Investigation of phytoplasma effector PHYL1Pn that interferes plant miRNA-mediated regulation for leafy flowering using an integrated omics approach

植物菌質體 (Phytoplasma) 為絕對寄生型維管束局限性病原細菌,感染其指示 植物日日春 (Catharanthus roseus) 後,於花部會產生花器綠化及花器葉化等特殊 病徵,而此葉花花器已被報導與植物菌質體之效應蛋白 SAP54 有關。本研究首次 觀察到直徑小於 1 μm 且多樣化的 PnWB 細胞形態,並顯示其 PHYL1Pn 效應蛋 白為 SAP54 之同源蛋白,可造成植物產生花器葉片化病徵。PHYL1Pn 轉基因阿拉 伯芥花器葉化表現型與 SHORT VEGETATIVE PHASE (SVP) 之轉基因植物相似, 其 AtSVP 表現量顯著提升,導致開花時間延後,而原位雜交實驗則證明 PHYL1Pn 會導致 SVP 在花瓣的累積表現。此外,PHYL1Pn 轉基因植物之 miR396 表現量明 顯減少,造成植株根部延長。本研究推測 PHYL1Pn 干擾 SVP 與 miR396 在植物 內之表現量,進而導致葉狀花的產生。除此之外,前人研究中日日春健康花器 (HF) 與 PnWB 感染第四型葉狀花 (S4) 之轉錄體及小分子 RNA 之次世代定序結果 則透過客制化微型核酸晶片進行其基因表現量之驗證。再者,降解組定序則用來 驗證微型核酸與目標基因間配對之預測結果,並藉此尋找新的受微型核酸所調控 之基因。本研究所建立之整合性高通量分析流程配合生物實驗,期望未來能廣泛 應用於非模式生物於分子生物學領域之研究。Phytoplasma is a cell wall-less bacteria that obligate parasites in phloem and causes virescence and phyllody on flowers. In this study, we confirm the peanut witches’ broom (PnWB) phytoplasma infection in Catharanthus roseus and first report the morphology and size of PnWB phytoplasma. In addition, the PHYL1Pn, an effector of PnWB phytoplasma, causes virescence and phyllody phenotypes in Arabidopsis (herein leafy flower), which similar to SHORT VEGATATIVE PHASE (SVP) over-expression plants. In PHYL1Pn plants, the miR396 was down-regulated whereas the AtSVP was up-regulated and the leaf and root phenotypes of the transgenic plants were agreed with miR396 and AtSVP expression patterns. We suggested that PHYL1Pn may induce leafy flower transition through interfering miR396 and AtSVP expression. In this study, we built up an intergraded omics pipeline for a non-model organism research. The three high-throughput omics was used to verify the transcriptome and predicted miRNA targets profiles of C. roseus, and also used to identify novel miRNA targets in growth, development and metabolism related genes in C. roseus. The experimental methods including transgenic and mutant lines, northern blot and in situ hybridization were applied in confirming phytoplasma effector affecting miRNA/target regulation.口試委員審定書............................................................................................................... I 誌謝 .................................................................................................................................. II 中文摘要........................................................................................................................ IV Abstract ............................................................................................................................V Contents......................................................................................................................... VII Introduction .......................................................................................................................1 Materials and Methods....................................................................................................10 Plant materials and growth condition......................................................................10 Phytoplasma inoculation .........................................................................................10 RNA extraction .......................................................................................................11 Transmission electron microscope observation of PnWB ......................................11 Transgenic plants ....................................................................................................12 Northern blot for miR396 detection........................................................................13 Real-time RT-PCR ..................................................................................................13 Floral tissue staining ...............................................................................................15 In situ hybridization ................................................................................................15 MicroRNA/target prediction ...................................................................................16 Microarray and degradome .....................................................................................17 Bioinformatics analysis...........................................................................................17 Results .............................................................................................................................19 PnWB observation ..................................................................................................19 The phenotype observation of PHYL1Pn plant.........................................................20 Anatomy of leafy flower .........................................................................................21 The AtSVP expression in PHYL1Pn plant ................................................................22 Flowering time and root length observation ...........................................................22 AtSVP signal detection ............................................................................................24 The integrated work flow of high-throughput omics ..............................................24 Comparison of gene expression level between NGS and microarray.....................25 MiRNA target validation.........................................................................................26 The expression profiles of confirmed miRNA target genes....................................28 MADS transcription factor gene expression levels.................................................29 Discussion .......................................................................................................................31 Morphological observation of PnWB .....................................................................31 PHYL1Pn effector triggers leafy flower formation..................................................32 PHYL1Pn affects the MADS genes expression in IM ..............................................33 The effect of PHYL1Pn on AtSVP and miRNA396 .................................................35 PHYL1Pn enhanced AtSVP expression in petal .......................................................36 Integrated omics approaches for prediction the bioinformatics accuracy...............37 Novel miRNA targets in C. roseus .........................................................................39 References .......................................................................................................................41 Tables ..............................................................................................................................59 Figures.............................................................................................................................66 Supplementary material and methods .............................................................................82 Plant tissue fixation .................................................................................................82 Tissue staining.........................................................................................................84 In situ hybridization ................................................................................................8