利用次世代定序對具病徵表現差異性之葉片黃化病罹病日日春進行轉錄體分析

Abstract

植物菌質體 (phytoplasma) 為寄生於植物篩管細胞中之無細胞壁細菌。在自然界中主要透過媒介昆蟲進行傳播，可引起葉片黃化、簇葉、花器綠化等病徵，造成多種經濟作物損失。因接種日日春葉片黃化病植物菌質體之日日春有低機率產生無病徵枝條，其外觀與健康枝條並無差異，而初步接菌試驗中顯示無病徵枝條對日日春葉片黃化病植物菌質體具有相當高之抗性，我們推測在無病徵枝條中可能有某種系統性抗病反應被啟動。本研究利用次世代定序對罹病及健康日日春進行全轉錄體分析，比較罹病株有病徵枝條、罹病株無病徵枝條及健康株枝條之間的基因表現量差異，試著找出無病徵枝條對植物菌質體產生抗病性的可能原因。將次世代定序所得之序列資訊進行de novo assembly，得到64607條平均長度為762 bp的轉錄體序列，並與NCBI non-redundant、Gene Ontology (GO) 以及Kyoto Encyclopedia of Genes and Genomes (KEGG) 等資料庫進行比對及註解，從中得知各個序列與其他物種的關聯性及功能性為何。在植物菌質體病害中，植物激素的失衡被認為是引起病徵的原因之一，同時植物激素在系統性抗病路徑中也扮演著重要角色，透過GO enrichment analysis，發現有病徵枝條對吉貝素的反應下降，對於水楊酸的反應則是兩者皆較健康株有所提升。而在實際測量植物激素的結果中，有病徵枝條中的吉貝素些微下降而水楊酸顯著提升，無病徵枝條中的水楊酸則是些微提升。另外在無病徵枝條中，有較多與葉綠體、醣類和肌醇的新陳代謝以及幾丁質分解相關的基因被提升表現，反之有病徵枝條卻有許多和葉綠體、光合作用相關的基因表現受到抑制。除此之外，有較多與逆境、抗性相關的基因在有病徵枝條中被提高表現，無病徵枝條則和健康植株較為相似。其中也發現協助SA訊息傳導之DIR1 (Defective in Induced Resistance1)及DIR1-like之基因表現量在無病徵枝條與有病徵枝條間有極端差異，這可能與無病徵枝條之形成有所關連。同時在本篇所討論到之部分基因皆以real-time RT-PCR對基因表現量作驗證，與次世代定序獲得的表現量結果具良好的一致性。為了能更進一步了解日日春之基因功能性，本研究嘗試在日日春上建立一個過量表現系統以利後續研究。根據前人及本篇研究獲得的結果顯示，日日春接種植物菌質體產生之無病徵枝條應與水楊酸誘導之抗性反應有所關聯，但這樣的抗性反應並不是每次都能成功抵禦植物菌質體之入侵，須更進一步地釐清其細節。期望在本研究中建立之日日春轉錄體資料庫及對於無病徵枝條系統性抗病之線索，可以提供未來更進一步研究植物對抗植物菌質體病害之方向。

Phytoplasmas are wall-less prokaryotic plant pathogens that are restricted in phloem. They are transmitted by insect vectors, and can cause severe symptoms including yellows, witches’ broom and virescence on many economically important crops, therefore result in yield loss worldwide. Periwinkle leaf yellowing (PLY) phytoplasma induce witches’ broom symptoms on infected periwinkle plants. Interestingly, there are low probability of developing non-symptomatic shoots on infected plants. Previous data showed that these shoots were highly resistant to PLY phytoplasma, which we presumed systemic resistance might be induced in these shoots. To understand the mechanism of this potential systemic resistance, we compared transcriptome differences among healthy, symptomatic and non-symptomatic shoots using RNA-seq. A de novo assembly was performed to assemble pair-end reads from all conditions, and finally, 64607 unigenes were obtained with average length of 762 bp. These unigenes were annotated by comparing these unigenes with the NCBI non-redundant (nr), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using BLAST and Blast2GO program. Phytohormone imbalance has been considered to be one of the main causes of the disease symptoms, it has also been demonstrated to play important roles in plant systemic resistances. Accordingly, we focused on phytohormone-related genes and found those genes related to the response to gibberellin stimulus were mostly down-regulated in symptomatic group. On the other hand, genes related to the response to salicylic acid stimulus were mostly induced in both symptomatic and non-symptomatic group. In the measurement results of GA and SA content in leave tissues, the GA content was decreased in symptomatic group and SA content was increased both in non-symptomatic and symptomatic group. After performing GO enrichment analysis by Fisher’s exact test, there were more up-regulated genes related to chloroplast, inositol and carbohydrate metabolic, chitin catabolic in non-symptomatic group. But in the down-regulated genes from symptomatic group, gene related to chloroplast and photosynthesis. Furthermore, the expressions of stress-related genes were mostly up-regulated in symptomatic group; in contrast, the expression levels of these genes in non-symptomatic group were more similar with healthy group. We found DIR1 (Defective in Induced Resistance1) and DIR1-like, which were related to SAR signal transportation, expressed differently between non-symptomatic shoot and symptomatic shoot. This difference may relate to non-symptomatic shoots formation. The expression values of these key genes that we discussed in this study were confirmed using real-time RT-PCR, the tendency between RSEM and real-time RT-PCR were consistent in most genes. For more realizing the gene functions of periwinkle, we also attempted to construct a transient overexpression system in periwinkle for further experiments. According to the results from previous and this studies, the formation of non-symptomatic shoots may relate to SA dependent defense pathway, but this defense response was not always defense successfully against phytoplasma and we need more information to dissect the details of this systemic resistance in periwinkle. Expecting the transcriptome database and our discoveries in periwinkle defense against PLY phytoplasma could provide novel insights for further research in defense to phytoplasma and other plant pathogens.