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Paul Schulze-Lefert

Publications and source records attributed to Paul Schulze-Lefert.

At least 19 recordsLinked to original sources
PubMed2025

Breaking the reproducibility barrier with standardized protocols for plant-microbiome research.

Inter-laboratory replicability is crucial yet challenging in microbiome research. Leveraging microbiomes to promote soil health and plant growth requires understanding underlying molecular mechanisms using reproducible experimental systems. In a global collaborative effort involving five laboratories, we aimed to help advance reproducibility in microbiome studies by testing our ability to replicate synthetic community assembly experiments. Our study compared fabricated ecosystems constructed using two different synthetic bacterial communities, the model grass Brachypodium distachyon, and sterile EcoFAB 2.0 devices. All participating laboratories observed consistent inoculum-dependent changes in plant phenotype, root exudate composition, and final bacterial community structure, where Paraburkholderia sp. OAS925 could dramatically shift microbiome composition. Comparative genomics and exudate utilization linked the pH-dependent colonization ability of Paraburkholderia, which was further confirmed with motility assays. The study provides detailed protocols, benchmarking datasets, and best practices to help advance replicable science and inform future multi-laboratory reproducibility studies.

Plants↗
PubMed2006

Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses.

Plant immune responses are triggered by pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. We show that in barley, intracellular mildew A (MLA) R proteins function in the nucleus to confer resistance against the powdery mildew fungus. Recognition of the fungal avirulence A10 effector by MLA10 induces nuclear associations between receptor and WRKY transcription factors. The identified WRKY proteins act as repressors of PAMP-triggered basal defense. MLA appears to interfere with the WRKY repressor function, thereby de-repressing PAMP-triggered basal defense. Our findings reveal a mechanism by which these polymorphic immune receptors integrate distinct pathogen signals.

Amino Acid Sequence↗
PubMed2006

Chemical interference of pathogen-associated molecular pattern-triggered immune responses in Arabidopsis reveals a potential role for fatty-acid synthase type II complex-derived lipid signals.

We describe an experimental setup using submerged cultures of Arabidopsis seedlings in 96-well microtiter plates that permits chemical intervention of rapid elicitor-mediated immune responses. Screening of a chemical library comprising 120 small molecules with known biological activities revealed four compounds reducing cellulysin- or flg22-activated gene expression of the early pathogen-associated molecular patterns (PAMP)-responsive ATL2 gene. One chemical, oxytriazine, was found to induce ATL2 gene expression in the absence of PAMP. By monitoring additional flg22-triggered immediate early plant responses, we present evidence that two compounds, triclosan and fluazinam, interfere with the accumulation of reactive oxygen species and internalization of the activated plasma membrane resident FLS2 immune receptor. Using triclosan structure types and enzyme activity inhibition assays, Arabidopsis MOD1 enoyl-acyl carrier protein reductase, a subunit of the fatty-acid synthase type II (FAS II) complex, was identified as a likely cellular target of triclosan. Inhibition of all tested elicitor-triggered early immune responses by triclosan indicates a potential role for signaling lipids in flg22-triggered immunity. Chemical profiling of eca mutants, each showing deregulated ATL2 gene expression, with the identified compounds revealed mutantspecific response patterns and allowed us to deduce tentative action sites of ECA genes relative to the compound targets.

Acetyltransferases↗
PubMed2006

Plant pathogens trick guard cells into opening the gates.

The stomata of plants regulate gas exchange and water transpiration in response to changing environmental conditions. New work reveals that stomata also have an important role in host defense. In this issue of Cell, Melotto et al. (2006) show that stomata close upon detection of potential microbial pathogens to prevent the infection of the leaf interior. Moreover, pathogenic bacteria have evolved strategies to suppress the closure of stomata.

Amino Acids↗
PubMed2006

Mapped Ds/T-DNA launch pads for functional genomics in barley.

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.

Base Composition↗
PubMed2006

Conserved requirement for a plant host cell protein in powdery mildew pathogenesis.

In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.

Arabidopsis↗
PubMed2006

Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration.

Arabidopsis thaliana is a host to the powdery mildew Erysiphe cichoracearum and nonhost to Blumeria graminis f. sp hordei, the powdery mildew pathogenic on barley (Hordeum vulgare). Screening for Arabidopsis mutants deficient in resistance to barley powdery mildew identified PENETRATION3 (PEN3). pen3 plants permitted both increased invasion into epidermal cells and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall and intracellularly. pen3 mutants were compromised in resistance to the necrotroph Plectosphaerella cucumerina and to two additional inappropriate biotrophs, pea powdery mildew (Erysiphe pisi) and potato late blight (Phytophthora infestans). Unexpectedly, pen3 mutants were resistant to E. cichoracearum. This resistance was salicylic acid-dependent and correlated with chlorotic patches. Consistent with this observation, salicylic acid pathway genes were hyperinduced in pen3 relative to the wild type. The phenotypes conferred by pen3 result from the loss of function of PLEIOTROPIC DRUG RESISTANCE8 (PDR8), a highly expressed putative ATP binding cassette transporter. PEN3/PDR8 tagged with green fluorescent protein localized to the plasma membrane in uninfected cells. In infected leaves, the protein concentrated at infection sites. PEN3/PDR8 may be involved in exporting toxic materials to attempted invasion sites, and intracellular accumulation of these toxins in pen3 may secondarily activate the salicylic acid pathway.

ATP-Binding Cassette Transporters↗
PubMed2006

Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins.

The Arabidopsis (Arabidopsis thaliana) genome contains 15 genes encoding protein homologs of the barley mildew resistance locus o (MLO) protein biochemically shown to have a seven-transmembrane domain topology and localize to the plasma membrane. Towards elucidating the functions of MLOs, the largest family of seven-transmembrane domain proteins specific to plants, we comprehensively determined AtMLO gene expression patterns by a combination of experimental and in silico studies. Experimentation comprised analyses of transgenic Arabidopsis lines bearing promoter::Beta-glucuronidase (GUS) transcriptional fusions as well as semi-quantitative determination of transcripts by reverse transcription coupled to polymerase chain reaction (RT-PCR). These results were combined with information extracted from public gene profiling databases, and compared to the expression patterns of genes encoding the heterotrimeric G-protein subunits. We found that each AtMLO gene has a unique expression pattern and is regulated differently by a variety of biotic and/or abiotic stimuli, suggesting that AtMLO proteins function in diverse developmental and response processes. The expression of several phylogenetically closely-related AtMLO genes showed similar or overlapping tissue specificity and analogous responsiveness to external stimuli, suggesting functional redundancy, co-function, or antagonistic function(s).

Arabidopsis Proteins↗
PubMed2006

Nonhost resistance in Arabidopsis-Colletotrichum interactions acts at the cell periphery and requires actin filament function.

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.

Actin Cytoskeleton↗
PubMed2006

Metabolic consequences of susceptibility and resistance (race-specific and broad-spectrum) in barley leaves challenged with powdery mildew.

In a compatible interaction biotrophic fungi often lower the yield of their hosts by reducing photosynthesis and altering the fluxes of carbon within the infected leaf. In contrast, comparatively little is known about the metabolic consequences of activating resistance responses. In this study we investigated the hypothesis that the activation of both race-specific (Mla12) and broad-spectrum (mlo) resistance pathways in barley leaves infected with Blumeria graminis represents a cost to the plant in terms of carbon production and utilization. We have shown, using quantitative imaging of chlorophyll fluorescence, that during a susceptible interaction, photosynthesis was progressively reduced both in cells directly below fungal colonies and in adjacent cells when compared with uninoculated leaves. The lower rate of photosynthesis was associated with an increase in invertase activity, an accumulation of hexoses and a down-regulation of photosynthetic gene expression. During both Mla12- and mlo-mediated resistance, photosynthesis was also reduced, most severely inhibited in cells directly associated with attempted penetration of the fungus but also in surrounding cells. These cells displayed intense autofluorescence under ultraviolet illumination indicative of the accumulation of phenolic compounds and/or callose deposition. The depression in photosynthesis was not due only to cell death but also to an alteration in source-sink relations and carbon utilization. Apoplastic (cell wall-bound) invertase activity increased more rapidly and to a much greater extent than in infected susceptible leaves and was accompanied by an accumulation of hexoses that was localized to areas of the leaf actively exhibiting resistance responses. The accumulation of hexoses was accompanied by a down-regulation in the expression of Rubisco (rbcS) and chlorophyll a/b binding protein (cab) genes (although to a lesser extent than in a compatible interaction) and with an up-regulation in the expression of the pathogenesis-related protein 1 (PR-1). These results are consistent with a role for invertase in the generation of hexoses, which may supply energy for defence reactions and/or act as signals inducing defence gene expression.

Ascomycota↗
PubMed2005

Pre- and postinvasion defenses both contribute to nonhost resistance in Arabidopsis.

Nonhost resistance describes the immunity of an entire plant species against nonadapted pathogen species. We report that Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi that in nature colonize grass and pea species. The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism. Postinvasion fungal growth is blocked by a separate resistance layer requiring the EDS1-PAD4-SAG101 signaling complex, which is known to function in basal and resistance (R) gene-triggered immunity. Concurrent impairment of pre- and postinvasion resistance renders Arabidopsis a host for both nonadapted fungi.

Arabidopsis↗
PubMed2005

Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain.

Many fungal pathogens must enter plant cells for successful colonization. Barley mildew resistance locus o (Mlo) is required for host cell invasion upon attack by the ascomycete powdery mildew fungus, Blumeria graminis f.sp. hordei, and encodes the founder of a family of heptahelical integral membrane proteins unique to plants. Recessively inherited loss-of-function mutant alleles (mlo) result in effective penetration resistance to all isolates of the biotrophic parasite. We used noninvasive fluorescence-based imaging to show that fluorescently tagged MLO protein becomes redistributed in the plasma membrane (PM) and accumulates beneath fungal appressoria coincident with the initiation of pathogen entry into host cells. Polarized MLO accumulation occurs once upon attack and appears to be independent of actin cytoskeleton function. Likewise, barley ROR2 syntaxin, a genetically defined component of penetration resistance to B. graminis f.sp. hordei, and a subset of predicted PM-resident proteins become redistributed to fungal entry sites. We previously identified calmodulin, a cytoplasmic calcium sensor, as an interactor and positive regulator of MLO activity and demonstrate here by FRET microscopy an increase in MLO/calmodulin FRET around penetration sites coincident with successful host cell entry. Our data provide evidence for the formation of a pathogen-triggered PM microdomain that is reminiscent of membrane microdomains (lipid rafts) induced upon attempted entry of pathogenic bacteria in animal cells.

Calmodulin↗
PubMed2005

Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.

We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Amino Acid Sequence↗
PubMed2005

Barley Rom1 reveals a potential link between race-specific and nonhost resistance responses to powdery mildew fungi.

The Rar1 gene, identified in the context of race-specific powdery mildew resistance mediated by the Hordeum vulgare (barley) resistance (R) gene Mla12, is required for the function of many R-mediated defense responses in mono- and dicotyledonous plant species. Mla resistance is associated with an oxidative burst and a subsequent cell death reaction of attacked cells. Rar1 mutants are impaired in these responses and, to identify genetic elements which negatively regulate the Mla12-triggered response, we have screened mutagenized Mla12 rar1 mutant populations for restoration of the resistance response. Here we describe the restoration of Mla12-specified resistance (rom1) mutant that restores features of disease resistance to a Blumeria graminis f. sp. hordei isolate expressing the avirulence gene AvrMla12 and retains susceptibility to an isolate lacking AvrMla12. Histochemical analyses show that, in rom1 mutant plants, a whole-cell oxidative burst and cell death response in attacked epidermal cells is restored in the incompatible interaction. Defense responses against tested inappropriate powdery mildews, B. graminis f. sp. tritici and Golovinomyces orontii, were diminished in rar1 mutant plants and enhanced in rom1 mutant plants relative to the wild type. These findings indicate antagonistic activities of Rar1 and Rom1 and reveal their contribution to nonhost and race-specific resistance responses.

Carrier Proteins↗
PubMed2004

Conserved ERAD-like quality control of a plant polytopic membrane protein.

The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism.

Amino Acid Substitution↗
PubMed2004

RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance.

The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.

Arabidopsis Proteins↗
PubMed2004

A barley cultivation-associated polymorphism conveys resistance to powdery mildew.

Barley (Hordeum vulgare) has played a pivotal role in Old World agriculture since its domestication about 10,000 yr ago. Barley plants carrying loss-of-function alleles (mlo) of the Mlo locus are resistant against all known isolates of the widespread powdery mildew fungus. The sole mlo resistance allele recovered so far from a natural habitat, mlo-11, was originally retrieved from Ethiopian landraces and nowadays controls mildew resistance in the majority of cultivated European spring barley elite varieties. Here we use haplotype analysis to show that the mlo-11 allele probably arose once after barley domestication. Resistance in mlo-11 plants is linked to a complex tandem repeat array inserted upstream of the wild-type gene. The repeat units consist of a truncated Mlo gene comprising 3.5 kilobases (kb) of 5'-regulatory sequence plus 1.1 kb of coding sequence. These generate aberrant transcripts that impair the accumulation of both Mlo wild-type transcript and protein. We exploited the meiotic instability of mlo-11 resistance and recovered susceptible revertants in which restoration of Mlo function was accompanied by excision of the repeat array. We infer cis-dependent perturbation of transcription machinery assembly by transcriptional interference in mlo-11 plants as a likely mechanism leading to disease resistance.

Alleles↗