Initial responses of the trap-crop, Solanum sisymbriifolium, to Globodera pallida invasions.

Many researchers today are looking for mechanisms underlying plant defenses against nematodes by identifying differentially expressed genes in domesticated hosts. In order to provide a different perspective, we analyzed the root transcriptome of an undomesticated non-host species, Solanum sisymbriifolium Lamark (SSI) before and after Globodera pallida infection. Utilizing RNAseq analyses, we identified changes in the expression of 277 transcripts. Many of these genes were not annotated; however, the annotated set included peroxidases, reactive oxygen species-producing proteins, and regulators of cell death. Importantly, 60% of the nematode-responsive genes did not respond to physical damage to root tissues, or to exogenous treatments with either salicylic acid or methyl jasmonate. Based on this, we speculate that the majority of changes in SSI gene expression were promoted by either nematode effectors, pathogen-associated molecular patterns (PAMPs), or by exposure to untested endogenous signaling molecules such as ethylene, or by exposure to multiple stimuli. This study incorporates our findings into a model that accounts for part of this plant's unusual resistance to nematodes.

Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium.

Solanum sisymbriifolium, also known as "Litchi Tomato" or "Sticky Nightshade," is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did.

Assessment of Globodera pallida RNA Extracted from Solanum Roots.

The introduction of high-throughput sequencing technologies has made transcriptome analyses of plant-pathogen interactions almost routine. Nevertheless, it is still challenging to obtain RNA from populations made up of two species. An RNA extraction method that worked well on free-living Caenorhabditis elegans failed when applied to isolated Globodera pallida J2 larva. Furthermore, alternative protocols that extracted RNA from free-living J2 larva produced less satisfactory results once the animals entered their hosts' roots. We have compared several extraction procedures to ascertain whether a single protocol was capable of recovering high-quality, high-molecular-weight RNA from newly hatched J2 larva as well as from larva embedded in roots of both potatoes (Solanum tuberosum L. cv. Desiree) and a very distantly related species, Solanum sisymbriifolium. Although it was possible to recover large amounts of RNA from J2 larvae using Proteinase K treatments, this protocol failed to yield high-quality nematode RNA from infected roots. By comparison, mechanical disruption procedures yielded lower amounts of RNA from infected roots, but what was recovered was of higher quality. We conclude that different extraction protocols need to be developed to sample mixed populations of organisms.

Proximity-dependent inhibition in Escherichia coli isolates from cattle.

We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-µm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.

Bibersteinia trehalosi inhibits the growth of Mannheimia haemolytica by a proximity-dependent mechanism.

Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time ( approximately 10 min versus approximately 27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-microm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 mum that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.

Use of a site-specific recombination-based biosensor for detecting bioavailable toluene and related compounds on roots.

We constructed and characterized a plasmid-based genetic system that reports the expression of a toluene-responsive promoter (PtbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the PtbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site-specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80-100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 micro m toluene for one to three hours. Compared to the response of A506 containing a plasmid-borne PtbuA1-gfp fusion, the recombination-based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the PtbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high PtbuA1-promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant- or rhizosphere microbial community-derived inducers of the PtbuA1 promoter. This recombination-based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment.

Site-specific recombination-based genetic system for reporting transient or low-level gene expression.

We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 microM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment.