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Paper Sharing丨 Discovery of stress mechanism in plants under environmental stress

Title: Phytocytokine signalling reopens stomata in plant immunity and water loss

Magazine: Nature

IF: 49.962

Author and Unit: Texas A&M University-Libo Shan, Ping He and Shandong Jianzhu University-Shuguo Hou are the co-corresponding authors of this article.

Vazyme Collaborative Products: ClonExpress® II One Step Cloning Kit (Cat.No. C112, Ligase-Independent Single Fragment Rapid Cloning Kit)

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Background

Plants face the challenges of pathogen infection and extreme external environments during their growth and development. They can sense these stresses and respond to stress to protect themselves. The dynamic regulation of plant surface stomata is an important way of immune regulation. Studies have found that plants prevent water loss by closing the stomata on the surface and limit the entry of pathogenic bacteria1, 2. However, closing the stomata for a long time reduces photosynthesis and transpiration. This will lead to increased water content and temperature in the plant, thereby promoting the colonization of pathogenic bacteria. At present, it is unclear whether there is an immune mechanism against the virulent effects of pathogens in plants.

Paper Review

On May 4, 2022, the team of Ping He and Libo Shan from Texas A&M University and the team of Shuguo Hou from Shandong Jianzhu University published a research paper titled "Phytocytokine signalling reopens stomata in plant immunity and water loss" online in "Nature". A new class of immune-regulating plant cytokines Small Phytocytokines Regualting Defense and Water Loss (SCREWs) and the cognate receptor kinase Plant SCREW Unresponsive Reportor (NUT) was reported for the first time. The mechanism involved in plant immune regulation was revealed. The immune cell regulator SCREWs acts on ABI, a key factor in the abscisic acid (ABA) signaling pathway, through the SCREW-NUT pathway, and promotes the dephosphorylation of Open Stomata 1 (OST1) by ABA Insensitive 1 (ABI1), thereby inhibiting stomatal closure and restarting stomatal opening. The schematic diagram is as Figure 1:0616-2

Figure 1. Schematic diagram of the role of SCREW-NUT pathway
(Picture comes from the paper. If it is an infringement of your copyrights, please contact us.)

This study has changed the previous understanding of the interaction between plants and the environment, and is of great significance for further revealing plant immunity and even promoting ecological virtuous circles and food security issues.

Methods & Results

The process of discovering SCREWs and its mechanism of plant immune regulation:

1. Analyze the Arabidopsis transcriptome induced by pathogenic bacteria, screen out SCREWs, and analyze its activity.

The 31 conserved amino acids at the carboxyl terminal of SCREWs can induce typical immune responses such as MAPK activation and plant growth inhibition. This small peptide forms a disulfide bond through two conserved cysteines, which plays a crucial role in SCREW immunity induction.

2. Construct SCREWs overexpression lines to verify gene function.

SCREW1 overexpression induces senescence in Arabidopsis thaliana and up-regulates the expression of immune-related gene PR1; the double-gene knockout strains of SCREW1 and SCREW2 are more susceptible to Pseudomonas syringae than wild type. The results showed that SCREWs could induce plant immune responses and enhance plant resistance to pathogens.

3. Verify that SCREW-NUT pathway regulates plant immunity by surface plasmon resonance and co-immunoprecipitation techniques.

MUT is the receptor of SCREW. SCREW recognizes and induces the polymerization of LRR-RK NAK1 and SERK4, triggering the downstream immune response independent of intracellular receptor kinase BIK1.

4. Detect the stomatal opening and closing, stomatal conductance and water potential of plants. Verify the mechanism of SCREW-NUT pathway regulating stomatal movement and plant water loss by immunoblotting experiments.

The SCREW-NUT pathway acts on ABI, a key factor in the ABA signaling pathway, and promotes the dephosphorylation of OST1 by ABI1, thereby inhibiting stomatal closure and restarting stomatal opening.

Vazyme Helps

The SCREW1, SCREW2, NUT and ABI2 fragments were recombined into pMDC32-35S::GFP or pMDC32-35S::HA vectors that had been digested by BamHI and StuI, respectively, to construct a gene-overexpressing vector for studying gene function. During this process, the researchers used Vazyme’s rapid clone product (Vazyme #C112). This product is simple, fast and efficient, suitable for any vector, regardless of the restriction site carried by the insert, and can efficiently clone DNA fragments of 50 bp - 10 kb. Linearized cloning vectors and PCR products can be cloned without purification. No ligase, no self-ligation, and the positive rate is > 95%. In addition, Vazyme offers online primer design software CE Design when using this product.

 

Figure 2. Application of Vazyme’s product in the paper
(Picture comes from the paper. If it is an infringement of your copyrights, please contact us.)

Vazyme’s Cloning Products

ClonExpress technology is a simple, fast, and efficient DNA seamless cloning technology that enables directional cloning of inserts into any vector at any site. ClonExpress II is a next-generation recombinant cloning kit containing the enhanced recombinase Exnase II. Linearize the vector using any methods, and then introduce the end sequence of the linearized vector at the 5' end of the insert forward/reverse PCR primers. In this way, the 5' and 3' ends of the PCR product have sequences (15 - 20 bp) identical to the two ends of the linearized vector, respectively. After the PCR product with the vector end sequences at both ends and the linearized vector are mixed in a certain proportion, under the catalysis of Exnase II recombinase, the transformation can be carried out in only 30 minutes, and the positive rate can reach > 95%.

The following are the rapid cloning kits we can provide (click on the cat.no. to see the detail of each product) :

Product Name Cat.No. Volume
ClonExpress® II One Step Cloning Kit C112-01/02 25/50 rxn
ClonExpress® MultiS One Step Cloning Kit C113-01/02 10/25 rxn
ClonExpress® Ultra One Step Cloning Kit C115-01/02 25/50 rxn

Since the establishment in 2012, Vazyme has been dedicated to our mission "Science and Technology Make a Healthier Life" to focus on technology innovation and continuously expand the application fields of core technologies in life science. As a R&D based company, we have been holding ourselves to the highest standards of ethics, accountability and professionalism. Now, we have a variety of product lines based on our self-developed technologies.

 

1. Hsu, P. K., Dubeaux, G., Takahashi, Y., & Schroeder, J. I. (2021). Signaling mechanisms in abscisic acid-mediated stomatal closure. The Plant journal : for cell and molecular biology, 105(2), 307–321. https://doi.org/10.1111/tpj.15067

2. Melotto, M., Underwood, W., & He, S. Y. (2008). Role of stomata in plant innate immunity and foliar bacterial diseases. Annual review of phytopathology, 46, 101–122. https://doi.org/10.1146/annurev.phyto.121107.104959