一、來自TRIM 突變庫水稻不稔實突變株sstl之研究 二、水稻黃斑駁病毒RYMV 中P1蛋白感染寄主的作用功能

Abstract

水稻是世界上主要的糧食作物之一，直到2025年將會有超過39億人口以稻米作為主要食物來源。特別是在亞洲和非洲地區，由於人口的急劇增加，水稻的產量遠遠落後於人口的需要，同時因食品安全的問題與日益嚴重的環境變化，使得調整作物適應氣候變遷，建立永續農業，解決餵養世界人口等成為迫切關注之議題。為此，我們在本論文中提出涵蓋兩個不同主題之研究，希望得以針對來自水稻不稔實突變體，瞭解其不稔實之可能原因。另外亦利用水稻黃斑駁病毒 (RYMV) ，瞭解其病毒P1蛋白在感染寄主的作用功能。 一、來自TRIM突變庫水稻不稔實突變株sstl之研究 水稻未發育的花藥或異常花粉導致雄性不稔實，進而引起產量嚴重銳減。因此了解水稻雄性不育機制是一重要研究課題。我們根據水稻發育中具有不稔實之花穗形態，找出一T-DNA未標記的插入突變體sst1。而在其後代分離族群中，完全不稔實的突變株 (sstl-s) 更是表現出其花粉嚴重缺陷與毫無萌發活力。此外在不稔實sstl-s的花藥轉錄體學分析中也透露，因生物代謝途徑的顯著差異，例如細胞壁代謝、脂質、二級代謝物和澱粉合成相關基因的異常抑制表現，造成sstl-s花藥的形態具有不規則的花粉外壁、沒有花粉內壁結構、花粉中毫無澱粉累積、與花藥中因缺乏黃酮類化合物而導致的白色花藥。此外在sstl-s也觀察到因其小孢子囊的缺陷而導致異常的花藥室和中止小孢子發育。對此，藉由sstl-s我們闡述小孢子發育的重要性與脂質、澱粉和細胞壁合成相關基因的表達有關，進一步了解sstl-s孢子囊的缺失在花藥和功能性小孢子發育中，能關鍵決定花粉的不稔實。 二、水稻黃斑駁病毒RYMV中P1蛋白感染寄主的作用功能 水稻黃斑駁病毒RYMV遍布在非洲各國，對水稻造成嚴重破壞導致水稻產量大幅下降。研究RYMV中的病毒蛋白P1，能使我們更了解病毒感染的機制。P1是一病毒抑制子，屬新型鋅指蛋白，其能藉由氧化還原作用而調節與鋅之結合能力，進而改變其蛋白構造。由於P1在病毒傳播中的作用仍不甚清楚，為此在我們的研究中，將針對P1在病毒複製、運移與抵禦寄主的可能機制進行探討。我們研究指出，具突變的P1病毒RYMV仍可以被複製，但複製能力比正常病毒低。具有突變的P1 RYMV，複製病毒RNA的效率從原本24 hrs會下降至48 hrs，表明功能性P1不僅調節病毒複製，還可能維持病毒RNA的穩定性。此外突變的P1 RYMV在感染水稻植物後，沒有顯示出病毒累積也無病癥，表示P1可能會幫助感染後病毒的傳播，而P1蛋白也顯示位在與細胞內物質分泌相關的胞器內質網上。同時我們也觀察到P1蛋白可在兩個相鄰細胞之間運移傳遞，了解到P1可能具有移動能力。熱誘導轉基因水稻也顯示P1轉錄體在熱處理後，可從下部葉片移動到未經熱處理的上部葉片，這一證據強烈表明P1可能參與病毒的長距離的移動。成功的病毒感染除了病毒本身的致病力，還需由寄主防禦機制來決定。我們的結果顯示具有功能的P1病毒可以通過降低抵禦生物逆境相關基因如PR10A和CPR5的表現，來幫助病毒抵制寄主防禦。此外P1在易受RYMV感染的水稻品種中，也可藉其抑制DCL-like和AGO的表達量來影響寄主RNA靜默途徑，進而使寄主易感病。總結上述結果，我們瞭解病毒P1蛋白作為多功能角色，包含參與病毒複製、運動以及對寄主的反擊之重要性。

Rice (Oryza sativa) is one of the main crops in the world. By 2025, more than 3.9 billion people count rice as daily food energy especially in Asia and Africa. Because of the increased population, the production of rice is still far away behind requirements. Meanwhile, the food safety issue related to feed the world continuously, to adjust crop adaptation in climate change, to establish sustainable agriculture, etc. are urgent to be solved. My study in this thesis will cover two different topics described as followings: First, studies of a rice sterile mutant (semi-sterile; sstl) from the TRIM collection and the second investigation, multifunctional roles of rice yellow mottle virus (RYMV) P1 protein in host infection. I. Studies of a rice sterile mutant sstl from the TRIM collection Sterility significantly affects rice production and leads to yield defects. The undeveloped anthers or abnormal pollens represent serious defects in rice male sterility. Therefore, understanding the mechanism of male sterility is an important task. Here, we identified an untagged T-DNA insertion mutant sstl based on its semi-sterile morphology of rice development. We investigated the fully sterile mutants (sstl-s) of sstl segregated progeny showed defective pollens and abnormal anthers. Transcriptomic analysis of sterile sstl-s revealed significant differences in several biosynthesis pathways, such as downregulated cell wall, lipids, secondary metabolism, and starch synthesis. The downregulation of gene expression is consistent with the morphological characterization of sstl-s anthers with irregular exine, absence of intine, lack of starch accumulation in pollen grains and no accumulation of flavonoids in anthers. Moreover, defective microsporangia development led to abnormal anther locule and aborted microspores. The downregulated expression of lipids, starch, and cell wall synthesis-related genes resulted in loss of fertility. In summary, the analysis of sstl-s mutant pointed out the importance of microsporangia in the development of anthers and functional microspores. II. Multifunctional roles of RYMV P1 protein in host infection Rice yellow mottle virus (RYMV), a virus spread all over African countries, has caused significant damage to rice plant and resulted in dramatic decrease of yield in Africa. ORF1 of RYMV encodes P1 protein which belongs to a novel zinc finger family with its conformation regulated by redox switches. P1 protein was suggested as suppressor after virus infection; however, the function of P1 in virus transition is still unclear. In the present study, we explored the possible mechanisms of P1 in virus replication rate, movement ability, and suppression of host defense. We showed that mutated P1 RYMV still could replicate but at less replication rate than that of normal RYMV after transfection into rice protoplast. All replication rate with mutated P1 RYMV infection decreased from 24 hrs to 48 hrs after inoculation. This result indicated that functional P1 not only could regulate RYMV replication production but also maintain viral RNA transcription stability. In addition, mutated P1 RYMV showed no virus accumulation after infection into rice plant and none of the infected plant displayed virus accumulation. This indicated P1 could help regulate virus spread after infection. P1 protein co-localized with ER marker suggested this protein might participate in virus spread in the cell. Meanwhile, P1-eGFP fusion protein was transmitted between two adjacent cells and thus illustrating P1 mobility. Heat-inducible transgenic rice also displayed P1 transcript moving from lower leaf to non-heat treated upper leaf after heat treatment. This evidence strongly suggested that P1 RNA could be involved in virus long-distance movement. In additional to virus infection ability, the virulence can also be affected by host defense. Our data suggested functional P1 protein might help virus resist host defense by decreasing the expression of biotic-related genes such as PR10A and CPR5. Moreover, P1 could hijack host silencing pathway through down-regulating DCL-like and AGO expression to make host susceptible. Taken together, these results illustrate that P1 protein may serve as multifunctional roles to participate in virus replication, movement, as well as counterattack.