A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid.

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Long-Term Social Outcomes of Hyperfractionated Radiation on Childhood ALL Survivors.

BACKGROUND/OBJECTIVES: Childhood cranial radiation has irreversible neurocognitive effects. Hyperfractionated radiation therapy (HFX) for acute lymphoblastic leukemia (ALL) was randomized against conventionally fractionated radiation therapy (CFX) in the DFCI 87-01/91-01 trials attempting to minimize these effects. When neurocognitive testing 8-year posttreatment demonstrated no difference, this strategy was abandoned. The objective of this study was to evaluate late social outcomes among patients who received HFX compared to CFX as part of the DFCI 87-01/91-01 trials. METHODS: This retrospective chart review examined all patients treated according to the DFCI 87-01/91-01 trials at the McMaster Children's Hospital in Hamilton, Canada. Patients <18 years at diagnosis and who have attended follow-up clinic since January 1, 2000 were included in this study. Social outcomes and IQ test results were examined for trends. Demographics and outcomes were presented with descriptive statistics. RESULTS: We identified 57 DFCI 87-01/91-01 trial participants: 14 received HFX, 29 received CFX, and 14 received no radiation. There were no demographic differences between the groups. HFX survivors were more likely to be living independently (64% vs. 28%, P = 0.02) and engaged in long-term relationships (57% vs. 25%, P = 0.04) than CFX. Nonsignificant trends suggested that HFX survivors may be more financially independent, employed full-time, had fewer educational difficulties in school, and higher scores on neuropsychological testing. Data trends, although not significant, persisted in logistic regression analysis when accounting for age. CONCLUSION: Long-term social outcomes were better among ALL survivors who received HFX than CFX. A wider study involving all patients enrolled on DFCI 87-01/91-01 protocols should be conducted to reconsider radiation protocols for ALL.

Gene editing of the E3 ligase PIRE1 fine-tunes ROS production for enhanced bacterial disease resistance in tomato.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of Solanum lycopersicum (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using CRISPR-cas9, we generated mutants in two S. lycopersicum PIRE homologs (SlPIRE). SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens Pseudomonas syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and PIRE is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning PIRE as a promising target for improving overall plant health.

Single-cell profiling of Arabidopsis leaves to Pseudomonas syringae infection.

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We expose Arabidopsis to Pseudomonas syringae or mock treatment and profile >11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from both treatments identifies distinct pathogen-responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection reveals a continuum of disease progression from an immune to a susceptible state. Confocal imaging of promoter-reporter lines for transcripts enriched in immune cell clusters shows expression surrounding substomatal cavities colonized or in close proximity to bacterial colonies, suggesting that cells within immune clusters represent sites of early pathogen invasion. Susceptibility clusters exhibit more general localization and are highly induced at later stages of infection. Overall, our work shows cellular heterogeneity within an infected leaf and provides insight into plant differential response to infection at a single-cell level.