The plant pathogenic bacterium
- Award ID(s):
- 1725122
- NSF-PAR ID:
- 10381745
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Pseudomonas syringae pv tomato DC3000 (Pst DC3000) causes disease in tomato, in the model plantArabidopsis thaliana, and conditionally inNicotiana benthamiana. The pathogenicity ofPst DC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. ThePst DC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliana nonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging ofNicotiana benthamiana plants transiently co-expressingHopD1 fused to the epitope haemagglutinin (HopD1-HA ) withAtNHR2B fused to the red fluorescent protein (AtNHR2B-RFP ), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants. -
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Premise of the Study Datura stramonium is a pharmacologically and evolutionarily important plant species in the family Solanaceae. Stable transformation methodology of this species would be advantageous for future genetic studies.Methods In vitro plant regeneration and
Agrobacterium tumefaciens –mediated transformation techniques were developed forD. stramonium based on methods reported for tomato. A binary vector containingpAtUBQ 10::erGFP Results We recovered primary transformants harboring the green fluorescent protein (
GFP ) transgene that resulted in expression of fluorescence in all tissues analyzed. Transformants were allowed to self‐pollinate, and two of five progeny contained theGFP transgene and displayed fluorescence identical to the primary transformants.Discussion We have demonstrated the first stable transformation in the genus
Datura . This is a key first step to study the genetic basis of traits in this evolutionarily interesting species. -
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Summary GacS/GacA is a conserved two‐component system that functions as a master regulator of virulence‐associated traits in many bacterial pathogens, including
Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA‐regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogenPseudomonas aeruginosa , GacS/GacA negatively regulates the expression of type III secretion system (T3SS)‐encoding genes. However, in the plant pathogenic bacteriumPseudomonas syringae , strain‐to‐strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re‐evaluated the function of GacA inP. syringae pv.tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence insideArabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface‐inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulatingP. syringae virulence. -
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Summary Integration of
Agrobacterium tumefaciens transferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis , DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type and
polQ mutantArabidopsis and rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis ) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of
polQ mutants wasc. 20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQ mutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.
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Agriculture is facing new challenges, with global warming modifying the survival chances for crops, and new pests on the horizon. To keep up with these challenges, gene delivery provides tools to increase crop yields. On the other hand, gene delivery also opens the door for molecular farming of pharmaceuticals in plants. However, towards increased food production and scalable molecular farming, there remain technical difficulties and regulatory hurdles to overcome. The industry-standard is transformation of plants via Agrobacterium tumefaciens , but this method is limited to certain plants, requires set up of plant growth facilities and fermentation of bacteria, and introduces lipopolysaccharides contaminants into the system. Therefore, alternate methods are needed. Mechanical inoculation and spray methods have already been discussed in the literature – and here, we compare these methods with a newly introduced petiole injection technique. Because our interest lies in the development of plant viruses as immunotherapies targeting human health as well as gene delivery vectors for agriculture applications, we turned toward tobacco mosaic virus as a model system. We studied the effectiveness of three inoculation techniques: mechanical inoculation, Silwet-77 foliar spray and petiole injections. The foliar spray method was optimized, and we used 0.03% Silwet L-77 to induce infection using either TMV or a lysine-added mutant TMV-Lys. We developed a method using a needle-laden syringe to target and inject the plant virus directly into the vasculature of the plant – we tested injection into the stem and petiole. Stem inoculation resulted in toxicity, but the petiole injection technique was established as a viable strategy. TMV and TMV-Lys were purified from single plants and pooled leaf samples – overall there was little variation between the techniques, as measured by TMV or TMV-Lys yields, highlighting the feasibility of the syringe injection technique to produce virus nanoparticles. There was variation between yields from preparation to preparation with mechanical, spray and syringe inoculation yielding 40–141 mg, 36–56 mg, 18–56 mg TMV per 100 grams of leaves. Similar yields were obtained using TMV-Lys, with 24–38 mg, 17–28, 7–36 mg TMV-Lys per 100 grams of leaves for mechanical, spray and syringe inoculation, respectively. Each method has its advantages: spray inoculation is highly scalable and therefore may find application for farming, the syringe inoculation could provide a clean, aseptic, and controlled approach for molecular farming of pharmaceuticals under good manufacturing protocols (GMP) and would even be applicable for gene delivery to plants in space.more » « less
