Transformation of crown gall resistant and susceptible Vitis genotypes by Agrobacterium vitis

Transformation of crown gall-susceptible and -resistant Vitis genotypes by Agrobacterium vitis strain CG49 was studied using uidA (GUS) in the p35SGUSINT construct. When greenhouse-grown material propagated through tissue culture was inoculated with CG49(p35SGUSINT) in vitro, the highly crown gall-susceptible V. vinifera Cabernet Sauvignon displayed GUS activity on 53 % of inoculated explants vs. 5 % for the resistant V. amurensis and 0 % for the resistant Couderc 3309. Response of Cabernet Sauvignon suggested a strong effect of shoot polarity on transformation. Inoculation of basal vs. apical explant surface in Cabernet Sauvignon indicated transformation in 88 % of basal inoculated explants with no transformation from apical inoculation. Basal inoculations indicated no transformation of V. amurensis and transformation in 10 % of Couderc 3309 explants. Inoculation of intact plants with CG49(p35SGUSINT) produced GUS-positive sites at 56 % of inoculated sites in Cabernet Sauvignon, 10 % of V. amurensis inoculated sites and 9 % of Couderc 3309 inoculated sites. Resistance to crown gall in these genotypes appears to be due to reduced susceptibility to transformation by A. vitis rather than post-transformation phenomena. These studies were complicated by production of GUS-positive spots from in vitro inoculations using wild-type CG49. Resident microorganisms producing b-glucuronidase may proliferate after tissue degradation by A. vitis-induced cell disruption. Use of in vitro internodal explants from tissue culture-propagated vines greatly reduced GUS expression from control CG49 inoculations and these were readily distinguished (by appearance and location) from GUS-positive spots resulting from transformation with uidA

Endophytic Agrobacterium in crown gall-resistant and -susceptible Vitis genotypes

Several methods were used to study endophytic colonization of Vitis genotypes by Agrobacterium vitis (AV). AV was seldom detected except at inoculated sites, indicating little systemic movement of the bacterium under the conditions of these experiments. AV populations at inoculated sites were evaluated for 10 months following inoculation of crown gall-resistant and -susceptible genotypes. Two months after inoculation, V. amurensis selections had significantly smaller populations than V. vinifera (Cabernet Sauvignon) or V. riparia x V. rupestris (C3309). All crown gall-resistant genotypes had significantly lower populations than Cabernet Sauvignon 10 months after inoculation. Examination of vines one year after inoculation indicated that AV populations were much higher at inoculated sites when crown galls developed. However, even when no galls were apparent, Cabernet Sauvignon had significantly higher AV populations than V. amurensis 689 (6-fold higher) and C3309 (70-fold higher). Crown gall-resistant genotypes appear to support lower populations of AV than the crow gall-susceptible Cabernet Sauvignon. Freezing followed by a two-day incubation significantly increased recovery of Agrobacterium using vascular fluid displacement in naturally-infected and artifically-inoculated vines and therefore increased the sensitivity of indexing for AV in grapevines

Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus

Biological control measures to prevent or reduce Agrobacterium vitis-caused losses in grapevine cultures are a worldwide increasing challenge. In the present study, tumour development in grapevine (Vitis vinifera L.) was induced in the sensitive cv. Kerner by infection with Agrobacterium vitis strain K306, carrying the p35Sgus-int plasmid with the gus gene as marker for transformation by the wild-type T-DNA. Pre-inoculation with the non-tumorigenic A. vitis strain F2/5 prevented tumour induction by K306(p35gus-int). Strain M1154, a Tn5 mutant of F2/5 in the luxR-like aviR gene, partially reduced the biocontrol efficiency compared to the wild-type F2/5. GUS-labelling by K306gus was poor in grapevine in contrast to A. tumefaciens 281(p35gus-int)-induced tumours in Arabidopsis, indicating plant species-dependent variable gus expression. To use the more reliable direct mRNA expression assay by RTPCR, a new experimental plant/A. vitis system was established with Ricinus communis as model plant. Ricinus/A. vitis galls were available within one week after K306gus inoculation, reached diameters up to 5 cm, and contained more abundant GUS staining. An additional transformation marker, mRNA expression of the T-DNA-located iaaM oncogene, coding auxin synthesis, was apparent only in tumours induced by the wild-type A. vitis strain K306 in the absence of the gus construct, which is under the control of the strong 35S CaMV promoter. F2/5 pre-inoculation suppressed GUS staining and gus mRNA expression. DAPI staining revealed the loss of vital fluorescent cell nuclei in F2/5-inoculated grapevine tissue and thus inhibition of any successful T-DNA transfer into host cell nuclei. Differentiation of typical circular vessels in globular vascular bundles in M1154-pretreated galls suggests interference with plant auxin metabolism. In conclusion, together with successfully establishing a new experimental model system, Ricinus/A. vitis, pre-treatment of host tissue with the non-pathogenic strain F2/5 resulted in preventing the integration and expression of the oncogenic T-DNA of A. vitis strains by locally necrotizing host cell nuclei.

Biological control of Agrobacterium vitis using non-tumorigenic agrobacteria

The potential use of non-tumorigenic agrobacteria for the biological control of grapevine crown gall in Italy was investigated. Four Agrobacterium strains belonging to the species radiobacter and vitis were used to protect the susceptible cv. Malvasia Istriana grafted on the rootstock 420 A. Moreover, the effect of each treatment on grapevine vitality and growth was assessed, including the percentage of marketable vines, as determined by industry standards. Treatments with the antagonists clearly reduced tissue colonization by the pathogen, with a drop of more than 100-fold in pathogen populations in the samples collected at the graft point. Another important effect was the reduction of internal necrosis possibly induced by the high concentration of the nopaline strain CG 49 used in the experiments. According to viticultural and commercial parameters, treatments with the antagonists improved the quality of the vines, with fewer discards and a high percentage of marketable material. Therefore, these antagonists can be considered beneficial for grapevine

Xylella fastidiosa pil-chp operon is involved in regulating key structural genes of both type I and IV pili

Xylella fastidiosa is the causal agent of Pierce's disease (PD) in grapevines. It has type I and type IV pili, which are both virulence factors involved in the PD-associated processes of motility, aggregation, and biofilm formation. Many questions remain as to how the two pili are regulated. We previously identified a X. fastidiosa pil-chp chemosensory-like cluster as an operon composed of genes pilG-I-J-L-chpB-C. In this study, we deleted pilG (resulting in a ∆pilG-I strain) and pilJ and discovered that both mutants (∆pilG-I and ∆pilJ) had reduced virulence after 24 weeks post-inoculation, whereas ∆chpB and ∆chpC did not. Both ∆pilG-I and ∆pilJ lost motility and were impaired in biofilm formation in rich artificial media and xylem sap. Gene expression was significantly downregulated for representative fimbrial adhesin and motility genes in ∆pilG-I, and to a lesser extent in ∆pilJ. Our data suggest that Pil, but not Chp, proteins are virulence factors, and pilG-I-J are involved in transcriptional regulation of type I and IV pili virulence genes and therefore motility and biofilm formation. To our knowledge, this is the first report of a chemotaxis-like operon involved in the regulation of key structural genes of both type I and type IV pili

Preliminary analysis of an extensive one year survey of trace elements and compounds in the suspended particulate matter in Cleveland, Ohio

Beginning in 1971 a cooperative program has been carried on by the City of Cleveland Division of Air Pollution Control and NASA Lewis Research Center to study the trace element and compound concentrations in the ambient suspended particulate matter in Cleveland Ohio as a function of source, monitoring location and meteorological conditions. The major objectives were to determine the ambient concentration levels at representative urban sites and to develop a technique using trace element and compound data in conjunction with meteorological conditions to identify specific pollution sources which could be developed into a practical system that could be readily utilized by an enforcement agency

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN−) ligands. Extrinsic CO and CN−, however, inhibit hydrogenases. The mechanism by which CN− binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN−-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN− and CO ligands and to show that extrinsic CN− binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN− treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally

Hot-water treatment of dormant grape cuttings: Its effects on Agrobacterium tumefaciens and on grafting and growth of vine

Hot-water treatment (50°C for 20-30 min) was carried out to confirm its efficacy in eradicating Agrobacterium tumefaciens biovar 3 (AT3) in symptomless grape cuttings.After the forcing period, analyses of callus from cuttings of grape cvs Albana, Lambrusco Grasparossa, Rulander and Fortana, and from their graft combinations with the rootstocks 420A, 41B, 5BB and 1103P, revealed the low infection level in the grape material used. Dormant scion and rootstock cuttings treated identically in the U.S. gave similar results. Despite this, it was possible to confirm the efficacy of thermotherapy in eradicating the pathogen.An assessment was also made of the effect of treatment on growth parameters of grafted vines in the greenhouse and after 8 months in a field nursery. The effect of hot-water treatment on the vitality and growth of vines varied with the different scion-rootstock combinations. Treatment did not generally have detrimental effects on vitality; there were some negative effects on graft-take. The number and length of canes, as well as the diameter of the trunks, increased in most instances.The treatments and times usually did not affect bud survival and, in most cases, increased the level of callus formation at the base of cuttings.