利用VirB2變異體研究T-線毛 影響農桿菌介導植物轉型能力之角色;Functional characterization of VirB2 variants to study the role of T-pilus in Agrobacterium-mediated transformation

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题名:	利用VirB2變異體研究T-線毛影響農桿菌介導植物轉型能力之角色;Functional characterization of VirB2 variants to study the role of T-pilus in Agrobacterium-mediated transformation
作者:	許馨心;Hsu, Sin-Sin
贡献者:	生命科學系
关键词:	農桿菌;農桿菌介導轉型作用;第四型分泌系統;T-線毛;VirB2 蛋白;Agrobacterium;Agrobacterium-mediated transformation (AMT);T4SS;T-pilus;VirB2
日期:	2024-10-04
上传时间:	2025-04-09 16:09:05 (UTC+8)
出版者:	國立中央大學
摘要:	農桿菌是一種植物病原細菌，它能感知植物信號並啟動感染過程，通過第四型分泌系統（T4SS）將效應蛋白質和轉移DNA（T-DNA）運送至植物細胞內，進行短暫表達/轉型）或嵌入植物染色體造成穩定轉型。第四型分泌系統的結構包括由11個VirB蛋白質(VirB1-11)所組成的轉運通道以及負責招募T-DNA及效應蛋白質的VirD4。VirB1-11蛋白質也負責生成T-線毛，其中VirB2蛋白質為T-線毛的主要組成分。先前研究發現一些缺乏T-線毛的VirB2變異體雖然不能形成T-線毛，但仍然誘導腫瘤產生，然而這些變異體在阿拉伯芥幼苗中的短暫轉型效率卻顯著降低，故稱為這種突變株為解偶聯突變體（T-pilus-/Vir⁺ uncoupling mutants）。這表明T-線毛對T-DNA的轉移和病原性並非必需，但可能提高農桿菌介導的轉型（AMT）效率。T-線毛在農桿菌介導的轉型中的具體機制尚待深入研究。本研究使用了兩株T-pilus-/Vir⁺的解耦突變體(VirB2L94A和VirB2A110G），以探討T-線毛在農桿菌介導的轉型效率中的作用。我們同時檢測了解偶聯突變體在細菌生長、VirB2蛋白質表達和阿拉伯芥幼苗中植物定殖的能力。結果顯示，儘管解偶聯突變體的短暫轉型效率降低，其生長速率和定殖於植物的效率與野生型相似。在細菌培養中，解偶聯突變體的VirB2蛋白質累積量與野生型相同，但在與阿拉伯芥共培養時，VirB2L94A和 VirB2A110G其蛋白質累積量有所降低。VirB2L94A和VirB2A110G在阿拉伯芥花浸轉化中的穩定轉型效率也與野生型相似。總而言之，研究結果表明，T-線毛的存在並不影響穩定轉型效率，而細菌生長和植物定殖效率並不是VirB2L94A和VirB2A110G其短暫轉型效率降低的原因。;Agrobacterium is a phytopathogenic bacterium that transfers the effector proteins and transferred DNA (T-DNA) into the plant cells for transient expression/transformation or stable transformation. This transfer process is mediated by the type IV secretion system (T4SS), consisting of 11 VirB proteins (VirB1-11) forming transmembrane protein channel and a coupling protein VirD4. The VirB protein channel also mediates the biogenesis of a surface filament, T-pilus. VirB2 protein, as the major subunit of T-pilus, is essential for virulence. A previous study showed that some VirB2 variants deficient in T-pilus still possess wild-type levels of tumorigenesis but have highly attenuated transient transformation efficiency in Arabidopsis seedlings. However, the underlying mechanisms of T-pilus involved in Agrobacterium-mediated transformation (AMT) await future investigation. In this study, I employed two T-pilus-/Vir⁺ uncoupling mutants, VirB2L94A and VirB2A110G, that retained virulence without detectable extracellular VirB2/T-pilus, to study the role of T-pilus in AMT efficiency and the phenotype in bacterial growth, protein level, and plant colonization efficiency in Arabidopsis seedlings. The results showed that although the transient transformation efficiency of the uncoupling mutants was attenuated, the uncoupling mutants retained similar growth rate and colonization efficiency to the wild-type. The uncoupling mutants exhibited similar VirB2 protein levels to the wild-type in virulence induction medium but their protein levels are reduced when co-cultured with Arabidopsis seedlings. VirB2L94A and VirB2A110G showed similar stable transformation efficiency by Arabidopsis floral dip transformation. In summary, the findings suggest that the existence of T-pilus does not affect the stable transformation efficiency, and the bacterial growth and plant colonization efficiency of Agrobacterium VirB2 of the uncoupling mutants is not the cause of reduced transient transformation efficiency.
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