Determination of AtSCD1 mRNA and protein levels in the temperature sensitive mutant scd1-1

Abstract only availablePlant cells defend themselves against bacterial infection by first detecting bacterial flagellin outside the cell and then initiating defense responses within the cell. Our lab is interested in identifying and characterizing vesicular trafficking components that play a role in defense responses to bacterial flagellin and its active peptide derivative, flg22, in Arabidopsis thaliana. Our current research focuses on SCD1, a protein implicated in polarized vesicle secretion. Recent results in our lab also indicate that scd1-1 plants, which contain a point mutation in the SCD1 gene, exhibit reduced flg22-responses, notably in the production of reactive oxygen species (ROS). To gain a better understanding of the role of SCD1 in flg22-responses, we analyzed SCD1 mRNA and protein levels in scd1-1 plants compared to the wild-type (WT), Colgl1, plants using qRT-PCR and protein blot analysis, respectively. SCD1 levels were compared in plants grown continuously at 22°C, the non-permissive temperature, to plants grown initially at 22°C and then shifted for 9 days to the permissive temperature, 17°C. Data indicate that at both 22°C and 17°C, SCD1 mRNA levels were statistically similar in scd1-1 compared to WT plants. Conversely, SCD1 protein levels were significantly higher in scd1-1 plants shifted to 17°C compared to those grown at 22°C. An increase in SCD1 protein level also correlated with an increase in flg22-induced ROS production at 17°C. To further confirm a requirement for SCD1 in flg22-repsonses, we showed that in an scd1-1 mutant plant transformed with the SCD1 gene (scd1-1/pscd1::SCD1), SCD1 protein levels were similar to those in WT grown at 22°C. These results are consistent with the complemented line exhibiting flg22-induced ROS production similar to WT levels. Given these results, both the temperature shift and the complementation experiments support our hypothesis that SCD1 protein is required for full flg22-induced responses.Life Sciences Undergraduate Research Opportunity Progra

The role of the brassinosteroid associated kinase (BAK1) in plant cell defense [abstract]

Abstract only availablePlants are able to resist bacterial and fungal infection by recognizing invading organisms outside the cell and then activating defense responses inside the cell. The first line of defense against invading organisms is known as the innate immune response. It is based on the recognition of pathogen-associated molecular patterns (PAMPs) found on pathogens (such as bacteria and fungi) by the plant cell. PAMPs induce defense responses through interaction with specific receptor proteins that are located on the membrane surface of the plant host cell. A well known PAMP is flg22, a 22-amino acid peptide derived from flagellum, the building block of bacterial flagellum which is important for the bacteria's mobility that enables it to move to the inside of the cell. In A. thaliana, the Flagellin Sensing 2 (FLS2) protein is the cell surface receptor that recognizes the bacterial PAMP flg22. Recognition of flg22 by FLS2 causes the plant to produce reactive oxygen molecules (ROS), which is an early defense response. Other plant defense responses include seedling growth inhibition and deposition of defense factors in the plant cell wall such as callose. In collaboration with Dr. Rathjen's lab, our lab has recently shown that the cell surface protein BAK1 (Brassinosteroid associated kinase 1) forms a complex with FLS2 after elicitation with flg22. The goal of this study was to characterize the role of BAK1 in innate immune responses to gain a better understanding in how BAK1 contributes to plant innate immunity against pathogen infections. In the future, we hope to use the knowledge gained from our studies and translate it into crop species such as tomato or rice to make these crop species more resistant against pathogen infection.Food for 21st Century, University of Missour

Bacterial Effectors Target the Common Signaling Partner BAK1 to Disrupt Multiple MAMP Receptor-Signaling Complexes and Impede Plant Immunity

SummarySuccessful pathogens have evolved strategies to interfere with host immune systems. For example, the ubiquitous plant pathogen Pseudomonas syringae injects two sequence-distinct effectors, AvrPto and AvrPtoB, to intercept convergent innate immune responses stimulated by multiple microbe-associated molecular patterns (MAMPs). However, the direct host targets and precise molecular mechanisms of bacterial effectors remain largely obscure. We show that AvrPto and AvrPtoB bind the Arabidopsis receptor-like kinase BAK1, a shared signaling partner of both the flagellin receptor FLS2 and the brassinosteroid receptor BRI1. This targeting interferes with ligand-dependent association of FLS2 with BAK1 during infection. It also impedes BAK1-dependent host immune responses to diverse other MAMPs and brassinosteroid signaling. Significantly, the structural basis of AvrPto-BAK1 interaction appears to be distinct from AvrPto-Pto association required for effector-triggered immunity. These findings uncover a unique strategy of bacterial pathogenesis where virulence effectors block signal transmission through a key common component of multiple MAMP-receptor complexes

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

Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

BACKGROUND: Arabidopsis thaliana and Pseudomonas syringae pathovar tomato (Pto) provide an excellent plant-bacteria model system to study innate immunity. During pattern-triggered immunity (PTI), cognate host receptors perceive pathogen-associated molecular patterns (PAMPs) as non-self molecules. Pto harbors many PAMPs; thus for experimental ease, many studies utilize single synthesized PAMPs such as flg22, a short protein peptide derived from Pseudomonas flagellin. Flg22 recognition by Arabidopsis Flagellin Sensing 2 (FLS2) initiates a plethora of signaling responses including rapid production of apoplastic reactive oxygen species (ROS). Assessing flg22-ROS has been instrumental in identifying novel PAMP-signaling components; but comparably little is known whether in Arabidopsis, ROS is produced in response to intact live Pto and whether this response can be used to dissect genetic requirements of the plant host and live bacterial pathogens in planta. RESULTS: Here, we report of a fast and robust bioassay to quantitatively assess early ROS in Arabidopsis leaves, a tissue commonly used for pathogen infection assays, in response to living bacterial Pto strains. We establish that live Pto elicits a transient and dose-dependent ROS that differed in timing of initiation, amplitude and duration compared to flg22-induced ROS. Our control experiments confirmed that the detected ROS was dependent on the presence of the bacterial cells. Utilizing Arabidopsis mutants previously shown to be defective in flg22-induced ROS, we demonstrate that ROS elicited by live Pto was fully or in part dependent on RbohD and BAK1, respectively. Because fls2 mutants did not produce any ROS, flagellin perception by FLS2 is the predominant recognition event in live Pto-elicited ROS in Arabidopsis leaves. Furthermore using different Pto strains, our in planta results indicate that early ROS production appeared to be independent of the Type III Secretion System. CONCLUSIONS: We provide evidence and necessary control experiments demonstrating that in planta, this ROS bioassay can be utilized to rapidly screen different Arabidopsis mutant lines and ecotypes in combination with different bacterial strains to investigate the genetic requirements of a plant host and its pathogen. For future experiments, this robust bioassay can be easily extended beyond Arabidopsis-Pto to diverse plant-pathosystems including crop species and their respective microbial pathogens

Rapid Phosphorylation of a Syntaxin during the Avr9/Cf-9-Race-Specific Signaling Pathway

The tomato (Lycopersicon esculentum) resistance (R) gene Cf-9 is required for resistance to races of the fungal pathogen Cladosporium fulvum expressing the elicitor Avr9 and also confers responsiveness to Avr9 in Cf-9-containing transgenic tobacco (Nicotiana tabacum; Cf9 tobacco). Although protein phosphorylation is required for many early Avr9/Cf-9-signaling events, so far the only phosphorylation targets known in this race-specific signaling pathway are three kinases: the two mitogen-activated protein kinases, wound-induced protein kinase and salicylic acid-induced protein kinase, and the calcium-dependent protein kinase NtCDPK2. Here, we provide evidence that a tobacco syntaxin is rapidly and transiently phosphorylated after Avr9 elicitation. The syntaxin was detected with an antibody against NtSyp121, a plasma membrane-localized syntaxin implicated in abscisic acid responses and secretion. Consistent with the gene-for-gene hypothesis, syntaxin phosphorylation required the presence of both Avr9 and Cf-9. This phosphorylation event occurred either upstream of the pathway leading to reactive oxygen species production or in a parallel pathway. Interestingly, rapid syntaxin phosphorylation was triggered by the race-specific elicitor Avr9 but not by flg22(P.aer), a general elicitor capable of inducing other defense-related signaling events in Cf9 tobacco such as reactive oxygen species production, mitogen-activated protein kinase activation, and PR5 transcript up-regulation. Furthermore, NtSyp121 transcript levels were increased at 24 h after elicitation with Avr9 but not with flg22(P.aer). Because most other previously described Avr9- and flg22(P.aer)-elicited responses are similar, syntaxin phosphorylation and NtSyp121 transcript up-regulation may serve as novel early biochemical and late molecular markers, respectively, to elucidate further differences in the signaling responses between these two elicitors

Arabidopsis clathrin adaptor EPSIN1 but not MODIFIED TRANSPORT TO THE VACOULE1 contributes to effective plant immunity against pathogenic Pseudomonas bacteria

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses

Arabidopsis Dynamin-Related Protein AtDRP2A Contributes to Late Flg22-Signaling and Effective Immunity Against Pseudomonas syringae Bacteria

In eukaryotes, dynamins and dynamin-related proteins (DRPs) are high–molecular weight GTPases responsible for mechanochemical fission of organelles or membranes. Of the six DRP subfamilies in Arabidopsis thaliana, AtDRP1 and AtDRP2 family members serve as endocytic accessory proteins in clathrin-mediated endocytosis. Most studies have focused on AtDRP1A and AtDRP2B as critical modulators of plant pattern-triggered immunity (PTI) against pathogenic, flagellated Pseudomonas syringae pv. tomato DC3000 bacteria and immune signaling in response to the bacterial flagellin peptide flg22. Much less is known about AtDRP2A, the closely related paralog of AtDRP2B. AtDRP2A and AtDRP2B are the only classical, or bona fide, dynamins in Arabidopsis, based on their evolutionary conserved domain structure with mammalian dynamins functioning in endocytosis. AtDRP2B but not AtDRP2A is required for robust ligand-induced endocytosis of the receptor kinase FLAGELLIN SENSING2 for dampening of early flg22 signaling. Here, we utilized Arabidopsis drp2a null mutants to identify AtDRP2A as a positive contributor to effective PTI against P. syringae pv. tomato DC3000 bacteria, consistent with reduced PATHOGEN RELATED1 (PR1) messenger RNA accumulation. We provide evidence that AtDRP2A is a novel modulator of late flg22 signaling, contributing positively to PR1 gene induction but negatively to polyglucan callose deposition. AtDRP2A has no apparent roles in flg22-elicited mitogen-activated protein kinase defense marker gene induction. In summary, this study adds the evolutionary conserved dynamin AtDRP2A to a small group of vesicular trafficking proteins with roles as non-canonical contributors in immune responses, likely due to modulating one or both the localization and activity of multiple different proteins with distinct contributions to immune signaling. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license