Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated... Show moreAgrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF, an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium-mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2. Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2. Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium, suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection. Show less
Background The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome... Show moreBackground The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. Results We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in rideopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. Conclusions The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium. Show less
Schmitz, D.J.; Ali, Z.; Wang, C.; Aljedaani, F.; Hooykaas, P.J.J.; Mahfouz, M.; Pater, B.S. de 2020
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNAguided Cas9 endonuclease is usually... Show moreClustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNAguided Cas9 endonuclease is usually delivered into plant cells as a DNA construct encoding Cas9 and the single guide RNA (sgRNA). However, constitutive expression of nucleases may cause off target mutations. In addition, DNA constructs can integrate into the host genome, causing mutations and complicating regulatory approval. Instead of DNA, here we deliver Cas9 through the Agrobacterium T4SS, accomplished by fusion of the VirF T4SS translocation peptide to Cas9 (NCas9F). Co-cultivation of Agrobacteria expressing NCas9F with yeast (Saccharomyces cerevisiae) harboring a sgRNA targeting CAN1 showed that NCas9F was translocated via T4SS and induced targeted mutations in the yeast genome. Infiltration of Nicotiana benthamiana leaves with Agrobacteria expressing NCas9F and sgRNA-PHYTOENE DESATURASE (PDS) resulted in targeted modifications at the PDS locus, albeit at a very low rate. In order to increase the mutation frequency NCas9F protein was co-transported with a TDNA encoding sgRNA-PDS1. Next generation sequencing confirmed that this resulted in targeted mutations at the PDS locus with a similar distribution but at a 5-fold lower frequency as the mutations obtained with a T-DNA encoding both Cas9 and sgRNA-PDS1. Similarly, infection with Tobacco rattle virus (TRV) encoding sgRNA-PDS2 combined with NCas9F protein translocation resulted in an equally high frequency of PDS mutations in N. benthamiana compared to T-DNA encoded sgRNA-PDS1 combined with NCas9F protein translocation. Our results revealed that translocation of NCas9F protein via the Agrobacterium T4SS can be used for targeted mutagenesis in host cells instead of the permanent and constitutive expression of Cas9 from a T-DNA. Show less
The RNA guided endonuclease based on the CRISPR/Cas system of Streptococcus pyogenus is a potent new tool for genome engineering in plants. Delivery of this system is usually done by... Show moreThe RNA guided endonuclease based on the CRISPR/Cas system of Streptococcus pyogenus is a potent new tool for genome engineering in plants. Delivery of this system is usually done by transforming plants (and other organisms) with Agrobacterium tumefaciens which transfers a T-DNA encoding the required genes through its Type IV Secretion System (T4SS). Instead, here we report the delivery of the Cas9 protein of the CRISPR/Cas system of Streptococcus pyogenus through the T4SS of Agrobacterium to Nicotiana benthamiana and Saccharomyces cerevisiae. Show less
The development of methods for the genetic modification of plants a few decades ago has provided a tremendous boost for molecular plant science. Crop plants have been generated that are... Show moreThe development of methods for the genetic modification of plants a few decades ago has provided a tremendous boost for molecular plant science. Crop plants have been generated that are resistant to insects or herbicides, or that produce useful sugars or healthy nutrients. Although the ban on growing GM crops in Europe has considerably limited the application of GM technologies, they have still contributed considerably to fundamental plant science. Especially by using the natural and very efficient mechanism of DNA transfer by the soil born bacterium Agrobacterium tumefaciens, many collections of mutant lines of model plant species such as Arabidopsis and rice have been generated, in which genes are disrupted or overexpressed by the insertion of an Agrobacterium transfer DNA (T-DNA) construct. These collections have been used in forward or reverse genetics studies to unravel the function of a gene or a family of genes in plant defense or development, and to identify the key regulators in these processes. The study described in this thesis focused on the use of one of these key regulators, the Arabidopsis AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 15/REJUVENATOR (AHL15/RJV), to alter developmental processes such as flowering, senescence and regeneration. Show less
The project focuses on visualizing Agrobacterium Mediated Transformation (AMT) of host cells by real time microscopy. With new visualization techniques the function of several proteins, which have... Show moreThe project focuses on visualizing Agrobacterium Mediated Transformation (AMT) of host cells by real time microscopy. With new visualization techniques the function of several proteins, which have recently been discovered in our lab to play a role during AMT, are studied. Show less
The VirB/D4 Type four secretion system (T4SS) is a bacterial multiprotein complex that spans the bacterial envelope, which mediates the translocation of T-DNA and effector virulence proteins into... Show moreThe VirB/D4 Type four secretion system (T4SS) is a bacterial multiprotein complex that spans the bacterial envelope, which mediates the translocation of T-DNA and effector virulence proteins into recipient cell. My research revealed that the Agrobacterium VirE3 and VirD2 proteins are effector proteins that are translocated by the Agrobacterium T4SS into host cells. The translocated effector protein VirF carries an N-terminal F-box domain. My research focused on the identification of the host proteins interacting with VirF. The capacity of VirF to interact in vivo with ASK1 and CUL1, indicates that VirF plays a role in planta as part of a SCF complex, most likely mediating the ubiquitination of proteins targeted for degradation via the proteasome. Five other protein interactors of VirF (PIFs) were identified using the yeast 2-hybrid system. These VirF interactors are involved in different cellular processes, such as in metabolic processes, and in defense and stress responses. This suggests that Agrobacterium may translocate an effector virulence protein into plant host cells, which mimics the function of an eukaryotic F-box protein (FBP) and thus can direct the host SCF E3 Ub-ligase to the relevant plant targets. Show less
Agrobacterium is the natures genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium... Show moreAgrobacterium is the natures genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the whole eukaryotic host genes which are involved in this event. For this, I used a collection of around 4800 homozygous diploid deletion mutants and the cDNA libraries of yeast Saccharomyces cerevisiae as the eukaryotic host and two strains of Agrobacterium. First, a large-scale AMT protocol for the transformation of yeast mutants was developed. Then, the yeast mutant collection was screened for the mutants with an increased or a decreased AMT. 249 deletion mutant strains were found which affect AMT. Of great importance were host chromatin remodeling and histone modifying complexes. Besides, using GFP technique I showed the nuclear localization of Agrobacteriums VirD2 in yeast. Yeast two-hybrid screens also identified 12 yeast putative interaction partners of VirD2 protein. Show less
