High-throughput transcriptome and small RNA analysis for studying phytoplasma infection on Catharanthus roseus using Next Generation Sequencing

植物菌質體是一種沒有細胞壁且無法純培養的絕對寄生菌，在世界各地上造成許多重要的經濟作物病害。花生簇葉病植物菌質體感染日日春後會造成花器發育不正常，包括花瓣褪色、花器綠化、葉片化等病徵。由於目前花生簇葉病植物菌質體和日日春皆缺乏完整基因體的資訊，因此對於植物菌質體如何造成這些花部型態改變的原因至今瞭解不多。本研究是利用次世代高通量定序 (Next Generation Sequencing, NGS) 的方式針對日日春於感染花生簇葉病植物菌質體後其花器RNA 及small RNA 表現差異進行分析，目的在探討植物菌質體感染之後日日春花器病徵造成的原因。次世代高通量定序在分析轉錄組 (transcriptome) 上具有快速和高通量的優勢，並且可以應用在非模式物種轉錄組的分析。利用NGS定序的結果顯示，經由重新重組 (de novo assembly) 的方式，在健康花器和罹病之花器的轉錄組中重組成功率達85.9%，總計重組出60,580 contigs，且平均長度為1kb多的基因序列。進一步將日日春轉錄組和阿拉柏芥轉錄蛋白資料庫經由BLAST比對進行基因註解，結果顯示日日春轉錄組比對到的序列長度分佈和阿拉柏芥資料庫很相似，日日春轉錄組基因序列之完整度也比日日春EST 資料庫的基因序列高。藉由阿拉柏芥gene ontology (GO) 的資料庫進行日日春轉錄組分類後，從中挑選出有差異性表現的基因，結果發現在罹病之花器中和抗病相關、煙中化合物 (karrikin)、葉綠體及光合作用相關基因之基因表現量會上升。在small RNA 分析部分，經由比較日日春健康花器和罹病之花器的small RNA長度分佈，結果發現在罹病花器中24-nt small RNA 的數量會大量下降。本研究統計出在日日春轉錄組中414 個基因可能和24-nt small RNA 經由RNA-directed DNA methylation (RdDM) 機制產生甲基化的功能有關，經由分類發現這些基因在罹病之花器表現量會上升而且主要和抗病及葉綠體的基因有關。進一步和罹病之花器的型態觀察做比較，結果發現這些基因上升的現象和罹病之花器具有光合作用能力和葉片化型態出現的特徵一致。另一方面在日日春轉錄組中發現有許多200-600 nt基因序列無法和阿拉柏芥轉錄蛋白資料庫比對到，推測可能為 non-coding RNA (ncRNA) 並具調控組蛋白甲基化功能的潛力。經由分析結果推測花生簇葉病植物菌質體的感染使得日日春轉錄體在24-nt small RNA下降，進而使得和RdDM 調控的有關基因表現量上升，表示植物菌質體可能藉由調控24-nt small RNA的表現，影響植物基因甲基化的能力，進而使得因為甲基化抑制的基因回復表現，此一推測未來仍需要進一步相關實驗進行驗證。由本研究結果得知利用次世代高通量定序進行的生物資訊分析策略，可以提供許多大量的基因資訊，對於研究植物和植物病原菌的交互作用關係是一種嶄新的研究方向。Phytoplasmas are special bacteria that are obligated parasites on plants and cause severe damage on economical crops worldwide. The peanut witches-broom (PnWB) phytoplasma were caused significant floral malformation such as virescence, phyllody and petal discoloration in a numerous phytoplasma ideal host plant periwinkle (Catharanthus roseus). Studying of phytoplasmas-host interaction, however, is limited due to the lack of genome information of periwinkle plant. Next generation sequencing (NGS) is a new high-throughput sequencing technology that is rapid and economic on the genomic project, especially on non-model species. In this study, the whole transcriptome and small RNA profiles of the flower organs of the healthy and PnWB phytoplasma-infected periwinkles were investigated by NGS. The 85.9% successful rate of de novo assembly from periwinkle sequence reads are generated 60,580 contigs. The sequence analysis results showed the periwinkle sequences are similar to those in Arabidopsis coding sequence (CDS) database and more complete compared with Catharanthus EST database. Based on the gene ontology (GO) on classification roles with Arabidopsis CDS database, gene related to defense, photosynthesis, chloroplast, and karrikin were highly expressed in transcriptome of PnWB phytoplasma-infected flower profiles. In addition, the total number of 24-nt small RNA displayed dramatic decrease in PnWB phytoplasma-infected flower. Moreover, there were 414 candidate genes in periwinkle transcriptome that were likely associated with the 24-nt small RNAs and RNA-directed DNA methylation (RdDM); those genes were highly expressed in diseased organs and were associated with defense and chloroplast development. This results were consistent with the morphological phenotype between healthy and PnWB phytoplasma-infected flower organs that photosynthesis and chloroplast were developed in the phyllody of floral organ. Furthermore, the 200-600 nt non-coding RNA (ncRNA) were indentified in contigs that might involve in the histone methlyation of periwinkle. The results suggested that PnWB phytoplasma might inhibit the 24-nt small RNA biogenesis that caused the suppression of RdDM pathway to reactative the methlyated genes. In conclusion, this study provides a new vision to understand the disease mechanism between host and phytoplasma in the periwinkle plant by bioinformatics with powerful and fast high throughput technique