Plant Pathogen Agrobacterium Tumefaciens Research Articles

Virus-induced Gene Silencing (VIGS) in <em>Nicotiana benthamiana</em> and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5. Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2. Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Type IV Secretion System of Anaplasma phagocytophilum and Ehrlichia chaffeensis

The intracellular bacterial pathogens Ehrlichia chaffeensis and Anaplasma phagocytophilum have evolved to infect leukocytes and hijack biological compounds and processes of these host defensive cells. Bacterial type IV secretion (T4S) system transports macromolecules across the membrane in an ATP-dependent manner and is increasingly recognized as a virulence factor delivery mechanism that allows pathogens to modulate eukaryotic cell functions for their own benefit. Genes encoding T4S system homologous to those of a plant pathogen Agrobacterium tumefaciens have been identified in E. chaffeensis and A. phagocytophilum. Upon interaction with new host cells, E. chaffeensis and A. phagocytophilum genes encoding the T4S apparatus are upregulated. The delivered macromolecules are referred to as T4S substrates, or effectors, because they affect and alter basic host cellular processes, resulting in disease development. Recently, A. phagocytophilum 160-kDa AnkA protein was to be delivered by T4S system into the host cytoplasm. Thus, dynamic signal transduction events are likely induced by T4S substrates in the host cells for successful establishment of intracellular infection. Further studies on Ehrlichia and Anaplasma T4S effectors cognate host cell molecules will undoubtedly advance our understanding of the complex interplay between obligatory intracellular pathogens and their hosts. Such data can be applied toward treatment, diagnosis, and control of ehrlichiosis and anaplasmosis.

Quorum Quenching in Agrobacterium tumefaciens : Chance or Necessity?

Cell-cell communication or “quorum sensing” (QS) between members of a population is an established phenomenon that has been described for many different bacterial species. A number of different types of QS systems have been discovered; however, a unifying theme is the synthesis of a small signal molecule, often called an autoinducer or pheromone, which activates a specific response when it accumulates to a threshold concentration within a population. A relatively new and exciting aspect of the field of QS that has received much recent attention is “quorum quenching” (QQ), or interference of a QS signaling system. This occurs through either the inhibition of a QS component or the depletion of the signal itself, resulting in an attenuation of the response. In the plant pathogen Agrobacterium tumefaciens, an enzyme (BlcC) that destroys the bacterium's QS signal has been recently described, prompting much speculation that this enzyme is specifically involved in the quenching of the QS system. A variety of explanations for the adaptive significance of QQ in the QS system of A. tumefaciens and implications for the bacterium's role as a plant pathogen have been suggested in the literature (for example, see references 4 and 27). However, the role of BlcC in QQ was never directly addressed. In A. tumefaciens, the QS system regulates Ti (tumor-inducing) plasmid conjugation. In this issue, Khan and Farrand (12) directly address the biological significance of BlcC by examining its effect on Ti plasmid conjugation both in culture and in planta. Their study has implications for our understanding of the possible roles in Agrobacterium and other bacteria of BlcC-like enzymes, which are generally thought to function as quorum quenchers of proteobacteria.

Rational design of inhibitors of VirA–VirG two-component signal transduction

VirA–VirG two-component system regulates the vir (virulence) operon in response to specific host factors (xenognosins) in the plant pathogen Agrobacterium tumefaciens. Using whole cell assays, stable inhibitors inspired by the labile natural benzoxazinone inhibitor HDMBOA are developed. It is found that aromatic aldehydes represent a minimal structural unit for activity. In particular, 3-hydroxy-4,6-dimethoxy-3 H-isobenzofuran-1-one (HDI) was found to have the highest activity, making it the most potent developed inhibitor of virulence gene expression in Agrobacterium.

마늘오일을 이용한 오이와 토마토 흰가루병 방제

4종류의 식물 추출 오일(마늘, 생강, 계피, 레몬그라스)에 대한 실내 항균효과를 5종의 식물병원세균과 4종의 식물병원진균에 대해 조사하였다. 4종의 오일중 마늘오일과 계피오일의 항균효과가 우수하였다. 특히, 마늘오일은 식물병원세균인 Agrobacterium tumefaciens, Erwinia carotovora, Ralstonia solanacearum, Xanthomonas campestris와 식물병원진균인 Phytophthora infestans, Fusarium oxysporum, Collectotricum sp.에 대해 강한 항균력을 나타내었다. 그러나, Acidovorax avenae와 Pythium sp.에 대해서는 항균효과가 없었다. 마늘오일의 오이 흰가루병(Sphaerotheca fusca)과 토마토 흰가루병(Erysiphe cichoracearum)에 대한 포장 방제효과 실험결과 각각 70.0-74.6%와 71.2%의 방제가를 나타내었다. The effectiveness of four plant oils (garlic, ginger, cinnamon and lemongrass) against a range of plant pathogenic organisms was tested in vitro. Of the four oils, two oils (garlic and cinnamon) showed relatively good antimicrobial activities. Activity of garlic oil was shown against the plant pathogenic bacteria Agrobacterium tumefaciens, Ewinia carotovora, Ralstonia solanacearum, Xanthomonas campestris, and the fungi Phytophthora infestans, Fusarium oxysporum, Collectotricum sp., whereas Acidovorax avenae and Pythium sp. showed the resistance towards garlic oil. Results from the planta bioassays under greenhouse conditions indicated that garlic oil significantly reduced the cucumber powdery mildew (Sphaerotheca fusca) and tomato powdery mildew (Erysiphe cichoracearum) showing control value 70.0-74.6% and 71.2%, respectively.

Structural biology of Helicobacter pylori type IV secretion system

Helicobacter pylori chronically infects the gastric mucosa of millions of people annually worldwide: it has been estimated that over 50% of the world population carries this infection. H. pylori has been associated with the development of several diseases, like chronic gastritis, gastric and duodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphoma [1-3]. The complete genome sequence of two different isolates of H. pylori (J99 and 26995) is known. The strains that contain a 37 kb foreign DNA region, called cag pathogenicity island (cag-PAI), cause the most severe form of virulence [4]. The cag-PAI encodes for a functional type IV secretion apparatus homologous to the VirB/D4 Type IV Secretion System (T4SS) of the plant pathogen Agrobacterium tumefaciens and other Gram-negative bacteria [5]. T4SSs are involved in conjugal DNA transfer, in the DNA delivery to (or uptake from) the environment, for instance the release of oncogenic DNA into infected plant cells by A. tumefaciens, or in the translocation of effector proteins [6,7]. The T4SS encoded by the cag-PAI of H. pylori is responsible for the translocation into the host cell of the protein CagA, a major antigenic virulence factor encoded within the cag-PAI. Once secreted into the gastric epithelial cells, CagA induces cellular modifications, such as elongation and spreading of host cells [8]. The aim of this structural genomic project is to determine the three-dimensional structure of most of the proteins encoded by the cag-PAI, a task that will allow to elucidate the function and the organization of the entire T4SS of such a relevant pathogenic bacterium [9].

Identification of amino acid residues of the Agrobacterium tumefaciens quorum‐sensing regulator TraR that are critical for positive control of transcription

The LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a two-domain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl- l-homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo, and for DNA binding activity in vitro. Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase alpha-subunit.

REP code: defining bacterial identity in extragenic space

Through the analysis of 57 bacterial genomes we have detected repetitive extragenic palindromic DNA sequences (REPs) in 11 species. For a sequence to be considered as REP, the following criteria should be met: (i) It should be extragenic, (ii) palindromic, (iii) of a length between 21 and 65 bases and (iv) should constitute more than 0.5% of the total extragenic space. Species-specific REPs have been found in human pathogens such as Escherichia coli, Salmonella enterica, Neisseria meningitidis, Mycobacterium tuberculosis, Rickettsia conorii and Pseudomonas aeruginosa, the plant pathogen Agrobacterium tumefaciens and the soil bacteria Deinococcus radiodurans, Pseudomonas putida and Sinorhizobium meliloti.

The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein ( Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a V max of 1.3 nmol phosphate released per minute per milligram DnaK, and a K m of 62 μM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.

Cell-signalling repression in bacterial quorum sensing.

In this paper we expand on two mathematical models for investigating the role of three distinct repression mechanisms within the so-called quorum sensing (QS) cell-signalling process of bacterial colonies growing (1) in liquid cultures and (2) in biofilms. The repression mechanisms studied are (i) reduction of cell signalling molecule (QSM) production by a constitutively produced agent degrading the messenger RNA of a crucial enzyme (QSE), (ii) lower QSM production rate due to a negative feedback process and (iii) loss of QSMs by binding directly to a constitutively produced agent; the first two mechanisms are known to be employed by the pathogenic bacterium Pseudomonas aeruginosa and the last is relevant to the plant pathogen Agrobacterium tumefaciens. The modelling approach assumes that the bacterial colony consists of two sub-populations, namely down- and up-regulated cells, that differ in the rates at which they produce QSMs, while QSM concentration governs the switching between sub-populations. Parameter estimates are obtained by curve-fitting experimental data (involving P. aeruginosa growth in liquid culture, obtained as part of this study) to solutions of model (1). Asymptotic analysis of the model (1) shows that mechanism (i) is necessary, but not sufficient, to predict the observed saturation of QSM levels in an exponentially growing colony; either mechanism (ii) or (iii) also needs to be incorporated to obtain saturation. Consequently, only a fraction of the population will become up-regulated. Furthermore, only mechanisms (i) and (iii) affect the main timescales for up-regulation. Repression was found to play a less significant role in a biofilms, but mechanisms (i)-(iii) were nevertheless found to reduce the ultimate up-regulated cell fraction and mechanisms (i) and (iii) to increase the timescale for substantial up-regulation and to decrease the wave speed of an expanding front of QS activity.

Broad-spectrum activity of the volatile phytoanticipin allicin in extracts of garlic ( Allium sativum L.) against plant pathogenic bacteria, fungi and Oomycetes

The volatile antimicrobial substance allicin is produced in garlic after cellular decompartmentalisation when the tissues are damaged and the substrate alliin mixes with the enzyme alliin-lyase (E.C.4.4.1.4). The effectiveness of garlic extract against a range of plant pathogenic organisms was tested in vitro and in planta in diseased tissues. Allicin in garlic extracts was quantified spectrophotometrically and a rapid bioassay was developed for routine use. The in vitro activity of allicin against a prototrophic E. coli isolate was compared with that of the conventional antibiotics ampicillin and kanamycin. Activity in vitro was shown against the plant pathogenic bacteria Agrobacterium tumefaciens, Erwinia carotovora, Pseudomonas syringae pv. maculicola, P.s. pv. phaseolicola, P.s. pv. tomato, Xanthomonas campestris pv. campestris, the fungi Alternaria brassisicola, Botrytis cinerea, Plectosphaerella cucumerina, Magnaporthe grisea, and the Oomycete Phytophthora infestans. Disease reduction in planta was shown for Magnaporthe grisea-infected rice, Hyaloperonospora parasitica-infected Arabidopsis thaliana and Phytophthora infestans-infected potato tubers. Significantly, the active principle was effective in reducing P. infestans spore germination in vitro and disease in blighted tubers via the vapour phase (fumigation) as well as by direct application at the inoculation site. In Arabidopsis the reduction in disease was apparently due to a direct action against the pathogen since no accumulation of salicylic acid (a marker for systemic acquired resistance, or SAR) was observed after application. The potential for developing preparations of garlic for use as an alternative to synthetic fungicides for organic food production is discussed.

Two-dimensional reference map of Agrobacterium tumefaciens proteins.

Proteomics based on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is a commonly used method for physiological studies. Physiological proteomics requires 2-D reference maps, on which most of the main proteins are identified. We present a reference map for the bacterial plant pathogen Agrobacterium tumefaciens proteins, which contains more than 300 entries with an isoelectric point (pI) between 4 and 7. The quantitative study of the proteins in the analytical window of the master gel demonstrated unique features, in comparison with other bacteria. In addition, a theoretical analysis of several protein parameters was performed and compared with the experimental results. A comparison of the theoretical molecular weight (MW) of the proteins and their theoretical pI with their vertical and horizontal migration distances, respectively, pointed out the existence of several proteins that strongly diverted from the graph trend-line. These proteins were clearly subjected to post-translational modifications, which changed their pI and/or MW. Additional support for post-translational modifications comes from the identification of multiple spots of the same gene products. Post-translational modifications appear to be more common than expected, at least for soluble proteins, as more than 10% of the proteins were associated with multiple spots.

Isolation of temperature sensitive mutants and their mapping in Agrobacterium tumefaciens.

The first temperature sensitive mutants of plant pathogenic bacterium Agrobacterium tumefaciens were isolated by Tn5 mutagenesis. Several genes responsible for mutants were identified and mapped on the circular and linear chromosomes in A. tumefaciens MAFF301001.

The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5′-phosphate from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate and AMP

The plant pathogen Agrobacterium tumefaciens produces cytokinins upon induction of the virulence genes by secondary metabolites from wounded plants, and these hormones are believed to stimulate the infection process. To study the biosynthetic pathway, the tzs gene, encoding the Tzs (trans-zeatin-synthesizing) protein from A. tumefaciens, was cloned and the protein was overproduced and purified. Analysis of its reactivity with radioactively labeled substrate demonstrated conversion of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate, a product of the deoxyxylulose phosphate pathway, with AMP to zeatin riboside 5′-phosphate. This suggests that A. tumefaciens uses an alternative pathway of cytokinin biosynthesis, which had previously been hypothesized to operate in plants.

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using β-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

Complete genome sequence of Caulobacter crescentus

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Microbial hazards in plant tissue and cell cultures

A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.

Genome analysis of Agrobacterium tumefaciens: linkage map and genetic features of the left region of the linear chromosome.

In addition to a unique tumor-inducing (Ti) plasmid, the plant pathogenic bacterium Agrobacterium tumefaciens has an unconventional chromosomal organization. Our previous studies on A. tumefaciens MAFF301001 revealed that it possesses a 2 Mb linear and a 2.8 Mb circular chromosome plus a 206.479 kbp Ti plasmid (pTi-SAKURA). In this study, a linkage map for the left half of its linear chromosome covering a 900 kbp region was constructed and the number of potential genes existing in the region was estimated. The linkage map consists of 31 BAC and 8 lambda phage recombinants without any gaps. It confirmed the size and all the structural landmarks indicated in the corresponding region of our previously constructed physical map for the linear chromosome. Sequencing analysis of the end-regions of each linking clone led to the identification of 6 genes and another 27 potential genes or ORFs, including genes and/or gene clusters responsible for homologous recombination (ruvB), trehalose/maltose sugar transport (thuR, thuG) and alanine catabolism (dadR). Two virulence-related gene homologues (attK and celB), previously reported in the circular chromosome of a different strain of A. tumefaciens were found in this region. These findings will provide a ready-to-use linkage map for further functional analysis of the linear chromosome.

Different toxicity of Escherichia coli and Agrobacterium tumefaciens against tobacco BY2 cells

Tobacco BY2 cells were withered away by the physical contact with intestinal bacterium Escherichia coli. In contrast, plant pathogenic bacterium Agrobacterium tumefaciens with/without tumor inducing Ti plasmid was not so toxic as E. coli. Mini-cells of E. coli decreased their toxicity.

An Agrobacterium catalase is a virulence factor involved in tumorigenesis.

Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component. However, the soil-borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T-DNA that causes crown gall tumours on many plant species. A. tumefaciens does not elicit a typical HR on those plants. Here, we report that inactivation of one of A. tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2. This provides the first genetic evidence that the Agrobacterium-plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen.