Expression and regulation analyses of virB genes ingrobacterium tumefaciens
Date Issued
2008
Date
2008
Author(s)
Chen, Yi-Chun
Abstract
Agrobacterium tumefaciens is a plant pathogenic bacterium, the causal agent of crown gall diseases on wide range of dicotyledons. The tumors are caused by transferring T-DNA (Transferred DNA) from bacterium into the host plant genome. A. tumefaciens is capable of sensing the plant-released signal molecules such as sugars and phenolic compounds (e.g. acetosyringone，AS) to activate the expression of virulence (vir) genes encoded by the tumor-inducing (Ti) plasmid. Among these, the virB operon encoding 11 VirB proteins and VirD4 comprises a type IV secretion system (T4SS) to transfer T-DNA and effectors from bacteria into the host plant cells. AS-induced vir gene expression is regulated at transcriptional level via VirA/VirG with maximal activity in acidic AB-MES minimal medium. However, it is not clear whether vir genes can be efficiently induced by AS when grown in rich medium or are regulated at posttranscriptional levels. In this study, we aim to understand whether virB genes are expressed and regulated differently when A. tumefaciens is grown in different culture media for AS induction. The virB gene expression was analyzed at transcriptional/translational levels and protein steady state when induced by AS in both acidic AB-MES minimal medium and acidic 523 rich medium. By transcriptional fusion to gfp (green fluorescent protein), the promoter activities of virB, virD, and virE are efficiently induced when grown in either AB-MES or 523 media although the promoter activities are higher in AB-MES medium than in 523 medium at early time points. To further investigate virB gene expression at protein levels, Western blotting and translational fusions were carried out. By Western blot analysis, three groups of VirB proteins are classified based on their protein accumulation patterns in both AB-MES and 523 cultures. VirB2, VirB7, and VirB9 belong to group I as they accumulate at higher levels at early time points (up to 16 hr) in AB-MES in comparison to those in 523. The second group of proteins include VirB1, VirB4, VirB5, VirB8, and VirB11 as they accumulate at higher levels at late time points (after 24 hr) in AB-MES in comparison to those in 523. The third group includes VirB3 and VirB10 as they accumulate at very low levels in 523, in contrast to those at much higher levels in AB-MES. Interestingly, several VirB-GFP translational fusions result in efficient AS-induced activity in both AB-MES and 523 media. By VirB/D4-mediated IncQ plasmid RSF1010 transfer between A. tumefaciens strains, only very little transfer events are detected when conjugation was performed on 523. In conclusions, our data suggest that virB is transcribed and translated efficiently when A. tumefaciens was induced by AS in both AB-MES and 523 media. However, while all tested VirB proteins accumulated stably when grown in AB-MES medium, certain VirB proteins are less stable when grown in 523 medium, which may lead to the failure in assembly of the functional T4SS for DNA transfer.
Subjects
Agrobacterium tumefaciens
acetosyringone
vir regulon
gene expression
gene regulation
type IV secretion system
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