Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule.

Induction of Agrobacterium tumefaciens virulence genes by plant phenolic compounds is essential for successful T-DNA transfer to a host plant. In Douglas fir needles, the major virulence region inducer is the glycoside coniferin (J. W. Morris and R. O. Morris, Proc. Natl. Acad. Sci. USA 87:3612-3618, 1990). Agrobacterium strains with high beta-glucosidase activity respond to coniferin and infect Douglas fir seedlings, whereas most strains with low beta-glucosidase activity fail to respond to coniferin and are avirulent on this host. We have cloned two beta-glucosidase genes from A. tumefaciens B3/73 and sequenced one of them, cbg1. It appears to be part of a polycistronic unit and shows a high bias for GC-rich codons. When expressed in Escherichia coli, Cbg1 beta-glucosidase hydrolyzes coniferin but not cellobiose. The 88-kDa predicted product of cbg1 is highly similar to one other bacterial beta-glucosidase and several fungal beta-glucosidases. There is little homology between Cbg1 and other bacterial beta-glucosidases, including an Agrobacterium cellobiase.     

Stimulation of Agrobacterium tumefaciens virulence with indole-3-acetic acid

The phytopathogen Agrobacterium tumefaciens incites the production of crown-gall on a wide range of dicotyledonous plants. Gall formation is dependent upon indole-3-acetic acid (IAA) and cytokinin production by the transformed plant cells. Upon incubation of Agrobacterium tumefaciens C58 with the plant hormone indole-3-acetic acid (IAA), bacterial virulence on cucumber plants was stimulated up to tenfold. Stimulation was maximized after exposure of bacteria to 50 or 100 μg ml^-1 IAA for 3 h. This was shown to be at the early log phase of bacterial growth.
The authors suggest that the excretion of IAA by the transformed plant cells stimulates bacterial virulence mechanism(s) encoded by the Ti plasmid, the chromosome, or both.     

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.     

Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA on bacterial gene expression and showed that SA inhibits induction of virulence (vir) genes and the repABC operon, and differentially regulates the expression of many other sets of genes. Using virus-induced gene silencing, we further demonstrate that plant genes involved in SA biosynthesis and signaling are important determinants for Agrobacterium infectivity on plants. Silencing of ICS (isochorismate synthase), NPR1 (nonexpresser of pathogenesis-related gene 1), and SABP2 (SA-binding protein 2) in N. benthamiana enhanced Agrobacterium infection. Moreover, plants treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid, a potent inducer of SAR, showed reduced disease symptoms. Our data suggest that SA and SAR both play a major role in retarding Agrobacterium infectivity.     

Expression of the Agrobacterium tumefaciens chvB virulence region in Azospirillum spp.

Inner membranes of Azospirillum brasilense incubated with UDP-glucose were unable to synthesize beta-(1-2) glucan and lacked the 235-kilodalton intermediate protein known to be involved in the synthesis of beta-(1-2) glucan in Agrobacterium tumefaciens and Rhizobium meliloti. Inner membranes of A. brasilense strains carrying a cosmid containing the chromosomal virulence genes chvA and chvB of Agrobacterium tumefaciens formed beta-(1-2) glucan in vitro and synthesized the 235-kilodalton intermediate protein. No DNA homology to the chvB region was found in different wild-type strains of A. brasilense, but the introduction of a cosmid containing the Agrobacterium tumefaciens chvA and chvB regions yielded strains in which DNA hybridization with the chvB region was detected, provided that the strains were grown under an antibiotic selective pressure.     

Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer

Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation.     

VirD4-independent transformation by CloDF13 evidences an unknown factor required for the genetic colonization of plants via Agrobacterium

Agrobacterium uses a mechanism similar to conjugation for trans-kingdom transfer of its oncogenic T-DNA. A defined VirB/VirD4 Type IV secretion system is responsible for such a genetic transfer. In addition, certain virulence proteins as VirE2 can be mobilized into host cells by the same apparatus. VirE2 is essential to achieve plant but not yeast transformation. We found that the limited host range plasmid CloDF13 can be recruited by the virulence apparatus of Agrobacterium for transfer to eukaryotic hosts. As expected the VirB transport complex was required for such trans-kingdom DNA transfer. However, unexpectedly, the coupling factor VirD4 turned out to be necessary for transfer to plants but not for transport into yeast. The CloDF13 encoded coupling factor (Mob) was essential for transfer to both plants and yeast though. This is interpreted by the different specificities of Mob and VirD4. Hence, Mob being required for the transport of the CloDF13 transferred DNA (to both plants and yeast) and VirD4 being required for transport of virulence proteins such as VirE2. Nevertheless, the presence of the VirE2 protein in the host plant was not sufficient to restore the deficiency for VirD4 in the transforming bacteria. We propose that Mob functions encoded by the plasmid CloDF13 are sufficient for DNA mobilization to eukaryotic cells but that the VirD4-mediated pathway is essential to achieve DNA nuclear establishment specifically in plants. This suggests that other Agrobacterium virulence proteins besides VirE2 are translocated and essential for plant transformation.     

农杆菌介导法在橡胶树遗传转化中的应用与展望

综述农杆菌介导法在巴西橡胶树遗传转化中的应用进展,分析影响农杆菌转化的关键因素,如植物基因型与外植体、菌株与载体类型、菌液浓度与侵染时间、vir诱导物、筛选剂与抑菌剂、培养基的组成和附加成分等,并对提高巴西橡胶树转化效率的策略进行探讨。     

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large- scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene -glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co- cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

Production and properties of an inhibitor of the Pseudomonas autoinducer by Pseudomonas aeruginosa

An inhibitor was found in the culture fluid of Pseudomonas aeruginosa PAO1, which could inhibit the activity of the Pseudomonas autoinducer (PAI). The maximal inhibitory activity occurred in stationary phase culture sup ernatant. The PAI inhibitor did not influence the cell growth and the PAI production by P. aeruginosa PAO1 when the PAI inhibitor was added into culture medium. The induced expression of lacZ in the reporter strain Agrobacterium tumefaciens NT1 was suppressed by this PAI inhibitor, whereas inhibition could be relieved by increasing the auto inducer concentration. The quorum sensing of P. aeruginosa was inhibited presumably by inhibiting the inducing activity of Pseudomonas autoinducer but not by inhibiting the production of Pseudomonas autoinducer. It was demonstrated that the structure of the PAI inhibitor was different from that of acyl-homoserine lactones.     

Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid

Forty-nine Tn3 and Tn5 transposition insertion mutations were introduced into the virulence region of the pTiA6NC plasmid of Agrobacterium tumefaciens. Five Tn5 transposition mutations from an earlier study (D. Garfinkel and E. Nester, J. Bacteriol. 144:732-743, 1980) were also mapped more accurately. These mutations defined five separate loci within the virulence region. Two Tn3 insertions into one of these loci, virA, result in a strain which is only weakly virulent; however, a Tn5 insertion into this locus eliminates virulence. One Tn5 insertion into another locus, virC, results in a strain which is weakly virulent. Two additional Tn5 insertions into this locus eliminate virulence. Insertions into the remaining three loci eliminate virulence entirely.