Agrobacterium rhizogenes-mediated transformation is widely used in different species with various purposes. The development of composite plants (wild-type shoot with transgenic roots) has been a milestone for functional characterization of genes. Previously, composite plants were generated by two steps from inducing of hairy roots to growing in the growth medium. Hairy roots were induced in an induction medium and the growth of composite plants generated were in another different growth medium. The composite plants produced was subject to transplanting. Here, we describe an improved and optimized protocol for generation of composite plant achieved by one-step in cucumber, which has not been reported previously in living plants. Incubation of explants post inoculation to induce transgenic roots and the growth of rooted explants were in the same medium. The primary root of 5-day-old seedling was excised and the slant cut of residual hypocotyl with 1 cm length was inoculated with A. rhizogenes harboring the desired gene construct followed by directly planted into a pot with wet sterile vermiculite. More than 90% of the infected seedlings can produce positive transgenic root. In addition, we further used the one-step transformation protocol to analyze the function of Arabidopsis YAO promoter. The result indicated that pYAO::GUS was highly conserved expression in whole root and high activity in the root tips. Therefore, a fast, expedient, high efficient, and one-step transformation method of composite cucumber produced is established, which is suitable for promoter functional analysis and other root-related events. Key messageA highly reliable, facile and one-step generation of composite cucumber plants (with transgenic roots and wild-type shoot) protocol performed effectively in different genotypes cucumber using Agrobacterium rhizogenes-mediated transformation.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

IMPORTANCERelatively little is known about the persistence of symptoms in patients with COVID-19 for more than 1 year after their acute illness. OBJECTIVE To assess the health outcomes among hospitalized COVID-19 survivors over 2 years and to identify factors associated with increased risk of persistent symptoms. DESIGN, SETTING, AND PARTICIPANTS This was a longitudinal cohort study of patients who survived COVID-19 at 2 COVID-19-designated hospitals in Wuhan, China, from February 12 to April 10, 2020. All patients were interviewed via telephone at 1 year and 2 years after discharge. The 2-year follow-up study was conducted from March 1 to April 6, 2022. Statistical analysis was conducted from April 20 to May 5, 2022. The severity of disease was defined by World Health Organization guideline for COVID-19. EXPOSURES COVID-19. MAIN OUTCOMES AND MEASURESThe main outcome was symptom changes over 2 years after hospital discharge. All patients completed a symptom questionnaire for evaluation of symptoms, along with a chronic obstructive pulmonary disease assessment test (CAT) at 1-year and 2-year followup visits. RESULTSOf 3988 COVID-19 survivors, a total of 1864 patients (median [IQR] age, 58.5 [49.0-68.0] years; 926 male patients [49.7%]) were available for both 1-year and 2-year follow-up visits. The median (IQR) time from discharge to follow-up at 2 years was 730 (719-743) days. At 2 years after hospital discharge, 370 patients (19.8%) still had symptoms, including 224 (12.0%) with persisting symptoms and 146 (7.8%) with new-onset or worsening of symptoms. The most common symptoms were fatigue, chest tightness, anxiety, dyspnea, and myalgia. Most symptoms resolved over time, but the incidence of dyspnea showed no significant change (1-year vs 2-year, 2.6% [49 patients] vs 2.0% [37 patients]). A total of 116 patients (6.2%) had CAT total scores of at least 10 at 2 years after discharge. Patients who had been admitted to the intensive care unit had higher risks of persistent symptoms (odds ratio, 2.69; 95% CI, 1.02-7.06; P = .04) and CAT scores of 10 or higher (odds ratio, 2.83; 95% CI, 1.21-6.66; P = .02). CONCLUSIONS AND RELEVANCEIn this cohort study, 2 years after hospital discharge, COVID-19 survivors had a progressive decrease in their symptom burden, but those with severe disease during hospitalization, especially those who required intensive care unit admission, had higher risks of persistent symptoms. These results are related to the original strain of the virus, and their relevance to infections with the Omicron variant is not known.

Background Agrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required. Results Here, we report a novel, low cost, and robust reporter for convenient, non-destructive, and directly visual selection of transgenic hairy roots by naked eye, which can be used in the study of rhizobia-legume symbiosis. The reporter gene AtMyb75 in Arabidopsis, encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated by A. rhizogenes, which induced purple/red colored anthocyanin accumulation in crop species like soybean (Glycine max (L.) Merr.) and two model legume species, Lotus japonicas and Medicago truncatula. Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability of AtMyb75 as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulation Rfg1 gene in the soybean PI377578 (Nod-) inoculated with Sinorhizobium fredii USDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin. Conclusions Anthocyanin is a reliable, user-friendly, convenient, non-destructive, low cost, directly visual reporter for studying symbiotic nitrogen-fixing nodule development and could be widely applied in broad leguminous plants.

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After nitrogen treatments, plant leaves become narrower and thicker, and the chlorophyll content increases. However, the molecular mechanisms behind these regulations remain unknown. Here, we found that the changes in leaf width and thickness were largely compromised in the shade avoidance 3 (sav3) mutant. The SAV3 gene encodes an amino-transferase in the auxin biosynthesis pathway. Thus, the crosstalk between shade and nitrogen in Arabidopsis leaf development was investigated. Both hypocotyl elongation and leaf expansion promoted by the shade treatment were reduced by the high-N treatment; high-N-induced leaf narrowing and thickening were reduced by the shade treatment; and all of these developmental changes were largely compromised in the sav3 mutant. Shade treatment promoted SAV3 expression, while high-N treatment repressed SAV3 expression, which then increased or decreased auxin accumulation in cotyledons/leaves, respectively. SAV3 also regulates chlorophyll accumulation and nitrogen assimilation and thus may function as a master switch responsive to multiple environmental stimuli.