Steffen Rietz

Ethylene Signaling Regulates Accumulation of the FLS2 Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.

Balanced Nuclear and Cytoplasmic Activities of EDS1 Are Required for a Complete Plant Innate Immune Response

An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat (NB-LRR) receptors recognizing specific microbial effectors. Signaling from activated receptors of the TIR (Toll/Interleukin-1 Receptor)-NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility1). In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes. EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus. By enhancing EDS1 export from inside nuclei (through attachment of an additional nuclear export sequence (NES)) or conditionally releasing EDS1 to the nucleus (by fusion to a glucocorticoid receptor (GR)) in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming. Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei. Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites. We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack.

Different roles of Enhanced Disease Susceptibility1 (EDS1) bound to and dissociated from Phytoalexin Deficient4 (PAD4) in Arabidopsis immunity

• Enhanced Disease Susceptibility1 (EDS1) is an important regulator of plant basal and receptor-triggered immunity. Arabidopsis EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), whose combined activities are essential for defense signaling. The different sizes and intracellular distributions of EDS1-PAD4 and EDS1-SAG101 complexes in Arabidopsis leaf tissues suggest that they perform nonredundant functions. • The nature and biological relevance of EDS1 interactions with PAD4 and SAG101 were explored using yeast three-hybrid assays, in vitro analysis of recombinant proteins purified from Escherichia coli, and characterization of Arabidopsis transgenic plants expressing an eds1 mutant (eds1(L262P) ) protein which no longer binds PAD4 but retains interaction with SAG101. • EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors. Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, consistent with the EDS1 physical association stabilizing PAD4. The dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci. By contrast, an EDS1-PAD4 complex is necessary for basal resistance involving transcriptional up-regulation of PAD4 itself and mobilization of salicylic acid defenses. • Different EDS1 and PAD4 molecular configurations have distinct and separable functions in the plant innate immune response.

Molecular Identification of Cytosolic, Patatin-Related Phospholipases A from Arabidopsis with Potential Functions in Plant Signal Transduction

Rapid activation of phospholipase A (PLA) by auxin or plant-pathogen interaction suggests a function in signal transduction for this enzyme, but the molecular identification of a cytosolic PLA carrying out this function remains open. We isolated four cDNA sequences from Arabidopsis (ecotype Columbia), AtPLA I,AtPLA IIA, AtPLA IVA, andAtPLA IVC, which are members of the patatin-related PLA gene family in plants and which are homologous to the animal Ca2+-independent PLA2 gene family. Expression was measured by reverse transcriptase-polymerase chain reaction, andAtPLA I transcripts were found preferentially in shoots,AtPLA IIA and AtPLA IVA in roots, andAtPLA IVC in flowers. Transient expression of the four PLA-green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) leaves showed they were located in the cytosol and not in the vacuoles. Surprisingly,AtPLA::green fluorescent protein was also localized to chloroplasts. The enzymatic activity of the purified recombinant AtPLA IVA toward phosphatidylcholine was dependent on Ca2+, saturated at 0.5 mm, and had a pH optimum of about 7.0. It had both PLA1 and PLA2 specificity. The enzyme showed in vitro highest sensitivity toward the PLA2 inhibitors palmitoyltrifluoromethyl ketone (PACOCF3,K i approximately 30 nm), arachidonyltrifluoromethyl ketone (AACOCF3,K i approximately 25 μm), and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (K i approximately 200 nm) and was also sensitive to other previously used inhibitors 5,8,11,14-eicosatetraynoic acid (K iapproximately 3 μm) and nordihydroguajaretic acid (K i approximately 15 μm). The influence of these PLA2 inhibitors on elongation in etiolated Arabidopsis seedlings was tested, and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one and 5,8,11,14-eicosatetraynoic acid inhibited hypocotyl elongation maximally at concentrations close to theirK i in vitro.

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast-derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo-oxidative stress and display EDS1-dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1-regulated SA and ROS by examining gene expression profiles, photo-oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA-biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast-derived O(2)(*-) that lead to SA-assisted H(2)O(2) accumulation as part of a mechanism limiting cell death. A combination of EDS1-regulated SA-antagonized and SA-promoted processes is necessary for resistance to host-adapted pathogens and for a balanced response to photo-oxidative stress. In contrast to SA, the apoplastic ROS-producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo-oxidative stress. Thus, chloroplastic O(2)(*-) signals are processed by EDS1 to produce counter-balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O(2)(*-) or O(2)(*-)-generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.

Perturbation of Arabidopsis Amino Acid Metabolism Causes Incompatibility with the Adapted Biotrophic Pathogen Hyaloperonospora arabidopsidis

This work identified two different Arabidopsis mutants that have reduced susceptibility to an infectious biotrophic pathogen due to overaccumulation of the amino acid Thr. This is detrimental for the host plant and the infecting pathogen but does not affect disease caused by some other pathogen species. Therefore, the host metabolic state can influence disease in quite a specific manner. Reliance of biotrophic pathogens on living plant tissues to propagate implies strong interdependence between host metabolism and nutrient uptake by the pathogen. However, factors determining host suitability and establishment of infection are largely unknown. We describe a loss-of-inhibition allele of ASPARTATE KINASE2 and a loss-of-function allele of DIHYDRODIPICOLINATE SYNTHASE2 identified in a screen for Arabidopsis thaliana mutants with increased resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa). Through different molecular mechanisms, these mutations perturb amino acid homeostasis leading to overaccumulation of the Asp-derived amino acids Met, Thr, and Ile. Although detrimental for the plant, the mutations do not cause defense activation, and both mutants retain full susceptibility to the adapted obligate biotrophic fungus Golovinomyces orontii (Go). Chemical treatments mimicking the mutants’ metabolic state identified Thr as the amino acid suppressing Hpa but not Go colonization. We conclude that perturbations in amino acid homeostasis render the mutant plants unsuitable as an infection substrate for Hpa. This may be explained by deployment of the same amino acid biosynthetic pathways by oomycetes and plants. Our data show that the plant host metabolic state can, in specific ways, influence the ability of adapted biotrophic strains to cause disease.

The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin‐regulated genes

AUXIN-BINDING PROTEIN 1 (ABP1) is not easily accessible for molecular studies because the homozygous T-DNA insertion mutant is embryo-lethal. We found that the heterozygous abp1/ABP1 insertion mutant has defects in auxin physiology-related responses: higher root slanting angles, longer hypocotyls, agravitropic roots and hypocotyls, aphototropic hypocotyls, and decreased apical dominance. Heterozygous plants flowered earlier than wild-type plants under short-day conditions. The length of the main root, the lateral root density and the hypocotyl length were little altered in the mutant in response to auxin. Compared to wild-type plants, transcription of early auxin-regulated genes (IAA2, IAA11, IAA13, IAA14, IAA19, IAA20, SAUR9, SAUR15, SAUR23, GH3.5 and ABP1) was less strongly up-regulated in the mutant by 0.1, 1 and 10 μm IAA. Surprisingly, ABP1 was itself an early auxin-up-regulated gene. IAA uptake into the mutant seedlings during auxin treatments was indistinguishable from wild-type. Basipetal auxin transport in young roots was slower in the mutant, indicating a PIN2/EIR1 defect, while acropetal transport was indistinguishable from wild-type. In the eir1 background, three of the early auxin-regulated genes tested (IAA2, IAA13 and ABP1) were more strongly induced by 1 μm IAA in comparison to wild-type, but eight of them were less up-regulated in comparison to wild-type. Similar but not identical disturbances in regulation of early auxin-regulated genes indicate tight functional linkage of ABP1 and auxin transport regulation. We hypothesize that ABP1 is involved in the regulation of polar auxin transport, and thus affects local auxin concentration and early auxin gene regulation. In turn, ABP1 itself is under the transcriptional control of auxin.

Structural Basis for Signaling by Exclusive EDS1 Heteromeric Complexes with SAG101 or PAD4 in Plant Innate Immunity.

Biotrophic plant pathogens encounter a postinfection basal resistance layer controlled by the lipase-like protein enhanced disease susceptibility 1 (EDS1) and its sequence-related interaction partners, senescence-associated gene 101 (SAG101) and phytoalexin deficient 4 (PAD4). Maintainance of separate EDS1 family member clades through angiosperm evolution suggests distinct functional attributes. We report the Arabidopsis EDS1-SAG101 heterodimer crystal structure with juxtaposed N-terminal α/β hydrolase and C-terminal α-helical EP domains aligned via a large conserved interface. Mutational analysis of the EDS1-SAG101 heterodimer and a derived EDS1-PAD4 structural model shows that EDS1 signals within mutually exclusive heterocomplexes. Although there is evolutionary conservation of α/β hydrolase topology in all three proteins, a noncatalytic resistance mechanism is indicated. Instead, the respective N-terminal domains appear to facilitate binding of the essential EP domains to create novel interaction surfaces on the heterodimer. Transitions between distinct functional EDS1 heterodimers might explain the central importance and versatility of this regulatory node in plant immunity.

Roles of Arabidopsis Patatin-Related Phospholipases A in Root Development Are Related to Auxin Responses and Phosphate Deficiency

Phospholipase A enzymes cleave phospho- and galactolipids to generate free fatty acids and lysolipids that function in animal and plant hormone signaling. Here, we describe three Arabidopsis patatin-related phospholipase A (pPLA) genes AtPLAIVA, AtPLAIVB, and AtPLAIVC and their corresponding proteins. Loss-of-function mutants reveal roles for these pPLAs in roots during normal development and under phosphate deprivation. AtPLAIVA is expressed strongly and exclusively in roots and AtplaIVA-null mutants have reduced lateral root development, characteristic of an impaired auxin response. By contrast, AtPLAIVB is expressed weakly in roots, cotyledons, and leaves but is transcriptionally induced by auxin, although AtplaIVB mutants develop normally. AtPLAIVC is expressed in the floral gynaecium and is induced by abscisic acid (ABA) or phosphate deficiency in roots. While an AtplaIVC-1 loss-of-function mutant displays ABA responsiveness, it exhibits an impaired response to phosphate deficiency during root development. Recombinant AtPLA proteins hydrolyze preferentially galactolipids and, less efficiently, phospholipids, although these enzymes are not localized in chloroplasts. We find that AtPLAIVA and AtPLAIVB are phosphorylated by calcium-dependent protein kinases in vitro and this enhances their activities on phosphatidylcholine but not on phosphatidylglycerol. Taken together, the data reveal novel functions of pPLAs in root development with individual roles at the interface between phosphate deficiency and auxin signaling.

Mutants of phospholipase A (pPLA‐I) have a red light and auxin phenotype

pPLA-I is the evolutionarily oldest patatin-related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA-I [ppla-I-1 in Wassilewskija (Ws) and ppla-I-3 in Columbia (Col) ] revealed pPLA-I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin-induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome-modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red-enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light-related phenotypes observed in both alleles point to an involvement of pPLA-I in phytochrome signalling.