Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Conservation and divergence of flg22, pep1 and nlp20 in activation of immune response and inhibition of root development.

Many pattern-recognition receptors (PRRs) and their corresponding ligands have been identified. However, it is largely unknown how similar and different these ligands are in inducing plant innate immunity and affecting plant development. In this study, we examined three well characterized ligands in Arabidopsis thaliana, namely flagellin 22 (flg22), plant elicitor peptide 1 (pep1) and a conserved 20-amino-acid fragment found in most necrosis and ethylene-inducing peptide 1-like proteins (nlp20). Our quantitative analyses detected the differences in amplitude in the early immune responses of these ligands, with nlp20-induced responses typically being slower than those mediated by flg22 and pep1. RNA sequencing showed the shared differentially expressed genes (DEGs) was mostly enriched in defense response, whereas nlp20-regulated genes represent only a fraction of those genes differentially regulated by flg22 and pep1. The three elicitors all inhibited primary root growth, especially pep1, which inhibited both auxin transport and signaling pathway. In addition, pep1 significantly inhibited the cell division and genes involved in cell cycle. Compared with flg22 and nlp20, pep1 induced much stronger expression of its receptor in roots, suggesting a potential positive feedback regulation in the activation of immune response. Despite PRRs and their co-receptor BAK1 were necessary for both PAMP induced immune response and root growth inhibition, bik1 mutant only showed impaired defense response but relatively normal root growth inhibition, suggesting BIK1 acts differently in these two biological processes.

Effectors of Puccinia striiformis f. sp. tritici Suppressing the Pathogenic-Associated Molecular Pattern-Triggered Immune Response Were Screened by Transient Expression of Wheat Protoplasts.

Puccinia striiformis f. sp. tritici (Pst) is an important pathogen of wheat (Triticum aestivum L.) stripe rust, and the effector protein secreted by haustoria is a very important component involved in the pathogenic process. Although the candidate effector proteins secreted by Pst haustoria have been predicted to be abundant, few have been functionally validated. Our study confirmed that chitin and flg22 could be used as elicitors of the pathogenic-associated molecular pattern-triggered immune (PTI) reaction in wheat leaves and that TaPr-1-14 could be used as a marker gene to detect the PTI reaction. In addition, the experimental results were consistent in wheat protoplasts. A rapid and efficient method for screening and identifying the effector proteins of Pst was established by using the wheat protoplast transient expression system. Thirty-nine Pst haustorial effector genes were successfully cloned and screened for expression in the protoplast. We identified three haustorial effector proteins, PSEC2, PSEC17, and PSEC45, that may inhibit the response of wheat to PTI. These proteins are localized in the somatic cytoplasm and nucleus of wheat protoplasts and are highly expressed during the infection and parasitism of wheat.

Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

Overexpression of a Tartary Buckwheat Gene, FtbHLH3, Enhances Drought/Oxidative Stress Tolerance in Transgenic Arabidopsis.

bHLH (basic helix-loop-helix) transcription factors play important roles in the abiotic stress response in plants, but their characteristics and functions in tartary buckwheat (Fagopyrum tataricum), a flavonoid-rich cereal crop with a strong stress tolerance, have not been fully investigated. Here, a novel bHLH gene, designated FtbHLH3, was isolated and characterized. Expression analysis in tartary buckwheat revealed that FtbHLH3 was mainly induced by polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA) treatments. Subcellular localization and a yeast one-hybrid assay indicated that FtbHLH3 has transcriptional activation activities. Overexpression of FtbHLH3 in Arabidopsis resulted in increased drought/oxidative tolerance, which was attributed to not only lower malondialdehyde (MDA), ion leakage (IL), and reactive oxygen species (ROS) but also higher proline (Pro) content, activities of antioxidant enzymes, and photosynthetic efficiency in transgenic lines compared to wild type (WT). Moreover, qRT-PCR analysis indicated that the expression of multiple stress-responsive genes in the transgenic lines was significantly higher than in WT under drought stress. In particular, the expression of AtNCED, a rate-limiting enzyme gene in ABA biosynthesis, was increased significantly under both normal and stress conditions. Additionally, an ABA-response-element (ABRE) was also found in the promoter regions. Furthermore, the transgenic Arabidopsis lines of the FtbHLH3 promoter had higher GUS activity after drought stress. In summary, our results indicated that FtbHLH3 may function as a positive regulator of drought/oxidative stress tolerance in transgenic Arabidopsis through an ABA-dependent pathway.

A basic phenylalanine-rich oligo-peptide causes antibody cross-reactivity.

Glycolate oxidase (GO) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) are the two enzymes that serve key functions in the photorespiration and photosynthesis of plants. A 2 kDa highly basic phenylalanine-rich oligo-peptide (BOP) binds to the surface of acidic GO via ionic and hydrophobic interactions, forming the GO-BOP complex (GC). Previously, RubisCO was thought to exist as a single species composed of a large (rbc L, 54 kDa) and a small subunit (rbc S, 14 kDa). Here we show for the first time, using 2-DE, SDS-PAGE, immunoassays and amino acid determination, that BOP also interacts with RubisCO and that many RubisCO-BOP complexes (RCs), differing in pI, hydrophobicity and activity, coexist in green leaves. GCs, RCs and crude extract from green leaves analyzed by SDS-PAGE Western blotting showed that BOP exists either in subunit-BOP complexes (GO subunit-BOP, rbc L-BOP and rbc S-BOP etc.), with a wide variation in the number and the position of BOPs bound to each subunit molecular, or alone without a binding partner. When rbc L-BOP and rbc S-BOP were assayed by SDS-PAGE, BOP was dissociated from the subunit and it self-assembled to form 37 different BOP polymers (basic phenylalanine-rich protein) whose molecular weights (M(r)s) ranged from 34.0 to 91.6 kDa, indicating that the M(r) of BOP is about 2 kDa. Thus, the addition of BOP changes the M(r) of the subunit-BOP complexes so minimally that the rbc L and rbc S run at their predicted M(r)s on SDS-PAGE. In summary, the results described here demonstrate that the presence of BOP in complexes (both subunit-BOP complex and protein-BOP complex) can cause cross-reactivity of antibodies against different proteins.