Receptor-like kinases and pathogen-associated molecular patterns perception in Arabidopsis

Comparable to the innate immune response in mammals and insects, plants possess highly specific and sensitive recognition systems for pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin. In Arabidopsis, perception of flagellin occurs via recognition of the most conserved domain in its N-terminus, represented by the peptide flg22. Perception of this elicitor-active domain depends on the LRR receptor kinase FLS2. Here, using transcriptome analysis in Arabidopsis, we first present evidences that treatment with flg22, bacteria or avirulence proteins are similar, and that virulent bacteria are likely to suppress flg22-induced responses (Chapter 1). Second, we demonstrated that flagellin perception contributes to the basal resistance against bacteria, as fls2 mutants are more susceptible to bacterial infection, proving for the first time that perception of a single PAMP is sufficient to restrict bacterial growth (Chapter 2). This study also showed that PAMPs other than flagellin trigger resistance against virulent bacteria. Wild-type and fls2 mutants both display enhanced resistance when treated with crude bacterial extracts, even devoid of elicitor-active flagellin, indicating the existence of functional perception systems for PAMPs other than flagellin. One of these novel PAMPs was just identified as the elongation factor EF-Tu, and the corresponding active peptidic epitope identified (Appendix 1). FLS2 constituted so far the only PAMP receptor identified in Arabidopsis. Using ATH1 Affymetrix arrays, we identified about 1000 genes whose expression was induced 30 minutes after flg22 treatment (Chapter 2). Strikingly, among these induced genes there were 106 RLK out of the 610 RLK genes present in Arabidopsis. We hypothesize that the perception of a single PAMP (e.g. flg22) could enhance the sensitivity of the plant to microbial stimuli sensing the presence of invading microorganisms. In particular, some of the induced RLKs might be involved in the recognition of other PAMPs. We generated a collection of mutants for the induced LRR-RLKs to test the implication of the corresponding proteins in innate immune responses (Chapter 3.1). This approach led to the discovery of the EF-Tu receptor (Chapter 3.2) and suggested a new role for the previously described BAK1 gene in flg22- and probably elf18-signaling (Chapter 3.3)

The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca(2+)) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca(2+) burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca(2+)-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca(2+) burst, supporting its role as a negative regulator of BIK1

The Arabidopsis NADPH oxidases RbohD and RbohF display differential expression patterns and contributions during plant immunity

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to β-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H2O2 accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity

Transgenic expression of Arabidopsis ELONGATION FACTOR-TU RECEPTOR (AtEFR) gene in banana enhances resistance against Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW) caused by Xanthomonas campestris pv. musacearum (Xcm) is a severe bacterial disease affecting banana production in East and Central Africa, where banana is cultivated as a staple crop. Classical breeding of banana is challenging because the crop is clonally propagated and has limited genetic diversity. Thus, genetic engineering serves as a viable alternative for banana improvement. Studies have shown that transfer of the elongation factor Tu receptor gene (AtEFR) from Arabidopsis thaliana to other plant species can enhance resistance against bacterial diseases. However, AtEFR activity in banana and its efficacy against Xcm has not been demonstrated. In this study, transgenic events of banana (Musa acuminata) cultivar dwarf Cavendish expressing the AtEFR gene were generated and evaluated for resistance against Xcm under greenhouse conditions. The transgenic banana events were responsive to the EF-Tu-derived elf18 peptide and exhibited enhanced resistance to BXW disease compared to non-transgenic control plants. This study suggests that the functionality of AtEFR is retained in banana with the potential of enhancing resistance to BXW under field conditions

Broad application of a simple and affordable protocol for isolating plant RNA

BACKGROUND: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner. FINDINGS: We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents. CONCLUSIONS: Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide

Arabidopsis translation initiation factor binding protein CBE1 negatively regulates accumulation of the NADPH oxidase respiratory burst oxidase homolog D

Cell surface pattern recognition receptors sense invading pathogens by binding microbial or endogenous elicitors to activate plant immunity. These responses are under tight control to avoid excessive or untimely activation of cellular responses, which may otherwise be detrimental to host cells. How this fine-tuning is accomplished is an area of active study. We previously described a suppressor screen that identified Arabidopsis thaliana mutants with regained immune signaling in the immunodeficient genetic background bak1-5, which we named modifier of bak1-5 (mob) mutants. Here, we report that bak1-5 mob7 mutant restores elicitor-induced signaling. Using a combination of map-based cloning and whole-genome resequencing, we identified MOB7 as CONSERVED BINDING OF EIF4E1 (CBE1), a plant-specific protein that interacts with the highly conserved eukaryotic translation initiation factor eIF4E1. Our data demonstrate that CBE1 regulates the accumulation of RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), the NADPH oxidase responsible for elicitor-induced apoplastic reactive oxygen species (ROS) production. Furthermore, several mRNA decapping and translation initiation factors co-localize with CBE1 and similarly regulate immune signaling. This study thus identifies a novel regulator of immune signaling and provides new insights into ROS regulation, potentially through translational control, during plant stress responses

An evergreen mind and a heart for the colors of fall

With the finest biochemical and molecular approaches, convincing explorative strategies, and long-term vision, Stefan Hörtensteiner succeeded in elucidating the biochemical pathway responsible for chlorophyll degradation. After having contributed to the identification of key chlorophyll degradation products in the course of the past 25 years, he gradually identified and characterized most of the crucial players in the PAO/phyllobilin degradation pathway of chlorophyll. He was one of the brightest plant biochemists of his generation, and his work opened doors to a better understanding of plant senescence, tetrapyrrole homeostasis, and their complex regulation. He sadly passed away on 5 December 2020, aged 57

Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response