Unlocking Precision in Callose Staining: Unveiling the Role of Sirofluor.

Callose is a vital component in plant biology, contributing to essential processes like pollen maturation and defense against pathogens. However, misconceptions surrounding callose staining persist, particularly regarding the role of aniline blue. It is now known that commercial aniline blue contains sirofluor, and it is this fluorophore, rather than aniline blue itself, that is responsible for the observed fluorescence during callose detection.

Marker and readout genes for defense priming in Pseudomonas cannabina pv. alisalensis interaction aid understanding systemic immunity in Arabidopsis.

Following localized infection, the entire plant foliage becomes primed for enhanced defense. However, specific genes induced during defense priming (priming-marker genes) and those showing increased expression in defense-primed plants upon rechallenge (priming-readout genes) remain largely unknown. In our Arabidopsis thaliana study, genes AT1G76960 (function unknown), CAX3 (encoding a vacuolar Ca2+/H+ antiporter), and CRK4 (encoding a cysteine-rich receptor-like protein kinase) were strongly expressed during Pseudomonas cannabina pv. alisalensis-induced defense priming, uniquely marking the primed state for enhanced defense. Conversely, PR1 (encoding a pathogenesis-related protein), RLP23 and RLP41 (both encoding receptor-like proteins) were similarly activated in defense-primed plants before and after rechallenge, suggesting they are additional marker genes for defense priming. In contrast, CASPL4D1 (encoding Casparian strip domain-like protein 4D1), FRK1 (encoding flg22-induced receptor-like kinase), and AT3G28510 (encoding a P loop-containing nucleoside triphosphate hydrolases superfamily protein) showed minimal activation in uninfected, defense-primed, or rechallenged plants, but intensified in defense-primed plants after rechallenge. Notably, mutation in only priming-readout gene NHL25 (encoding NDR1/HIN1-like protein 25) impaired both defense priming and systemic acquired resistance, highlighting its previously undiscovered pivotal role in systemic plant immunity.

Engineered coumarin accumulation reduces mycotoxin-induced oxidative stress and disease susceptibility.

Coumarins can fight pathogens and are thus promising for crop protection. Their biosynthesis, however, has not yet been engineered in crops. We tailored the constitutive accumulation of coumarins in transgenic Nicotiana benthamiana, Glycine max and Arabidopsis thaliana plants, as well as in Nicotiana tabacum BY-2 suspension cells. We did so by overexpressing A. thaliana feruloyl-CoA 6-hydroxylase 1 (AtF6'H1), encoding the key enzyme of scopoletin biosynthesis. Besides scopoletin and its glucoside scopolin, esculin at low level was the only other coumarin detected in transgenic cells. Mechanical damage of scopolin-accumulating tissue led to a swift release of scopoletin, presumably from the scopolin pool. High scopolin levels in A. thaliana roots coincided with reduced susceptibility to the root-parasitic nematode Heterodera schachtii. In addition, transgenic soybean plants were more tolerant to the soil-borne pathogenic fungus Fusarium virguliforme. Because mycotoxin-induced accumulation of reactive oxygen species and cell death were reduced in the AtF6'H1-overexpressors, the weaker sensitivity to F. virguliforme may be caused by attenuated oxidative damage of coumarin-hyperaccumulating cells. Together, engineered coumarin accumulation is promising for enhanced disease resilience of crops.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

Spotting priming-active compounds using parsley cell cultures in microtiter plates.

BACKGROUND: Conventional crop protection has major drawbacks, such as developing pest and pathogen insensitivity to pesticides and low environmental compatibility. Therefore, alternative crop protection strategies are needed. One promising approach treats crops with chemical compounds that induce the primed state of enhanced defense. However, identifying priming compounds is often tedious as it requires offline sampling and analysis. High throughput screening methods for the analysis of priming-active compounds have great potential to simplify the search for such compounds. One established method to identify priming makes use of parsley cell cultures. This method relies on measurement of fluorescence of furanocoumarins in the final sample. This study demonstrates for the first time the online measurement of furanocoumarins in microtiter plates. As not all plants produce fluorescence molecules as immune response, a signal, which is not restricted to a specific plant is required, to extend online screening methods to other plant cell cultures. It was shown that the breathing activity of primed parsley cell cultures increases, compared to unprimed parsley cell cultures. The breathing activity can by monitored online. Therefore, online identification of priming-inducing compounds by recording breathing activity represents a promising, straight-forward and highly informative approach. However, so far breathing has been recorded in shake flasks which suffer from low throughput. For industrial application we here report a high-throughput, online identification method for identifying priming-inducing chemistry. RESULTS: This study describes the development of a high-throughput screening system that enables identifying and analyzing the impact of defense priming-inducing compounds in microtiter plates. This screening system relies on the breathing activity of parsley cell cultures. The validity of measuring the breathing activity in microtiter plates to drawing conclusions regarding priming-inducing activity was demonstrated. Furthermore, for the first time, the fluorescence of the priming-active reference compound salicylic acid and of furanocoumarins were simultaneously monitored online. Dose and time studies with salicylic acid-treated parsley cell suspensions revealed a wide range of possible addition times and concentrations that cause priming. The online fluorescence measuring method was further confirmed with three additional compounds with known priming-causing activity. CONCLUSIONS: Determining the OTR, fluorescence of the priming-active chemical compound SA and of furanocoumarins in parsley suspension cultures in MTPs by online measurement is a powerful and high-throughput tool to study possible priming compounds. It allows an in-depth screening for priming compounds and a better understanding of the priming process induced by a given substance. Evaluation of priming phenomena via OTR should also be applicable to cell suspensions of other plant species and varieties and allow screening for priming-inducing chemical compounds in intact plants. These online fluorescence methods to measure the breathing activity, furanocoumarin and SA have the potential to accelerate the search for new priming compounds and promote priming as a promising, eco-friendly crop protection strategy.

High-throughput Screening for Defense Priming-inducing Compounds in Parsley Cell Cultures.

Defense priming describes the enhanced potency of cells to activate defense responses. Priming accompanies local and systemic immune responses and can be triggered by microbial infection or upon treatment with certain chemicals. Thus, chemically activating defense priming is promising for biomedicine and agriculture. However, test systems for spotting priming-inducing chemicals are rare. Here, we describe a high-throughput screen for compounds that prime microbial pattern-spurred secretion of antimicrobial furanocoumarins in parsley culture cells. For the best possible throughput, we perform the assay with 1-ml aliquots of cell culture in 24-well microtiter plates. The advantages of the non-invasive test over competitive assays are its simplicity, remarkable reliability, and high sensitivity, which is based on furanocoumarin fluorescence in UV light.

Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

Disclosure of salicylic acid and jasmonic acid-responsive genes provides a molecular tool for deciphering stress responses in soybean.

Hormones orchestrate the physiology of organisms. Measuring the activity of defense hormone-responsive genes can help understanding immune signaling and facilitate breeding for plant health. However, different from model species like Arabidopsis, genes that respond to defense hormones salicylic acid (SA) and jasmonic acid (JA) have not been disclosed in the soybean crop. We performed global transcriptome analyses to fill this knowledge gap. Upon exogenous application, endogenous levels of SA and JA increased in leaves. SA predominantly activated genes linked to systemic acquired resistance and defense signaling whereas JA mainly activated wound response-associated genes. In general, SA-responsive genes were activated earlier than those responding to JA. Consistent with the paradigm of biotrophic pathogens predominantly activating SA responses, free SA and here identified most robust SA marker genes GmNIMIN1, GmNIMIN1.2 and GmWRK40 were induced upon inoculation with Phakopsora pachyrhizi, whereas JA marker genes did not respond to infection with the biotrophic fungus. Spodoptera exigua larvae caused a strong accumulation of JA-Ile and JA-specific mRNA transcripts of GmBPI1, GmKTI1 and GmAAT whereas neither free SA nor SA-marker gene transcripts accumulated upon insect feeding. Our study provides molecular tools for monitoring the dynamic accumulation of SA and JA, e.g. in a given stress condition.

An illuminated respiratory activity monitoring system identifies priming-active compounds in plant seedlings.

BACKGROUND: Growing large crop monocultures and heavily using pesticides enhances the evolution of pesticide-insensitive pests and pathogens. To reduce pesticide use in crop cultivation, the application of priming-active compounds (PrimACs) is a welcome alternative. PrimACs strengthen the plant immune system and could thus help to protect plants with lower amounts of pesticides. PrimACs can be identified, for example, by their capacity to enhance the respiratory activity of parsley cells in culture as determined by the oxygen transfer rate (OTR) using the respiration activity monitoring system (RAMOS) or its miniaturized version, µRAMOS. The latter was designed for with suspensions of bacteria and yeast cells in microtiter plates (MTPs). So far, RAMOS or µRAMOS have not been applied to adult plants or seedlings, which would overcome the limitation of (µ)RAMOS to plant suspension cell cultures. RESULTS: In this work, we introduce a modified µRAMOS for analysis of plant seedlings. The novel device allows illuminating the seedlings and records the respiratory activity in each well of a 48-well MTP. To validate the suitability of the setup for identifying novel PrimAC in Arabidopsis thaliana, seedlings were grown in MTP for seven days and treated with the known PrimAC salicylic acid (SA; positive control) and the PrimAC candidate methyl 1-(3,4-dihydroxyphenyl)-2-oxocyclopentane-1-carboxylate (Tyr020). Twenty-eight h after treatment, the seedlings were elicited with flg22, a 22-amino acid peptide of bacterial flagellin. Upon elicitation, the respiratory activity was monitored. The evaluation of the OTR course reveals Tyr020 as a likely PrimAC. The priming-inducing activity of Tyr020 was confirmed using molecular biological analyses in A. thaliana seedlings. CONCLUSION: We disclose the suitability of µRAMOS for identifying PrimACs in plant seedlings. The difference in OTR during a night period between primed and unprimed plants was distinguishable after elicitation with flg22. Thus, it has been shown that the µRAMOS device can be used for a reliable screening for PrimACs in plant seedlings.

The Induced Resistance Lexicon: Do's and Don'ts.

To be protected from biological threats, plants have evolved an immune system comprising constitutive and inducible defenses. For example, upon perception of certain stimuli, plants can develop a conditioned state of enhanced defensive capacity against upcoming pathogens and pests, resulting in a phenotype called 'induced resistance' (IR). To tackle the confusing lexicon currently used in the IR field, we propose a widely applicable code of practice concerning the terminology and description of IR phenotypes using two main phenotypical aspects: local versus systemic resistance, and direct versus primed defense responses. Our general framework aims to improve uniformity and consistency in future scientific communication, which should help to avoid further misinterpretations and facilitate the accessibility and impact of this research field.

Priming for enhanced ARGONAUTE2 activation accompanies induced resistance to cucumber mosaic virus in Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a broad-spectrum disease resistance response that can be induced upon infection from pathogens or by chemical treatment, such as with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). SAR involves priming for more robust activation of defence genes upon pathogen attack. Whether priming for SAR would involve components of RNA silencing remained unknown. Here, we show that upon leaf infiltration of water, BTH-primed Arabidopsis thaliana plants accumulate higher amounts of mRNA of ARGONAUTE (AGO)2 and AGO3, key components of RNA silencing. The enhanced AGO2 expression is associated with prior-to-activation trimethylation of lysine 4 in histone H3 and acetylation of histone H3 in the AGO2 promoter and with induced resistance to the yellow strain of cucumber mosaic virus (CMV[Y]). The results suggest that priming A. thaliana for enhanced defence involves modification of histones in the AGO2 promoter that condition AGO2 for enhanced activation, associated with resistance to CMV(Y). Consistently, the fold-reduction in CMV(Y) coat protein accumulation by BTH pretreatment was lower in ago2 than in wild type, pointing to reduced capacity of ago2 to activate BTH-induced CMV(Y) resistance. A role of AGO2 in pathogen-induced SAR is suggested by the enhanced activation of AGO2 after infiltrating systemic leaves of plants expressing a localized hypersensitive response upon CMV(Y) infection. In addition, local inoculation of SAR-inducing Pseudomonas syringae pv. maculicola causes systemic priming for enhanced AGO2 expression. Together our results indicate that defence priming targets the AGO2 component of RNA silencing whose enhanced expression is likely to contribute to SAR.

Formaldehyde-assisted isolation of regulatory DNA elements from Arabidopsis leaves.

Eukaryotic gene transcription is associated with the eviction of nucleosomes and the formation of open chromatin, which enables the recruitment of transcriptional coactivators and other regulatory factors. Open chromatin is thus a hallmark of functional regulatory DNA elements in genomes. In recent years, formaldehyde-assisted isolation of regulatory elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes, particularly yeast, human, and mouse. However, it has proven challenging to adapt the FAIRE protocol for use on plant material, and the few available protocols all have their drawbacks (e.g., applicability only to specific developmental stages). In this Protocol Extension, we describe a reliable FAIRE protocol for mature Arabidopsis (Arabidopsis thaliana) leaves that adapts the original protocol for use on plants. The main differences between this protocol extension and the earlier FAIRE protocol are an increased formaldehyde concentration in the chromatin crosslinking buffer, application of a repeated vacuum to increase crosslinking efficiency, and altered composition of the DNA extraction buffer. The protocol is applicable to leaf chromatin of unstressed and stressed plants and can be completed within 1 week. Here, we also describe downstream analysis using qPCR and next-generation sequencing. However, this Protocol Extension should also be compatible with downstream hybridization to a DNA microarray. In addition, it is likely that only minor adaptations will be necessary to apply this protocol to other Arabidopsis organs or plant species.

Isolation of Open Chromatin Identifies Regulators of Systemic Acquired Resistance.

Upon local infection, plants activate a systemic immune response called systemic acquired resistance (SAR). During SAR, systemic leaves become primed for the superinduction of defense genes upon reinfection. We used formaldehyde-assisted isolation of regulatory DNA elements coupled to next-generation sequencing to identify SAR regulators. Our bioinformatic analysis produced 10,129 priming-associated open chromatin sites in the 5' region of 3,025 genes in the systemic leaves of Arabidopsis (Arabidopsis thaliana) plants locally infected with Pseudomonas syringae pv. maculicola Whole transcriptome shotgun sequencing analysis of the systemic leaves after challenge enabled the identification of genes with priming-linked open chromatin before (contained in the formaldehyde-assisted isolation of regulatory DNA elements sequencing dataset) and enhanced expression after (included in the whole transcriptome shotgun sequencing dataset) the systemic challenge. Among them, Arabidopsis MILDEW RESISTANCE LOCUS O3 (MLO3) was identified as a previously unidentified positive regulator of SAR. Further in silico analysis disclosed two yet unknown cis-regulatory DNA elements in the 5' region of genes. The P-box was mainly associated with priming-responsive genes, whereas the C-box was mostly linked to challenge. We found that the P- or W-box, the latter recruiting WRKY transcription factors, or combinations of these boxes, characterize the 5' region of most primed genes. Therefore, this study provides a genome-wide record of genes with open and accessible chromatin during SAR and identifies MLO3 and two previously unidentified DNA boxes as likely regulators of this immune response.

The Arabidopsis non-host defence-associated coumarin scopoletin protects soybean from Asian soybean rust.

The fungus Phakopsora pachyrhizi (Pp) causes Asian soybean rust (SBR) disease which provokes tremendous losses in global soybean production. Pp is mainly controlled with synthetic fungicides to which the fungus swiftly develops fungicide resistance. To substitute or complement synthetic fungicides in Asian soybean rust control, we aimed to identify antifungal metabolites in Arabidopsis which is not a host for Pp. Comparative transcriptional and metabolic profiling of the Pp-inoculated Arabidopsis non-host and the soybean host revealed induction of phenylpropanoid metabolism-associated genes in both species but activation of scopoletin biosynthesis only in the resistant non-host. Scopoletin is a coumarin and an antioxidant. In vitro experiments disclosed fungistatic activity of scopoletin against Pp, associated with reduced accumulation of reactive oxygen species (ROS) in fungal pre-infection structures. Non-antioxidant and antioxidant molecules including coumarins with a similar structure to scopoletin were inactive or much less effective at inhibiting fungal accumulation of ROS and germination of Pp spores. When sprayed onto Arabidopsis leaves, scopoletin also suppressed the formation of Pp pre-infection structures and penetration of the plant. However, scopoletin neither directly activated defence nor did it prime Arabidopsis for enhanced defence, therefore emphasizing fungistatic activity as the exclusive mode of action of scopoletin against Pp. Because scopletin also protected soybean from Pp infection, the coumarin may serve as a natural fungicide or as a lead for the development of near-to-nature fungicides against Asian soybean rust.

Parallel online determination of ethylene release rate by Shaken Parsley cell cultures using a modified RAMOS device.

BACKGROUND: Ethylene is an important plant hormone that controls many physiological processes in plants. Conventional methods for detecting ethylene include gas chromatographs or optical mid-infrared sensors, which are expensive and, in the case of gas chromatographs, are hardly suitable for automated parallelized online measurement. Electrochemical ethylene sensors are cheap but often suffer from poor resolution, baseline drifting, and target gas oxidation. Thus, measuring ethylene at extremely low levels is challenging. RESULTS: This report demonstrates the integration of electrochemical ethylene sensors into a respiration activity monitoring system (RAMOS) that measures, in addition to the oxygen transfer rate, the ethylene transfer rate in eight parallel shake flasks. A calibration method is presented that is not prone to baseline drifting and considers target gas oxidation at the sensor. In this way, changes in ethylene transfer rate as low as 4 nmol/L/h can be resolved. In confirmatory experiments, the overall accuracy of the method was similar to that of gas chromatography-mass spectrometry (GC/MS) measurements. The RAMOS-based ethylene determination method was exemplified with parsley suspension-cultured cells that were primed for enhanced defense by pretreatment with salicylic acid, methyl jasmonate or 4-chlorosalicylic acid and challenged with the microbial pattern Pep13. Ethylene release into the headspace of the shake flask was observed upon treatment with salicylic acid and methyl jasmonate was further enhanced, in case of salicylic acid and 4-chlorosalicylic acid, upon Pep13 challenge. CONCLUSION: A conventional RAMOS device was modified for simultaneous measurement of the ethylene transfer rate in eight parallel shake flasks at nmol/L/h resolution. For the first time electrochemical sensors are used to provide a medium-throughput method for monitoring ethylene release by plants. Currently, this can only be achieved by costly laser-based detection systems and automated gas chromatographs. The new method is particularly suitable for plant cell suspension cultures. However, the method may also be applicable to intact plants, detached leaves or other plant tissues. In addition, the general principle of the technology is likely extendable to other volatiles or gases as well, such as nitric oxide or hydrogen peroxide.

Innate immune memory: An evolutionary perspective.

Over the last decades, there was increasing evidence for the presence of innate immune memory in living organisms. In this review, we compare the innate immune memory of various organisms with a focus on phylogenetics. We discuss the acquisition and molecular basis of immune memory and we describe the innate immune memory paradigm and its role in host defense during evolution. The molecular characterization of innate immunological memory in diverse organisms and host-parasite systems reconciles mechanisms with phenomena and paves the way to molecular comprehension of innate immune memory. We also revise the traditional classification of innate and adaptive immunity in jawed vertebrates. We emphasize that innate immune responses have the capacity to be "primed" or "trained", thereby exerting a yet unknown type of immunological memory upon re-infection.

Sulforaphane Modifies Histone H3, Unpacks Chromatin, and Primes Defense.

Modern crop production calls for agrochemicals that prime plants for enhanced defense. Reliable test systems for spotting priming-inducing chemistry, however, are rare. We developed an assay for the high-throughput search for compounds that prime microbial pattern-induced secretion of antimicrobial furanocoumarins (phytoalexins) in cultured parsley cells. The screen produced 1-isothiocyanato-4-methylsulfinylbutane (sulforaphane; SFN), a secondary metabolite in many crucifers, as a novel defense priming compound. While elucidating SFN's mode of action in defense priming, we found that in Arabidopsis (Arabidopsisthaliana) the isothiocyanate provokes covalent modification (K4me3, K9ac) of histone H3 in the promoter and promoter-proximal region of defense genes WRKY6 and PDF12, but not PR1 SFN-triggered H3K4me3 and H3K9ac coincide with chromatin unpacking in the WRKY6 and PDF12 regulatory regions, primed WRKY6 expression, unprimed PDF12 activation, and reduced susceptibility to downy mildew disease (Hyaloperonospora arabidopsidis). Because SFN also directly inhibits Harabidopsidis and other plant pathogens, the isothiocyanate is promising for the development of a plant protectant with a dual mode of action.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis.

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis mlo2 mlo6 mlo12 Triple Mutant.

Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O (MLO) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.

Respiration activity monitoring system for any individual well of a 48-well microtiter plate.

BACKGROUND: Small-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) µRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced µRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures. RESULTS: A technical solution for the required miniaturized valve and sensor implementation for an MTP-based µRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated. CONCLUSIONS: The introduced MTP-based µRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.