Abstract
We previously reported that the Agrobacterium virulence protein VirD5 possesses transcriptional activation activity, binds to a specific DNA element D5RE, and is required for Agrobacterium-mediated stable transformation, but not for transient transformation. However, direct evidence for a role of VirD5 in plant transcriptional regulation has been lacking. In this study, we found that the Arabidopsis gene D5RF (coding for VirD5 response F-box protein, At3G49480) is regulated by VirD5. D5RF has two alternative transcripts of 930 bp and 1594 bp that encode F-box proteins of 309 and 449 amino acids, designated as D5RF.1 and D5RF.2, respectively. D5RF.2 has a N-terminal extension of 140 amino acids compared to D5RF.1, and both of them are located in the plant cell nucleus. The promoter of the D5RF.1 contains two D5RE elements and can be activated by VirD5. The expression of D5RF is downregulated when the host plant is infected with virD5 deleted Agrobacterium. Similar to VirD5, D5RF also affects the stable but not transient transformation efficiency of Agrobacterium. Some pathogen-responsive genes are downregulated in the d5rf mutant. In conclusion, this study further confirmed Agrobacterium VirD5 as the plant transcription activator and identified Arabidopsis thaliana D5RF.1 as the first target gene of VirD5 in regulation.
Highlights
Agrobacterium (Agrobacterium tumefaciens) is a soil-borne pathogen that transforms plant cells into tumor cells by the delivery of an oncogenic piece of DNA from its Ti plasmid [1,2]
By searching the 500 bp sequence region upstream of ATG of the Arabidopsis database, we found a large number of genes in the Arabidopsis genome that contain the D5RE element
In order to select more believable candidates to determine the host target genes regulated by VirD5, we set two screening conditions: (1) the 500 bp promoter region contains at least two D5RE elements; (2) The annotated function of the candidate gene is involved in the host plant disease resistance/sensibility or the process of Agrobacterium infection
Summary
Agrobacterium (Agrobacterium tumefaciens) is a soil-borne pathogen that transforms plant cells into tumor cells by the delivery of an oncogenic piece of DNA from its Ti (tumor-inducing) plasmid [1,2]. We found that VirD5 has different functions It interacts with Arabidopsis VIP1, forms ternary complex with AtVIP1 and VirE2 in the plant cell nucleus, and competes with VBF for binding to AtVIP1 to prevent VirE2 and AtVIP1 from being rapidly degraded via UPS (ubiquitin proteasome system). It has transcriptional activation activity in yeast, forms homodimers in vivo and in vitro, and binds to a specific DNA element (D5RE, CCGCNC/GNGCGG). Its expression is downregulated upon deletion of virD5 It encodes a F-box protein with RNI-like/FBD-like domains and is located in the plant cell nucleus. Genetic evidence strongly indicated that D5RF is necessary for Agrobacterium-mediated transformation
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