Proteolysis in plant immunity.

Compared with transcription and translation, protein degradation machineries can act faster and be targeted to different subcellular compartments, enabling immediate regulation of signaling events. It is therefore not surprising that proteolysis has been used extensively to control homeostasis of key regulators in different biological processes and pathways. Over the past decades, numerous studies have shown that proteolysis, where proteins are broken down to peptides or amino acids through ubiquitin-mediated degradation systems and proteases, is a key regulatory mechanism to control plant immunity output. Here, we briefly summarize the roles various proteases play during defense activation, focusing on recent findings. We also update the latest progress of ubiquitin-mediated degradation systems in modulating immunity by targeting plant membrane-localized pattern recognition receptors (PRRs), intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs), and downstream signaling components. Additionally, we highlight recent studies showcasing the importance of proteolysis in maintaining broad-spectrum resistance without obvious yield reduction, opening new directions for engineering elite crops that are resistant to a wide range of pathogens with high yield.

Arabidopsis Tubby domain-containing F-box proteins positively regulate immunity by modulating PI4Kß protein levels.

The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5-bisphosphate. Arabidopsis has 11 Tubby domain-containing proteins referred to as Tubby-Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N-terminal F-box domain, which can interact with SKP-like proteins and form SKP1-Cullin-F-box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby-like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4-kinase ß proteins (PI4Kßs), PI4Kß1 and PI4Kß2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kß1,2 mutant, while TLP6 overexpression also leads to increased PI4Kß2 ubiquitination and reduction in its protein level in a proteasome-dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kß1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kßs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kßs protein levels.

Activation of NLR-Mediated Autoimmunity in Arabidopsis Early in Short Days 4 Mutant.

From a reverse genetic screen using CRISPR/Cas9 gene editing tool, we unintentionally identified an autoimmune mutant. Map-based cloning and whole-genome sequencing revealed that it contains a deletion in SMALL UBIQUITIN-RELATED MODIFIER (SUMO) protease encoding gene EARLY IN SHORT DAYS 4 (ESD4). Previous studies reported that esd4 mutants accumulate elevated levels of plant defense hormone salicylic acid (SA). However, upregulated PATHOGENESIS-RELATED GENE 1 (PR1) expression in esd4 only partly relies on SA level. In this study, we show that plant metabolite N-hydroxypipecolic acid (NHP) biosynthetic genes are upregulated in esd4, and NHP biosynthesis mutant flavin-dependent-monooxygenase 1 (fmo1) partially suppresses the autoimmune phenotypes of esd4, suggestive of a requirement of NHP signaling for the autoimmunity in esd4. As activation of nucleotide-binding leucine-rich repeat immune receptors (NLRs) are associates with the biosynthesis of SA and NHP and lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is a key component downstream of many NLRs, we examined the relationship between EDS1 and ESD4 by analyzing the eds1 esd4 double mutant. We found that eds1 largely suppresses esd4 autoimmunity and blocks the elevated expressions of SA and NHP biosynthesis-related genes in esd4. Overall, our study provides evidence supporting the hypothesis that SUMO protease ESD4 likely targets a yet to be identified guardee of NLR by removing its SUMO modification to avoid recognition by the cognate NLR. Loss of ESD4 results in activation of NLR-mediated autoimmunity.

SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious soilborne fungal pathogen that causes serious economic losses globally. The necrosis and ethylene-inducible peptide 1 (NEP1)-like proteins (NLPs) were previously shown to play an important role in pathogenicity in fungal and oomycete pathogens. Here, we generated S. sclerotiorum necrosis and ethylene-inducible peptide 2 (SsNEP2) deletion mutant through homologous recombination and found that SsNEP2 contributes to the virulence of S. sclerotiorum without affecting the development of mycelia, the formation of appressoria, or the secretion of oxalic acid. Although knocking out SsNEP2 did not affect fungal sensitivity to oxidative stress, it did lead to decreased accumulation of reactive oxygen species (ROS) in S. sclerotiorum. Furthermore, Ssnlp24SsNEP2 peptide derived from SsNEP2 triggered host mitogen-activated protein kinase (MAPK) activation, increased defense marker gene expression, and enhanced resistance to Hyaloperonospora arabidopsidis Noco2. Taken together, our data suggest that SsNEP2 is involved in fungal virulence by affecting ROS levels in S. sclerotiorum. It can serve as a pathogen-associated molecular pattern (PAMP) and trigger host pattern triggered immunity to promote the necrotrophic lifestyle of S. sclerotiorum.

A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanization Pathway.

Most plant fungal pathogens that cause worldwide crop losses are understudied, due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here, we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen Sclerotinia sclerotiorum and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanization-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanization of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanization is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Receptor-like kinases MDS1 and MDS2 promote SUMM2-mediated immunity.

Disruption of the MEKK1-MKK1/MKK2-MPK4 kinase cascade leads to activation of immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) immune receptor SUMM2, which monitors the phosphorylation status of CRCK3. Here we report that two receptor-like kinases (RLKs), MDS1, and MDS2, function redundantly to promote SUMM2-mediated immunity. Activation of SUMM2-mediated immunity is dependent on MDS1, and to a less extent on MDS2. MDS1 associates with CRCK3 in planta and can phosphorylate CRCK3 in vitro, suggesting that it may target CRCK3 to positively regulate SUMM2-mediated signaling. Our finding highlights a new defense mechanism where RLKs promote NLR-mediated immunity.

Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity.

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.

Chromosomal translocation-derived aberrant Rab22a drives metastasis of osteosarcoma.

Osteosarcoma is a type of aggressive malignant bone tumour that frequently metastasizes to lungs, resulting in poor prognosis. However, the molecular mechanisms of lung metastasis of osteosarcoma remain poorly understood. Here we identify exon-intron fusion genes in osteosarcoma cell lines and tissues. These fusion genes are derived from chromosomal translocations that juxtapose the coding region for amino acids 1-38 of Rab22a (Rab22a1-38) with multiple inverted introns and untranslated regions of chromosome 20. The resulting translation products, designated Rab22a-NeoFs, acquire the ability to drive lung metastasis of osteosarcoma. The Rab22a1-38 moiety governs the function of Rab22a-NeoFs by binding to SmgGDS-607, a GTP-GDP exchange factor of RhoA. This association facilitates the release of GTP-bound RhoA from SmgGDS-607, which induces increased activity of RhoA and promotes metastasis. Disrupting the interaction between Rab22a-NeoF1 and SmgGDS-607 with a synthetic peptide prevents lung metastasis in an orthotopic model of osteosarcoma. Our findings may provide a promising strategy for a subset of osteosarcoma patients with lung metastases.

Author Correction: Chromosomal translocation-derived aberrant Rab22a drives metastasis of osteosarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Redundant CAMTA Transcription Factors Negatively Regulate the Biosynthesis of Salicylic Acid and N-Hydroxypipecolic Acid by Modulating the Expression of SARD1 and CBP60g.

Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. CALMODULIN-BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2, and CAMTA3 are known redundant negative regulators of plant immunity, but the underlying mechanism also remains largely unknown. In this study, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3, which also negatively regulates the expression of SARD1, presumably via an indirect effect. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two single mutants defective in NHP biosynthesis. Interestingly, a suppressor screen conducted in the camta1/2/3 triple mutant background yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response.

Loss-of-function of Arabidopsis receptor-like kinase BIR1 activates cell death and defense responses mediated by BAK1 and SOBIR1.

The Arabidopsis receptor-like kinase (RLK) BIR1 (BAK1-INTERACTING RECEPTOR-LIKE KINASE 1) functions as a negative regulator of plant immunity. Previous work showed that loss-of-function of BIR1 leads to constitutive activation of cell death and defense responses. These autoimmune phenotypes are partially dependent on another RLK, SOBIR1. In order to identify additional components involved in the BIR1-regulated plant defense signaling pathway, a suppressor screen was carried out in the bir1-1 pad4-1 mutant background. Mutations in the suppressor mutants were identified by genetic mapping and re-sequencing of the mutant genomes. A number of suppressor mutants were found to carry mutations in an additional RLK, BAK1, indicating that BAK1 is required for activation of cell death and defense responses in bir1-1. Co-immunoprecipitation analysis revealed that BAK1 and SOBIR1 associate with each other in planta when the function of BIR1 is compromised. Although BAK1 was previously characterized as a negative regulator of cell death, our study highlights a novel role of BAK1 in promoting cell death and defense responses in conjunction with SOBIR1.

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

UNLABELLED: Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. IMPORTANCE: Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.

Heterotrimeric G proteins serve as a converging point in plant defense signaling activated by multiple receptor-like kinases.

In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G protein α-subunit1), one Gß subunit (Arabidopsis G protein ß-subunit1 [AGB1]), and three Gγs subunits (Arabidopsis G protein γ-subunit1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) suppressor of BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing suppressor of BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, flagellin-sensitive2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and chitin elicitor receptor kinase1 (CERK1), respectively. By contrast, G protein α-subunit1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals.