柑橘鱗砧類病毒小片段RNA於番茄上病程進展和機制探討

Abstract

類病毒為裸露、單股的環狀RNA分子，大小約為246至433個核苷酸，且不轉譯任何蛋白質。類病毒影響病徵產生之機制尚未明瞭。研究顯示，21至24個核苷酸大小的類病毒小片段RNA (viroid-derived small RNA, vd-sRNA) 會以寄主的messenger RNA (mRNA) 作為目標並切割，引起寄主mRNA的RNA靜默 (RNA silencing)。有一假說推定：vd-sRNA會抑制寄主mRNA的表現，進而產生病徵。柑橘鱗砧類病毒 (Citrus exocortis viroid, CEVd) 為主要危害柑橘的類病毒，其亦可感染茄科作物番茄。CEVd於番茄上可造成植株矮化、葉片黃化、扭曲無法伸展及向下捲曲之病徵。本研究自行製備番茄 (Solanum lycopersicum) Argonaute蛋白1a (SlAGO1a) 及番茄Argonaute蛋白1b (SlAGO1b) 之抗體，並自健康及感染CEVd之番茄植株進行SlAGO1a之免疫沈澱 (immunoprecipitation, IP)。於IP產物中萃取RNA，並與健康及感染CEVd之總量RNA (total RNA) 一同進行小RNA次世代定序，藉以得知CEVd是否會產生vd-sRNA及vd-sRNA是否會進入AGO蛋白中進行RNA靜默。我們於感病植株之total RNA及SlAGO1a-IP產物中皆發現序列與CEVd基因組相同的小片段RNA。SlAGO1a-IP中發現22,216種小片段RNA共讀取4,303,121次 (reads)，其中有950種，409,550讀取次數的小片段來自CEVd的基因組。比較total RNA與SlAGO1a-IP之vd-sRNA可發現兩者熱點分佈並不完全相同。此外，以psRNATarget軟體預測，950種的vd-sRNA可產生1,540個候選標靶基因。降解組定序 (Degradome sequencing) 將可證明這些候選標靶基因是否確實被vd-sRNA切割，而體外RNA誘導沈默複合體 (RNA-induced silencing complex, RISC) 活性分析將可為vd-sRNA調控寄主基因表現提供直接的證據。

Viroids are a 246- to 433-nts of non-coding, naked, single-stranded circular RNA. How viroid causes symptoms that remains unclear. A hypothesis proposes viroid-derived small RNAs (vd-sRNAs) (21- to 24-nts in length) might target and cleavage host mRNAs, resulting in gene silencing and leading to symptom development. CEVd-infected tomatoes show stunting, epinasty, and leaf distortion symptoms. We generated antibodies of tomato Solanum lycopersicum Argonaute 1a (SlAGO1a) and SlAGO1b. We performed immunoprecipitation with SlAGO1a IgG from CEVd-infected tomato plants and identified vd-sRNAs by small RNA deep sequencing. There are 4,303,121 reads in 22,216 species of small RNAs were found in SlAGO1a-IP profile, whereas 409,550 reads in 950 species belong to vd-sRNAs of CEVd. The hot-spot distributions of the vd-sRNAs between total small RNA and SlAGO1a-IP indicated that the critical vd-sRNAs which were loaded into SlAGO1a have different hot-spots. Based on the target prediction, 1,540 mRNAs of tomato plants are predicted by 950 species of vd-sRNAs. We are performing the degradome assay to evaluate these candidate targets and the in vitro RISC activity assay will perform with these IP SlAGO1s to confirm the vd-sRNA-mediated host gene cleavage. Uncovering predicted target mRNAs and the interaction between small RNAs will provide more insight information on the pathogenic CEVd-derived small RNAs.