Biological systems of the host cell involved in Agrobacterium infection

Genetic transformation of plants by Agrobacterium , which in nature causes neoplastic growths, represents the only known case of trans -kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell–cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity – thought to participate in the Agrobacterium -mediated genetic transformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75425/1/j.1462-5822.2006.00830.x.pd

Extracellular VirB5 Enhances T-DNA Transfer from Agrobacterium to the Host Plant

VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5—by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell—enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell

Movement Protein Pns6 of Rice dwarf phytoreovirus Has Both ATPase and RNA Binding Activities

Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5′- and 3′- terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions

RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins

Advances in structural and biochemical properties of carmovirus movement proteins (MPs) have only been obtained in p7 and p9 from Carnation mottle virus (CarMV). Alignment of carmovirus MPs revealed a low conservation of amino acid identity but interestingly, similarity was elevated in regions associated with the functional secondary structure elements reported for CarMV which were conserved in all studied proteins. Nevertheless, some differential features in relation with CarMV MPs were identified in those from Melon necrotic virus (MNSV) (p7A and p7B). p7A was a soluble non-sequence specific RNA-binding protein, but unlike CarMV p7, its central region alone could not account for the RNA-binding properties of the entire protein. In fact, a 22-amino acid synthetic peptide whose sequence corresponds to this central region rendered an apparent dissociation constant (K(d)) significantly higher than that of the corresponding entire protein (9 mM vs. 0.83-25.7 microM). This p7A-derived peptide could be induced to fold into an alpha-helical structure as demonstrated for other carmovirus p7-like proteins. Additionally, in vitro fractionation of p7B transcription/translation mixtures in the presence of ER-derived microsomal membranes strongly suggested that p7B is an integral membrane protein. Both characteristics of these two small MPs forming the double gene block (DGB) of MNSV are discussed in the context of the intra- and intercellular movement of carmovirus

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

10.1371/journal.ppat.1003948PLoS Pathogens103

ANK, a Host Cytoplasmic Receptor for the Tobacco mosaic virus Cell-to-Cell Movement Protein, Facilitates Intercellular Transport through Plasmodesmata

Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters

Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs

Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectible levels of genetic transformation of mouse organs

Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island

Background: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Methodology/Principal Findings: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg 2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity

High-Throughput Construction of Intron-Containing Hairpin RNA Vectors for RNAi in Plants

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics

The Tobacco mosaic virus movement protein associates with but does not integrate into biological membranes

Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative -helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components.This work was supported by grants BFU2009-08401 and BFU2012-39482 (to I. M.) and BIO2011-25018 (to V. P.) from the Spanish MINECO. A. P. is the recipient of a JAE predoc position (CSIC).Peiró Morell, A.; Martínez-Gil, L.; Tamborero, S.; Pallás Benet, V.; Sanchez Navarro, JA.; Mingarro, I. (2014). The Tobacco mosaic virus movement protein associates with but does not integrate into biological membranes. Journal of Virology. 88(5):3016-3026. https://doi.org/10.1128/JVI.03648-13S3016302688