利用酵母菌雙雜交法鑑定Argonaute4蛋白與冰花E3 ligase McCPN1具有交互作用

Abstract

冰花（Mesembryanthemum crystallinum L.; ice plant）是一種研究耐鹽機制的模式植物，在特定的生長期間若遭受到高鹽或是乾旱逆境，會誘導特定的反應以適應逆境。McCPN1是從冰花中鑑定出來的一種植物特有的RING-type copine蛋白，其蛋白結構包含在N端負責蛋白間交互作用的copine vWA domain，以及具有ubiquitin E3 ligase活性的really interesting new gene（RING）-finger domain。McCPN1與鹽誘導蛋白McSKD1具有交互作用，藉由ubiquitination修飾McSKD1間接調控蛋白的運輸。與McCPN1 高度相似的AtRGLG2蛋白具有調節阿拉伯芥對乾旱及鹽逆境的反應，為了瞭解McCPN1參與冰花鹽逆境適應的過程，利用全長的McCPN1進行酵母菌雙雜交試驗，在鹽處理冰花根部的cDNA基因庫篩選有交互作用的候選基因。共挑選120個候選基因與McCPN1具有交互作用，並且利用營養篩選與β-galactosidase活性測試，進一步挑選出E3、E9、E35、E36、E45、E46、E65與E67八個候選基因。其中E3與E9比對到Argonaute 4（AGO4），參與了小分子RNA干擾基因表現的機制；E35是具有galactose-binding domain-like與Sad 1/ UNC-like（SUN）C-terminal domain的半乳糖結合蛋白(galactose-binding protein)，利用domain分析得知，E35可能參與了細胞吸附、增殖、凋亡、細胞核的固著與移動，以及mRNA的剪接；E36是xyloglucan endotransglucosylase/hydrolases調控細胞壁的延展性並且參與組織脫落、果實的成長、成熟與老化；E45是具有DCD（Development and Cell Death）domain的蛋白，它可以藉由結合細胞骨架蛋白在細胞中移動，並且參與內質網逆境與滲透逆境下造成的程序性細胞凋亡過程；E46在DNA序列比對結果是26S ribosomal RNA而在蛋白質序列上則是比對到玉米的一個未知功能的蛋白；E65是熱休克蛋白70，負責蛋白的摺疊、再摺疊、運輸與透過溶酶體或蛋白酶體的方式降解蛋白，並且避免蛋白的凝聚；E67是SPIKE1蛋白，它調節了細胞骨架的排列，維持細胞骨架調控的細胞型態，並且參與了從內質網到高爾基氏體囊泡運輸之早期分泌路徑的動態平衡。根據酵母菌雙雜交試驗的結果推測，鹽逆境下McCPN1可藉由ubiquitination修飾候選蛋白來控制細胞型態、生長與死亡過程，以及協助蛋白運輸、在摺疊與降解。使用增強型螢光蛋白結合上McCPN1的方式來偵測蛋白累積的位置，結果在原生質膜、內質網與高爾基氏體有螢光蛋白的累積，也發現McCPN1會散佈在細胞質，此現象說明McCPN1廣泛參與細胞各部位的蛋白ubiquitin修飾過程。進一步挑選候選基因AGO4（E3與E9）進行深入的分析，利用快速擴增cDNA片段的方式（rapid amplification of cDNA ends, RACE）鑑定出全長的McAGO4。McAGO4蛋白具有N端DUF1785 domain、位於中間的PAZ domain與C端的PIWI domain。經由成對酵母菌雙雜交試驗（pair-wise Y2H）、pull-down assay、原生質體雙雜交測試（protoplast two-hybrid）與雙分子螢光互補方法（bimolecular fluorescence complementation, BiFC）等方式檢測McCPN1與McAGO4的交互作用，結果證實全長的McCPN1與McAGO4具有交互作用但是強度不高，推測可能是蛋白立體空間上排列位置所造成的結果。根據文獻中AGO4的研究結果，AGO4主要累積在細胞核並且參與了RNA主導的DNA甲基化（RNA-directed DNA methylation, RdDM），而BiFC結果發現McCPN1與McAGO4共同累積在細胞核，顯示McCPN1會移動到核內參與McAGO4所催化的反應。綜合以上結果得知，冰花McCPN1可能參與了許多鹽逆境下的反應，其中之一McCPN1藉由ubiquitination的修飾來調節McAGO4的功能，並且透過RdDM方式調控鹽逆境下的基因表現，進而增加植物對於鹽逆境的忍受度。

Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerance mechanism in higher plants. The unique responses for salinity tolerance of ice plant can be induced at specific developmental stages. McCPN1, a plant-specific RING-type copine, is identified from halophyte Mesembryanthemum crystallinum L. and has a copine vWA domain at the N-terminus for protein-protein interaction and a C-terminal really interesting new gene (RING)-finger domain which contains ubiquitin E3 ligase activity. McCPN1 interacts and ubiquitinates a salt-induced protein McSKD1, a protein involving in protein trafficking. McCPN1 has high similarity to AtRGLG2, an E3 ligase mediates auxin transport and salt and drought stress response in Arabidopsis. To explore the role of McCPN1 in the salt stress adaptation process of ice plant, full-length McCPN1 was used as a bait to perform yeast two hybrid (Y2H) screen in the cDNA library constructed from roots of salt-treated ice plant. There were 120 candidate proteins identified from Y2H screening and eight candidates, E3, E9, E35, E36, E45, E46, E65 and E67 were chosen to determine the DNA sequences, after nutrition screening and β-galactosidase activity assays. Candidate E3 and E9 were Argonaute 4 (AGO4) which participates in small RNAs interference-mediated regulation of genes expression. E35 was a galactose-binding protein with galactose-binding domain-like and Sad 1/ UNC-like (SUN) C-terminal domain and, based on domain analysis, involved in cell adhesion, proliferation, apoptosis, nuclear anchoring and migration, and pre-mRNA splicing. E36 was xyloglucan endotransglucosylase/hydrolases and participates in cell wall extensibility, abscission, fruit growth, ripening and softening. E45 was DCD (Development and Cell Death) domain protein which interacts with cytoskeleton proteins for its movement and participates in programmed cell death under endoplasmic reticulum stress and osmotic stress. E46 was 26S ribosomal RNA in DNA blasting and unknown function protein of maize in protein sequence searching. E65 was heat shock protein 70 and responsible for proteins folding, refolding, transport and degradation in lysosomes or proteasomes and preventing protein aggregation. E67 was SPIKE1 protein which mediates tissue organization, cell morphology by controlling cytoskeleton microtubule, vesicle trafficking from the ER to Golgi in early secretory pathway homeostasis. According to Y2H result, McCPN1 can interact with candidate proteins to regulate cell morphology, development, death processes, and help protein trafficking, refolding and degradation after salt stress through ubiquitination. Cellular localization of McCPN1-YFP fusion protein was found in the plasma membrane, ER and Golgi, and distributed over the cytoplasm. The result suggests that McCPN1 widely distributes in several compartments and participates the ubiquitination process in these campartments. Candidates E3 and E9 blasted as AGO4 were chosen for further analyses. Full-length McAGO4 was identified by rapid amplification of cDNA ends (RACE). The domain analysis showed it has an amino-terminal domain DUF1785, a central PAZ domain and PIWI domain at C-terminus. Full-length McCPN1 and full-length McAGO4 have weak interaction as shown by pair-wise Y2H, pull-down assay, protoplast two-hybrid and bimolecular fluorescence complementation (BiFC). The weak protein-protein interaction might be the result of steric hindrance between two proteins. It is known that AGO4 locates at the nucleus and is involved in RNA-directed DNA methylation (RdDM). The BiFC results showed McCPN1 and McAGO4 colocalized at the nucleus suggesting that cytoplasmic localized McCPN1 moves to the nucleus to participate McAGO4-mediated gene silencing. In conclusion, McCPN1 is involved in many salt stress responses and one of them is to modulate McAGO4 activity by ubiquitination and changes gene expression profiles to increase salt tolerance of ice plant.