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.

Orosomucoid proteins limit endoplasmic reticulum stress in plants.

Sphingolipids are cell membrane components and signaling molecules that induce endoplasmic reticulum (ER) stress responses, but the underlying mechanism is unknown. Orosomucoid proteins (ORMs) negatively regulate serine palmitoyltransferase activity, thus helping maintain proper sphingolipid levels in humans, yeast, and plants. In this report, we explored the roles of ORMs in regulating ER stress in Arabidopsis thaliana. Loss of ORM1 and ORM2 function caused constitutive activation of the unfolded protein response (UPR), as did treatment with the ceramide synthase inhibitor Fumonisin B1 (FB1) or ceramides. FB1 treatment induced the transcription factor bZIP28 to relocate from the ER membrane to the nucleus. The transcription factor WRKY75 positively regulates the UPR and physically interacted with bZIP28. We also found that the orm mutants showed impaired ER-associated degradation (ERAD), blocking the degradation of misfolded MILDEW RESISTANCE LOCUS-O 12 (MLO-12). ORM1 and ORM2 bind to EMS-MUTAGENIZED BRI1 SUPPRESSOR 7 (EBS7), a plant-specific component of the Arabidopsis ERAD complex, and regulate its stability. These data strongly suggest that ORMs in the ER membrane play vital roles in the UPR and ERAD pathways to prevent ER stress in Arabidopsis. Our results reveal that ORMs coordinate sphingolipid homeostasis with ER quality control and play a role in stress responses.

Plasma membrane-nucleo-cytoplasmic coordination of a receptor-like cytoplasmic kinase promotes EDS1-dependent plant immunity.

Plants use cell-surface immune receptors to recognize pathogen-specific patterns to evoke basal immunity. ENHANCED DISEASE SUSCEPTIBILITY (EDS1) is known to be crucial for plant basal immunity, whereas its activation mechanism by pattern recognition remains enigmatic. Here, we show that the fungal pattern chitin induced the plasma membrane-anchored receptor-like cytoplasmic kinase PBS1-LIKE 19 (PBL19) to undergo nuclear translocation in Arabidopsis. The palmitoylation-deficient PBL19C3A variant constantly resided in the nucleus, triggering transcriptional self-amplification mainly through WRKY8 and EDS1-dependent constitutive immunity. Unexpectedly, the metacaspase-cleaved PBL19 lacking the N-terminal nuclear localization sequence specifically interacted with and phosphorylated EDS1 in the cytoplasm. Phosphodeficient EDS1 attenuated PBL19C3A-induced constitutive immunity, while phosphomimetic EDS1 complemented the loss of PBL19 for fungal resistance. Collectively, these findings reveal a compelling model wherein the plasma membrane, nuclear and cytoplasmic pools of PBL19 temporally coordinate distinct roles of immune signal receiver, amplifier and effector to boost plant antifungal immunity via EDS1.

Arabidopsis lysin motif/F-box-containing protein InLYP1 fine-tunes glycine metabolism by degrading glycine decarboxylase GLDP2.

Lysin motif (LysM) is a carbohydrate-binding module often found in secreted or transmembrane proteins in living organisms from prokaryotes to eukaryotes. Thus far, all characterized LysM-containing proteins in plants are plasma membrane-resident receptors or co-receptors playing roles in plant-microbe interactions. Here, we interrogate the Arabidopsis LysM/F-box-containing protein InLYP1 and reveal its function in glycine metabolism. InLYP1 was mainly expressed by vigorously growing tissues, encoding a nuclear-cytoplasmic protein. We validated InLYP1 as part of the SKP1-CULLIN1-F-box E3 complex for mediating protein degradation. The glycine decarboxylase P-protein 1 (GLDP1) was identified as an InLYP1-interacting protein by both immunoprecipitation/mass spectrometry and yeast two-hybrid library screening. InLYP1 could also interact with GLDP2, a paralog of GLDP1 with weaker catalytic activity, and could mediate the degradation of GLDP2 but not GLDP1. Interestingly, both GLDPs could be O-glycosylated and form homodimers or heterodimers. Overexpression of InLYP1L9A encoding a dominant-negative variant could cause seedling germination retardation on the medium containing glycine. Collectively, these results shed light on the function of plant intracellular LysM-containing proteins, and suggest that InLYP1 may deplete GLDP2 to facilitate glycine decarboxylation in Arabidopsis.

Multiplex and optimization of dCas9-TV-mediated gene activation in plants.

Synthetic gene activators consisting of nuclease-dead Cas9 (dCas9) for single-guide RNA (sgRNA)-directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9-TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9-TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA-sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000-fold in rice. Moreover, the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9-TV-refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.

The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity.

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely understudied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern-triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of FLS2 nanoclusters and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis, with a particular increase in the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to manipulate pathogen-associated molecular pattern-triggered host immunity.

Hide-and-Seek: Chitin-Triggered Plant Immunity and Fungal Counterstrategies.

Fungal pathogens are major destructive microorganisms for land plants and pose growing challenges to global crop production. Chitin is a vital building block for fungal cell walls and also a broadly effective elicitor of plant immunity. Here we review the rapid progress in understanding chitin perception and signaling in plants and highlight similarities and differences of these processes between arabidopsis and rice. We also outline moonlight functions of CERK1, an indispensable chitin coreceptor conserved across the plant kingdom, which imply potential crosstalk between chitin signaling and symbiotic or biotic/abiotic stress signaling in plants via CERK1. Moreover, we summarize current knowledge about fungal counterstrategies for subverting chitin-triggered plant immunity and propose open questions and future directions in this field.

l-Phenylalanine Alters the Privileged Secondary Metabolite Production in the Marine-Derived Fungus Trichoderma erinaceum F1-1.

The effects of a single-amino-acid culture strategy on secondary metabolite production in the marine-derived fungus Trichoderma erinaceum F1-1 were investigated by culturing the fungus in GPY medium supplemented or not supplemented with l-phenylalanine. A suite of secondary metabolites, including seven terpenoids (1-7) and one polyketide (8), among which are four new compounds, harziandione A (1), cyclonerodiols A and B (3, 4), and trichodermaerin A (6), were isolated from the GPY medium without l-phenylanine, whereas 18 aromatic compounds (9-26), including six new compounds, trichoderolides A-F (9, 10, and 14-17), were isolated from the culture grown in the GPY medium with l-phenylalanine. The structures of the new compounds were determined by high-resolution mass spectrometry, NMR spectroscopic analysis, optical rotation calculations, chemical methods, and X-ray crystallography. Compounds 10, 12, 13, and 26 exhibited cytotoxic activities against MDA-MB-435 human melanocyte cancer cells. Compound 26 was cytotoxic to A549 adenocarcinomic human alveolar basal epithelial cells.

BAK1-mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis.

Heterotrimeric G proteins consisting of Gα, Gß and Gγ are conserved signaling hubs in eukaryotes. Without analogs to canonical animal G protein-coupled receptors, plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα. Meanwhile, the exact role of canonical Gα in plant innate immunity remains controversial. Here, we report multiple immune deficiencies in the null allele of Arabidopsis Gα (GPA1) in response to bacterial flg22 elicitor, clarifying a positive regulatory role of GPA1 in flg22 signaling. We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr19 upon flg22 elicitation. Interestingly, flg22 could not induce phosphorylation of GPA1T19A and GPA1T19D , suggesting that the dynamic Thr19 phosphorylation is required for GPA1 to respond to flg22. Moreover, flg22-induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo, and BAK1 could phosphorylate GPA1 but not GPA1T19A in vitro at the phosphorylation sites identified in vivo, suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to flg22. Furthermore, the T19A mutation could promote flg22-induced association, rather than dissociation, between GPA1 and RGS1. Taken together, our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.

Split Nano luciferase complementation for probing protein-protein interactions in plant cells.

Deciphering protein-protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual-use system enabling the detection of PPIs through the Nluc-based SLC and co-immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA- or FLAG-tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium-transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc-based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.

Cross-Microbial Protection via Priming a Conserved Immune Co-Receptor through Juxtamembrane Phosphorylation in Plants.

Living organisms can be primed for potentiated responses to recurring stresses based on prior experience. However, the molecular basis of immune priming remains elusive in plants that lack adaptive immunity. Here, we report that bacterial challenges can prepare plants for fungal attacks by inducing juxtamembrane phosphorylation of CERK1, the co-receptor indispensable for signaling in response to the fungal elicitor chitin. This phosphorylation is mediated by BAK1, a co-receptor for signaling in response to multiple elicitors. BAK1 interacts with CERK1, and loss of BAK1 reduces priming phosphorylation of CERK1. Juxtamembrane phosphomimetic mutations of CERK1 confer accelerated chitin responses and fortified fungal resistance without triggering constitutive immunity, whereas juxtamembrane phosphodeficient mutations diminish bacteria-induced protection against fungal infection. These findings reveal that crosstalk between cell-surface immune co-receptors can prime defense and demonstrate that juxtamembrane phosphorylation of plant receptor-like kinases can occur independent of kinase activation to place the protein into a prime state.

Dynamic G protein alpha signaling in Arabidopsis innate immunity.

Heterotrimeric G proteins composed of Gα, Gß and Gγ subunits are evolutionarily conserved signaling modules involved in diverse biological processes in plants and animals. The role and action of Gα remain largely enigmatic in plant innate immunity. We have recently demonstrated that Arabidopsis Gα (GPA1) is a key component of a new immune signaling pathway activated by bacteria-secreted proteases. Here we show that GPA1 is also involved in the signaling network of Arabidopsis in response to the bacterial flagellin epitope flg22. Specifically, GPA1 plays a pivotal role in an immune pathway involving the flg22 receptor FLS2, co-receptor BAK1, Regulator of G Signaling 1 (RGS1), and Arabidopsis Gß (AGB1), in which flg22 elicits GPA1/AGB1 dissociation from the FLS2/BAK1/RGS1 receptor complex. Consequently, we observed flg22-induced degradation of FLS2, BAK1 and RGS1 but not GPA1 or AGB1. We also found that GPA1 constitutively interacts with the NADPH oxidase RbohD to potentiate flg22-induced ROS burst independently of the central cytoplasmic kinase BIK1. Taken together, our work sheds multiple novel insights into the functions and regulatory mechanisms of GPA1 in Arabidopsis innate immunity.

Engineering Artificial MicroRNAs for Multiplex Gene Silencing and Simplified Transgenic Screen.

Artificial microRNA (amiRNA) technology offers reversible and flexible gene inactivation and complements genome-editing technologies. However, obtaining transgenic plants with maximal gene silencing remains a major technical challenge in current amiRNA applications. Here, we incorporated an empirically determined feature of effective amiRNAs to the amiRNA design and in silico generated a database containing 533,429 gene-specific amiRNAs for silencing 27,136 genes in Arabidopsis (Arabidopsis thaliana), with a genome coverage of 98.87%. In both single-gene and multiple-gene silencing, we observed an overall improvement in performance by amiRNAs designed using our strategy in Arabidopsis protoplasts and transgenic plants. In addition, the endogenous tRNA-processing system was used to generate multiple amiRNAs from tRNA-pre-amiRNA tandem repeats for multiplex gene silencing. An intronic amiRNA-producing fluorescent reporter was explored as a visual screening strategy for transgenic Arabidopsis and rice (Oryza sativa) plants with maximal whole-plant or cell type-specific gene silencing. These improvements enable the amiRNA technology to be a functional gene knockout tool for basic and applied plant research.

A Tyrosine Phosphorylation Cycle Regulates Fungal Activation of a Plant Receptor Ser/Thr Kinase.

Plants initiate immunity by cell-surface pattern-recognition receptors (PRRs), which perceive non-self molecules. PRRs are predominantly receptor serine/threonine (Ser/Thr) kinases that are evolutionarily related to animal interleukin-1 receptor-associated kinase (IRAK)/Pelle-soluble kinases. However, how the activity of these receptor kinases is modulated remains poorly understood. We report that the Arabidopsis PRR chitin elicitor receptor kinase 1 (CERK1) is autophosphorylated in unstimulated cells at tyrosine428 (Tyr428), a modification that is required for CERK1 activation upon binding to the fungal cell wall component chitin. Upon chitin activation, CERK1 recruits the CERK1-interacting protein phosphatase 1 (CIPP1), a predicted Ser/Thr phosphatase, to dephosphorylate Tyr428 and dampen CERK1 signaling. CIPP1 subsequently dissociates from Tyr428-dephosphorylated CERK1, allowing CERK1 to regain Tyr428 autophosphorylation and return to a standby state. This work sheds light onto plant chitin signaling and shows that a receptor kinase and phosphatase can coordinately regulate signal transduction of a receptor kinase through a phosphorylation cycle.