The TGA Transcription Factors from Clade II Negatively Regulate the Salicylic Acid Accumulation in Arabidopsis

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone's accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress

The TGA Transcription Factors from Clade II Negatively Regulate the Salicylic Acid Accumulation in Arabidopsis

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone’s accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress

PROHIBITIN3 Forms Complexes with ISOCHORISMATE SYNTHASE1 to Regulate Stress-Induced Salicylic Acid Biosynthesis in Arabidopsis.

Salicylic acid (SA) is a major defense signal in plants. In Arabidopsis (Arabidopsis thaliana), the chloroplast-localized isochorismate pathway is the main source of SA biosynthesis during abiotic stress or pathogen infections. In the first step of the pathway, the enzyme ISOCHORISMATE SYNTHASE1 (ICS1) converts chorismate to isochorismate. An unknown enzyme subsequently converts isochorismate to SA. Here, we show that ICS1 protein levels increase during UV-C stress. To identify proteins that may play roles in SA production by regulating ICS1, we analyzed proteins that coimmunoprecipitated with ICS1 via mass spectrometry. The ICS1 complexes contained a large number of peptides from the PROHIBITIN (PHB) protein family, with PHB3 the most abundant. PHB proteins have diverse biological functions that include acting as scaffolds for protein complex formation and stabilization. PHB3 was reported previously to localize to mitochondria. Using fractionation, protease protection, and live imaging, we show that PHB3 also localizes to chloroplasts, where ICS1 resides. Notably, loss of PHB3 function led to decreased ICS1 protein levels in response to UV-C stress. However, ICS1 transcript levels remain unchanged, indicating that ICS1 is regulated posttranscriptionally. The phb3 mutant displayed reduced levels of SA, the SA-regulated protein PR1, and hypersensitive cell death in response to UV-C and avirulent strains of Pseudomonas syringae and, correspondingly, supported increased growth of P. syringae The expression of a PHB3 transgene in the phb3 mutant complemented all of these phenotypes. We suggest a model in which the formation of PHB3-ICS1 complexes stabilizes ICS1 to promote SA production in response to stress

A Open One-Step RT-qPCR for SARS-CoV-2 detection

<p>Sequences of the plasmids needed to purify the required enzymes for a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples. Supplementary Information</p><p>Filetype GB</p&gt

One-Step RT-qPCR reaction mix using M-MLV RT and Taq DNA pol.

One-Step RT-qPCR reaction mix using M-MLV RT and Taq DNA pol.</p

Comparative Cq data between a commercial RT-qPCR kit and an Open RT-qPCR method based on homebrew M-MLV RT and Pfu-Sso7d.

Comparative Cq data for the TaqPath One-Step RT-qPCR kit and the dye-based open RT-qPCR reaction mix. Assigned sample number (# Sample) and clinical sample identifier (ID) are displayed. The clinical reports of the samples before they were re-tested by the two kits are also indicated in parentheses. The samples whose reports were altered are denoted in bold. (-): negative samples, (+): positive samples, ND: non-detected. (DOCX)</p

CDC SARS-CoV-2 N1 and N2 One-Step RT-qPCR assays performed with synthetic RNA using homebrew M-MLV RT and Pfu-Sso7d.

(A-B) Representative amplification curves using EvaGreen as DNA intercalating dye. Each curve represents a specific dilution of SARS-CoV-2 synthetic N RNA used as template: 1.4 x 107 copies approximately (red line), 1.4 x 106 (yellow line), 1.4 x 105 (green line), 1.4 x 104 (light blue line), 1.4 x 103 (blue line), 1.4 x 102 (purple line) and no template control (NTC, gray line). Characteristic Cq values are indicated on the upper left side of each panel in order to evaluate if amplification curves correspond to single amplicons (peaks); N1 and N2 melting curve analyses are shown in panels C and D, respectively. (TIF)</p

Primers used in One-Step qRT-PCR reactions.

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.</div