Loss of the arabidopsis protein kinases ANPs affects root cell wall composition, and triggers the cell wall damage syndrome

The Arabidopsis NPK1-related Protein kinases ANP1, ANP2 and ANP3 belong to the MAP kinase kinase kinase (MAPKKK) superfamily and were previously described to be crucial for cytokinesis, elicitor-induced immunity and development. Here we investigate the basis of their role in development by using conditional β-estradiol-inducible triple mutants to overcome lethality. In seedlings, lack of ANPs causes root cell bulging, with the transition zone being the most sensitive region. We uncover a role of ANPs in the regulation of cell wall composition and suggest that developmental defects of the triple mutants, observed at the cellular level, might be a consequence of the alterations of the pectic and cellulosic cell wall components. Lack of ANPs also induced a typical cell wall damage syndrome (CWDS) similar to that observed in plants treated with the cellulose biosynthesis inhibitor isoxaben (ISX). Moreover, anp double mutants and plants overexpressing single ANPs (ANP1 or ANP3) respectively showed increased and reduced accumulation of jasmonic acid and PDF1.2 transcripts upon ISX treatment, suggesting that ANPs are part of the pathway targeted by this inhibitor and play a role in cell wall integrity surveillance

GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding

Conserved microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) act as danger signals to activate the plant immune response. These molecules are recognized by surface receptors that are referred to as pattern recognition receptors. Oligogalacturonides (OGs), DAMPs released from the plant cell wall homogalacturonan, have also been proposed to act as local signals in the response to wounding. The Arabidopsis Wall-Associated Kinase 1 (WAK1), a receptor of OGs, has been described to form a complex with a cytoplasmic plasma membrane-localized kinase-associated protein phosphatase (KAPP) and a glycine-rich protein (GRP-3) that we find localized mainly in the cell wall and, in a small part, on the plasma membrane. By using Arabidopsis plants overexpressing WAK1, and both grp-3 and kapp null insertional mutant and overexpressing plants, we demonstrate a positive function of WAK1 and a negative function of GRP-3 and KAPP in the OG-triggered expression of defence genes and the production of an oxidative burst. The three proteins also affect the local response to wounding and the basal resistance against the necrotrophic pathogen Botrytis cinerea. GRP-3 and KAPP are likely to function in the phasing out of the plant immune response

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced in response to pathogen invasion attempts at the infection site and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). In this protocol the detection and quantification of camalexin produced in Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea is described

Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves

In Arabidopsis thaliana, one of the very early immune-related responses induced after elicitor perception is the oxidative burst, i.e. reactive oxygen species (ROS) generation including superoxide anion and hydrogen peroxide (H2O2). ROS production plays different roles in a wide range of biotic and abiotic stress responses, including the closure of stomata and the regulation of cell expansion. In particular, elicitor-induced H2O2 is produced mainly by the membrane localized NAD(P)H oxidases RESPIRATORY BURST OXIDASE HOMOLOGUE D and F. In this protocol, we describe a simple and reproducible luminol/peroxidase-based assay to detect and evaluate immunity-related accumulation of H2O2 produced in Arabidopsis leaf discs treated with immunity elicitors, such as oligogalacturonides (OGs), flagellin (flg22) or the elongation factor-thermo-unstable (EF-Tu - elf18). This method is based on the detection of the luminescence released by excited-luminol molecules generated after the horseradish peroxidase (HRP)-catalyzed oxidation of luminol molecules in the presence of H2O2. Levels as well as duration of the luminescence are proportional to the amount of H2O2 produced by elicited leaf discs

Tissue-specific expression of PvPGIP2 to improve wheat resistance against Fusarium graminearum

Fusarium Head Blight (FHB) is one of the most important wheat diseases caused by some fungi of the genus Fusarium. The pathogen infects the spike at flowering time and causes severe yield losses and deterioration of grain quality due to the secretion of mycotoxins during infection. The understanding of the precise mode of pathogen entering and the subsequent floral tissue colonize is a crucial point to control FHB. Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases (PGs) secreted by microbial pathogens and insects. The constitutive expression of the bean PvPGIP2 limits FHB symptoms and reduces mycotoxin accumulation in wheat. To better understand the spike tissues that play a role in limiting Fusarium infection, we have produced transgenic wheat plants expressing PvPGIP2 or in the endosperm or simultaneously in lemma, palea, anthers and rachis. We showed that this latter approach reduced FHB symptoms caused by F. graminearum compared to control non transgenic plants. The extent of disease symptom reduction was similar to what obtained when PvPGIP2 was expressed constitutively. We showed also that different level of PvPGIP2 accumulation produced similar level of protection. Conversely, the expression of PvPGIP2 only in the endosperm did not affect FHB symptom development, indicating that when the pathogen has reached the endosperm, inhibition of the polygalacturonase (PG) activity of the pathogen is ineffective to prevent fungal spread. Probably the rich source of the endosperm tissue makes the PG activity dispensable for pathogen colonization. Alternatively, the massive growth of the fungus at this stage produces a large amount of PG that is not inhibited by the available PGIP

Tissue specific expression of PvPGIP2 to improve wheat resistance against Fusarium graminearum

Fusarium Head Blight (FHB) is one of the most important wheat diseases caused by Fusarium spp.. The pathogen infects the spike at flowering time and causes severe yield losses and deterioration of grain quality due to the secretion of mycotoxins during infection. The understanding of the precise mode of pathogen entering and the subsequent floral tissue colonize is a crucial point to control FHB. Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases (PGs) secreted by pathogens. The constitutive expression of the bean PvPGIP2 limits FHB symptoms and reduces mycotoxin accumulation in wheat. To better understand which spike tissues plays a role in limiting Fusarium infection, we have produced transgenic wheat plants expressing PvPGIP2 in the endosperm or simultaneously in lemma, palea, anthers and rachis. This latter approach reduced FHB symptoms, whereas the expression of PvPGIP2 only in the endosperm did not affect FHB development, indicating that when the pathogen has reached the endosperm, inhibition of pathogen PGs ineffective to prevent fungal spread

Corrigendum: Loss of the Arabidopsis Protein Kinases ANPs Affects Root Cell Wall Composition, and Triggers the Cell Wall Damage Syndrome

The Arabidopsis NPK1-related Protein kinases ANP1, ANP2 and ANP3 belong to the MAP kinase kinase kinase (MAPKKK) superfamily and were previously described to be crucial for cytokinesis, elicitor-induced immunity and development. Here we investigate the basis of their role in development by using conditional β-estradiol-inducible triple mutants to overcome lethality. In seedlings, lack of ANPs causes root cell bulging, with the transition zone being the most sensitive region. We uncover a role of ANPs in the regulation of cell wall composition and suggest that developmental defects of the triple mutants, observed at the cellular level, might be a consequence of the alterations of the pectic and cellulosic cell wall components. Lack of ANPs also induced a typical cell wall damage syndrome (CWDS) similar to that observed in plants treated with the cellulose biosynthesis inhibitor isoxaben (ISX). Moreover, anp double mutants and plants overexpressing single ANPs (ANP1 or ANP3) respectively showed increased and reduced accumulation of jasmonic acid and PDF1.2 transcripts upon ISX treatment, suggesting that ANPs are part of the pathway targeted by this inhibitor and play a role in cell wall integrity surveillance.Highlights: The loss of ANP function affects cell wall composition and leads to typical cell wall damage-induced phenotypes, such as ectopic lignification and jasmonic acid accumulation

Ligand-induced monoubiquitination of BIK1 regulates plant immunity

The detection of microorganism-associated ligands by plant cells activates a signalling cascade in which the kinase BIK1 is monoubiquinated, released from the FLS2-BAK1 complex, and internalized by endocytosis. Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens(1-3). Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants(4). The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling

EXPRESSION OF BEAN PGIP2 UNDER CONTROL OF THE BARLEY LEM1 PROMOTER LIMITS FUSARIUM GRAMINEARUM INFECTION IN WHEAT

Fusarium Head Blight (FHB) caused by Fusarium graminearum is one of the most destructive fungal diseases of wheat worldwide. The pathogen infects the spike at flowering time and causes severe yield losses, deterioration of grain quality, and accumulation of mycotoxins. Better understanding of the means of pathogen entry and colonization of floral tissue is crucial to providing effective protection against FHB. Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the activity of polygalacturonases (PGs), a class of pectin-depolymerizing enzymes secreted by microbial pathogens, including Fusaria. The constitutive expression of a bean PGIP (PvPGIP2) under control of the maize Ubi1 promoter limits FHB symptoms and reduces mycotoxin accumulation in wheat grain [Janni et al. 2008 Molec. Plant Microb. Interact. 21:171]. To better understand which spike tissues play major roles in limiting F. graminearum infection, we explored the use of PvPGIP2 to defend specific spike tissues by expressing it under control of the barley Lem1 promoter [Somleva and Blechl 2005 Cer. Res. Comm. 33:665]. We show here that the expression of PvPGIP2 in lemma, palea, rachis and anthers reduced FHB symptoms caused by F. graminearum compared to symptoms in infected nontransgenic plants. However, the expression of PvPGIP2 only in the endosperm under control of a HMW-glutenin gene promoter did not affect FHB symptom development, indicating that once the pathogen has reached the endosperm, inhibition of the pathogen\u2019s PG activity is not effective in preventing its further spread

Proteolytic processing of SERK3/BAK1 regulates plant immunity, development and cell death

Plants have evolved many receptor-like kinases (RLKs) to sense extrinsic and intrinsic cues. The signaling pathways mediated by multiple leucine-rich repeat (LRR) RLK (LRR-RLK) receptors require ligand-induced receptor-coreceptor heterodimerization and transphosphorylation with BAK1/SERK family LRR-RLKs. Here we reveal an additional layer of regulation of BAK1 via a Ca2+-dependent proteolytic cleavage process that is conserved in Arabidopsis thaliana, Nicotiana benthamiana and Saccharomyces cerevisiae . The proteolytic cleavage of BAK1 is intrinsically regulated in response to developmental cues and immune stimulation. The surface-exposed aspartic acid (D287) residue of BAK1 is critical for its proteolytic cleavage and plays an essential role in BAK1-regulated plant immunity, growth hormone brassinosteroid-mediated responses and cell death containment. BAK1D287A mutation impairs BAK1 phosphorylation on its substrate BIK1, and its plasma membrane (PM) localization. Intriguingly, it aggravates BAK1 overexpression-triggered cell death independent of BIK1, suggesting that maintaining homeostasis of BAK1 through a proteolytic process is crucial to control plant growth and immunity. Our data reveal that in addition to layered transphosphorylation in the receptor complexes, the proteolytic cleavage is an important regulatory process for the proper functions of the shared co-receptor BAK1 in diverse cellular signaling pathways