai, X. D., Correa, V. R., Toruno, T. Y., Ammar, E. D., Kamoun, S. & Hogenhout, S. A. 2009. AY-WB phytoplasma secretes a protein that targets plant cell nuclei. Mol. Plant Microbe Interact. 22:18-30.

Bartel, D. P. 2007. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 131:11-29.

Baucher, M., Moussawi, J., Vandeputte, O. M., Monteyne, D., Mol, A., Perez-Morga, D. & El Jaziri, M. 2013. A role for the miR396/GRF network in specification of organ type during flower development, as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco. Plant Biol. 15:892-898.