農桿菌 VirB2 蛋白質之功能鑑定與發展一種用於基因功能性分析之高效率轉型方法

Abstract

Agrobacterium tumefaciens is a Gram-negative plant pathogenic bacterium with the unique ability to transfer DNA into plant genomes. This transfer is mediated by an evolutionarily conserved type IV secretion system (T4SS) comprising VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. In this study, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2-) and tumorigenesis on tomato stems (Vir+ or Vir-), the mutants could be classified into three groups: ExB2-/Vir-, ExB2-/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2-/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation and therefore renamed as T-pilus-/Vir+ mutants. Interestingly, although the five T-pilus-/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency. In view of the potential applications of the transient transformation assay using the Arabidopsis seedlings in gene functional studies, further systematic investigations of various biological factors and growth variances were performed to define a combination of key factors that maximize the high transient transformation and reporter gene expression efficiency in Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium transient transformation assay, we found that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in high transient expression levels in all infected young Arabidopsis seedlings. The presence of AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced transient expression levels, which are significantly higher than two existing methods. Importantly, the highly increased transient expression efficiency is not only achieved in the immune receptor mutant efr-1 but also in wild type Col-0 seedlings. Finally, this high transformation efficiency enabled the versatile applicability of the method for examining transcription factor actions and circadian reporter-gene regulation, as well as protein subcellular localization and protein–protein interactions in physiological contexts. This simple, fast, reliable, and robust transient expression system is named as AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings and offers a new system to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.