Publications - Stress and Develop Biology

BackgroundCalcium, as a second messenger, transduces extracellular signals into cellular reactions. A rise in cytosolic calcium concentration is one of the first plant responses after exposure to microbe-associated molecular patterns (MAMPs). We reported previously the isolation of Arabidopsis thaliana mutants with a “changed calcium elevation” (cce) response to flg22, a 22-amino-acid MAMP derived from bacterial flagellin.ResultsHere, we characterized the cce2 mutant and its weaker allelic mutant, cce3. Besides flg22, the mutants respond with a reduced calcium elevation to several other MAMPs and a plant endogenous peptide that is proteolytically processed from pre-pro-proteins during wounding. Downstream defense-related events such flg22-induced mitogen-activated protein kinase activation, accumulation of reactive oxygen species and growth arrest are also attenuated in cce2/cce3. By genetic mapping, next-generation sequencing and allelism assay, CCE2/CCE3 was identified to be ALG3 (Asparagine-linked glycosylation 3). This encodes the α-1,3-mannosyltransferase responsible for the first step of core oligosaccharide Glc3Man9GlcNAc2 glycan assembly on the endoplasmic reticulum (ER) luminal side. Complementation assays and glycan analysis in yeast alg3 mutant confirmed the reduced enzymatic function of the proteins encoded by the cce2/cce3 alleles – leading to accumulation of M5ER, the immature five mannose-containing oligosaccharide structure found in the ER. Proper protein glycosylation is required for ER/Golgi processing and trafficking of membrane proteins to the plasma membrane. Endoglycosidase H-insensitivity of flg22 receptor, FLS2, in the cce2/cce3 mutants suggests altered glycan structures in the receptor.ConclusionProper glycosylation of MAMP receptors (or other exported proteins) is required for optimal responses to MAMPs and is important for immune signaling of host plants.

Immunity against pathogen infection depends on a host's ability to sense invading pathogens and to rapidly trigger defence reactions that block pathogen proliferation. Both plants and animals detect conserved structural motifs of microbe‐specific compounds, so‐called microbe‐associated molecular patterns (MAMPs), through germline‐encoded immune sensors, which are accordingly termed pattern recognition receptors (PRRs) (Akira et al., 2006; Boller and Felix, 2009). Activated PRRs initiate signal transduction and trigger innate immune responses. MAMPs are generally derived from elements essential for microbial fitness and are conserved across species, thus enabling the host to detect a range of potential pathogens. In mammals, innate immune sensing of MAMPs is not only crucial for basal immune responses but is also tightly connected with and required for a subsequent adaptive, antibody‐mediated immunity (Akira et al., 2006; Janeway and Medzhitov, 2002). Plants, lacking an adaptive immune system, have apparently evolved a greater capacity to detect a broader repertoire of MAMPs. Different plant species possess distinct sets of highly specific PRRs, but the downstream signalling pathways are rather conserved and converge on common signalling steps. This allows the transfer of PRRs, even to different plant families, whilst maintaining their functionality and specificity (Zipfel, 2014). This also enables researchers to use well‐studied, genetically amenable model systems for the identification of MAMPs and their respective PRRs. Several examples of interfamily PRR transfer have demonstrated that the introduction of novel PRRs into plant species can confer relevant levels of resistance to otherwise susceptible plants (e.g. Afroz et al., 2011; Hao et al., 2015; Lacombe et al., 2010; Mendes et al., 2010; Schoonbeek et al., 2015; Tripathi et al., 2014). Hence, MAMP sensing by PRRs has great potential for the engineering of disease resistance in crop plants. In recent years, it has therefore become a major task to identify and isolate MAMPs from a range of microorganisms, and their respective PRRs, to study their role in innate immunity and their application potential.

Ca2+ is a secondary messenger involved in early signaling events triggered in response to a plethora of biotic and abiotic stimuli. In plants, environmental cues that induce cytosolic Ca2+ elevation include touch, reactive oxygen species, cold shock, and salt or osmotic stress. Furthermore, Ca2+ signaling has been implicated in early stages of plant–microbe interactions of both symbiotic and antagonistic nature. A long-standing hypothesis is that there is information encoded in the Ca2+ signals (so-called Ca2+ signatures) to enable plants to differentiate between these stimuli and to trigger the appropriate cellular response. Qualitative and quantitative measurements of Ca2+ signals are therefore needed to dissect the responses of plants to their environment. Luminescence produced by the Ca2+ probe aequorin upon Ca2+ binding is a widely used method for the detection of Ca2+ transients and other changes in Ca2+ concentrations in cells or organelles of plant cells. In this chapter, using microbe-associated molecular patterns (MAMPs), such as the bacterial-derived flg22 or elf18 peptides as stimuli, a protocol for the quantitative measurements of Ca2+ fluxes in apoaequorin-expressing seedlings of Arabidopsis thaliana in 96-well format is described.