Preventing crown gall disease using oncogene silencing or VirE1-expressing transgenics

Crown gall disease is caused by the ubiquitous soil bacterium Agrobacterium tumefaciens which transfers a portion if DNA (T-DNA) into the plant cell. Preventing infection by using the biocontrol strain Agrobacterium radiobacter K84 is currently the only defense for crown gall. Two different resistance strategies were examined in this work. The first was the use of VirE1-expressing transgenic Arabidopsis thaliana. VirE1 is the secretory chaperone of VirE2. VirE2 is exported into plant cells during infection and is essential for tumorigenesis. Once in plant cells VirE2, a single-stranded DNA binding protein, protects the single-stranded T-strand and also helps localize it to the nucleus. Since the interaction of VirE1 and VirE2 is favored over a VirE2 and T-strand interaction we hypothesized that plant expressed VirE1 would prevent VirE2 from binding the T-strand. One plant line exhibited 3.5-fold fewer tumors than wild-type plants. Some amino acid residues of VirE1 needed for interaction with VirE2 have also been identified in this work. This research provides further information about the VirE1-VirE2 interaction that may be used in future constructs for VirE1-mediated crown gall resistance. The other resistance strategy examined in this work is gene silencing of the A. tumefaciens oncogenes iaaM and ipt. These oncogenes are part of the T-DNA and once in the cells express auxin and cytokinin which cause gall growth. By generating constructs that made double-stranded RNA and creating transgenic apple tissue that expressed these constructs, sequence specific degradation of the target mRNA was initiated. Six apple lines were completely resistant to crown gall disease using gene silencing. These methods may be further improved and even combined to eventually create crown gall resistant transgenics for commercial production

Regulatory and DNA Repair Genes Contribute to the Desiccation Resistance of Sinorhizobium meliloti Rm1021 ▿

Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of S. meliloti to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that uvr gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance

Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5′ end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease

Complete genome of the switchgrass endophyte Enterobacter clocace P101

TheEnterobacter cloacaecomplex is genetically very diverse. The increasing number of complete genomic sequences ofE. cloacaeis helping to determine the exact relationship among members of the complex.E. cloacaeP101 is an endophyte of switchgrass (Panicum virgatum) and is closely related to otherE. cloacaestrains isolated from plants. The P101 genome consists of a 5,369,929 bp chromosome. The chromosome has 5,164 protein-coding regions, 100 tRNA sequences, and 8 rRNA operons

Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome▿

The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed