Oomycete intracellular effectors: specialised weapons targeting strategic plant processes.

Oomycete phytopathogens have adapted to colonise plants using effectors as their molecular weapons. Intracellular effectors, mostly proteins but also small ribonucleic acids, are delivered by the pathogens into the host cell cytoplasm where they interfere with normal plant physiology. The diverse host processes emerging as 'victims' of these 'specialised bullets' include gene transcription and RNA-mediated silencing, cell death, protein stability, protein secretion and autophagy. Some effector targets are directly involved in defence execution, while others participate in fundamental metabolisms whose alteration collaterally affects defences. Other effector targets are susceptibility factors (SFs), that is host components that make plants vulnerable to pathogens. SFs are mostly negative regulators of immunity, but some seem necessary to sustain or promote pathogen colonisation.

OXR2 Increases Plant Defense against a Hemibiotrophic Pathogen via the Salicylic Acid Pathway.

Arabidopsis (Arabidopsis thaliana) OXIDATION RESISTANCE2 (AtOXR2) is a mitochondrial protein belonging to the Oxidation Resistance (OXR) protein family, recently described in plants. We analyzed the impact of AtOXR2 in Arabidopsis defense mechanisms against the hemibiotrophic bacterial pathogen Pseudomonas syringae oxr2 mutant plants are more susceptible to infection by the pathogen and, conversely, plants overexpressing AtOXR2 (oeOXR2 plants) show enhanced disease resistance. Resistance in these plants is accompanied by higher expression of WRKY transcription factors, induction of genes involved in salicylic acid (SA) synthesis, accumulation of free SA, and overall activation of the SA signaling pathway. Accordingly, defense phenotypes are dependent on SA synthesis and SA perception pathways, since they are lost in isochorismate synthase1/salicylic acid induction deficient2 and nonexpressor of pathogenesis-related genes1 (npr1) mutant backgrounds. Overexpression of AtOXR2 leads to faster and stronger oxidative burst in response to the bacterial flagellin peptide flg22 Moreover, AtOXR2 affects the nuclear localization of the transcriptional coactivator NPR1, a master regulator of SA signaling. oeOXR2 plants have increased levels of total glutathione and a more oxidized cytosolic redox cellular environment under normal growth conditions. Therefore, AtOXR2 contributes to establishing plant protection against infection by P. syringae acting on the activity of the SA pathway.

Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1.

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.

Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production.

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC- and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector.

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.

Arabidopsis Proline Dehydrogenase Contributes to Flagellin-Mediated PAMP-Triggered Immunity by Affecting RBOHD.

Plants activate different defense systems to counteract the attack of microbial pathogens. Among them, the recognition of conserved microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) by pattern-recognition receptors stimulates MAMP- or PAMP-triggered immunity (PTI). In recent years, the elicitors, receptors, and signaling pathways leading to PTI have been extensively studied. However, the contribution of organelles to this program deserves further characterization. Here, we studied how processes altering the mitochondrial electron transport chain (mETC) influence PTI establishment. With particular emphasis, we evaluated the effect of proline dehydrogenase (ProDH), an enzyme that can load electrons into the mETC and regulate the cellular redox state. We found that mETC uncouplers (antimycin or rotenone) and manganese superoxide dismutase deficiency impair flg22-induced responses such as accumulation of reactive oxygen species (ROS) and bacterial growth limitation. ProDH mutants also reduce these defenses, decreasing callose deposition as well. Using ProDH inhibitors and ProDH inducers (exogenous Pro treatment), we showed that this enzyme modulates the generation of ROS by the plasma membrane respiratory burst NADPH oxidase homolog D. In this way, we contribute to the understanding of mitochondrial activities influencing early and late PTI responses and the coordination of the redox-associated mitochondrial enzyme ProDH with defense events initiated at the plasma membrane.

Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex.

Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)-triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression.

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence.

In planta effector competition assays detect Hyaloperonospora arabidopsidis effectors that contribute to virulence and localize to different plant subcellular compartments.

The genome of the pathogenic oomycete Hyaloperonospora arabidopsidis is predicted to encode at least 134 high-confidence effectors (HaRxL) carrying the RxLR motif implicated in their translocation into plant cells. However, only four avirulence genes (ATR1, ATR13, ATR5, and ATR39) have been isolated. This indicates that identification of HaRxL effectors based on avirulence is low throughput. We aimed at rapidly identifying H. arabidopsidis effectors that contribute to virulence by developing methods to detect and quantify multiple candidates in bacterial mixed infections using either Illumina sequencing or capillary electrophoresis. In these assays, referred to here as in planta effector competition assays, we estimate the contribution to virulence of individual effectors by calculating the abundance of each HaRxL in the bacterial population recovered from leaves 3 days after inoculation relative to abundance in the initial mixed inoculum. We identified HaRxL that enhance Pseudomonas syringae pv. tomato DC3000 growth in some but not all Arabidopsis accessions. Further analysis showed that HaRxLL464, HaRxL75, HaRxL22, HaRxLL441, and HaRxL89 suppress pathogen-associated molecular pattern-triggered immunity (PTI) and localize to different subcellular compartments in Nicotiana benthamiana, providing evidence for a multilayered suppression of PTI by pathogenic oomycetes and molecular probes for the dissection of PTI.

Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.

Nanoemulsions of synthetic rhamnolipids act as plant resistance inducers without damaging plant tissues or affecting soil microbiota.

Plant pathogens and pests can cause significant losses in crop yields, affecting food security and the global economy. Many traditional chemical pesticides are used to combat these organisms. This can lead to the development of pesticide-resistant strains of pathogens/insects and negatively impact the environment. The development of new bioprotectants, which are less harmful to the environment and less likely to lead to pesticide-resistance, appears as a sustainable strategy to increase plant immunity. Natural Rhamnolipids (RL-Nat) are a class of biosurfactants with bioprotectant properties that are produced by an opportunistic human pathogen bacterium. RL-Nat can act as plant resistance inducers against a wide variety of pathogens. Recently, a series of bioinspired synthetic mono-RLs produced by green chemistry were also reported as phytoprotectants. Here, we explored their capacity to generate novel colloidal systems that might be used to encapsulate bioactive hydrophobic compounds to enhance their performance as plant bioprotectants. The synthetic mono-RLs showed good surfactant properties and emulsification power providing stable nanoemulsions capable of acting as bio-carriers with good wettability. Synthetic RLs-stabilized nanoemulsions were more effective than RLs suspensions at inducing plant immunity, without causing deleterious effects. These nanoemulsions were innocuous to native substrate microbiota and beneficial soil-borne microbes, making them promising safe bio-carriers for crop protection.

Loss of compatibility might explain resistance of the Arabidopsis thaliana accession Te-0 to Golovinomyces cichoracearum.

BACKGROUND: The establishment of compatibility between plants and pathogens requires compliance with various conditions, such as recognition of the right host, suppression of defence mechanisms, and maintenance of an environment allowing pathogen reproduction. To date, most of the plant factors required to sustain compatibility remain unknown, with the few best characterized being those interfering with defence responses. A suitable system to study host compatibility factors is the interaction between Arabidopsis thaliana and the powdery mildew (PM) Golovinomyces cichoracearum. As an obligate biotrophic pathogen, this fungus must establish compatibility in order to perpetuate. In turn, A. thaliana displays natural variation for susceptibility to this invader, with some accessions showing full susceptibility (Col-0), and others monogenic dominant resistance (Kas-1). Interestingly, Te-0, among other accessions, displays recessive partial resistance to this PM. RESULTS: In this study, we characterized the interaction of G. cichoracearum with Te-0 plants to investigate the basis of this plant resistance. We found that Te-0's incompatibility was not associated with hyper-activation of host inducible defences. Te-0 plants allowed germination of conidia and development of functional haustoria, but could not support the formation of mature conidiophores. Using a suppressive subtractive hybridization technique, we identified plant genes showing differential expression between resistant Te-0 and susceptible Col-0 plants at the fungal pre-conidiation stage. CONCLUSIONS: Te-0 resistance is likely caused by loss of host compatibility and not by stimulation of inducible defences. Conidiophores formation is the main constraint for completion of fungal life cycle in Te-0 plants. The system here described allowed the identification of genes proposed as markers for susceptibility to this PM.

The N-terminal domain of Arabidopsis proline dehydrogenase affects enzymatic activity and protein oligomerization.

Proline dehydrogenase (ProDH) is a flavoenzyme that catalyzes the oxidation of proline (Pro) into Δ1-pyrroline-5-carboxylate (P5C). In eukaryotes, ProDH coordinates with different Pro metabolism enzymes to control energy supply or stress responses signaling. Heterologous expression and crystallization of prokaryotic enzymes provided key data on their active center, folding capacity and oligomerization status. In contrast, eukaryotic ProDHs have not been crystallized so far, and their study as recombinant proteins remains limited. Plants contain two isoforms of ProDH with non-redundant functions. To contribute to the study of these enzymes, we describe the modeling, expression in E. coli, purification, and characterization of the Arabidopsis isoenzymes, AtProDH1 and AtProDH2. The 3D model suggested that both proteins adopt a distorted barrel structure (ßα) with a cap formed by N-terminal α helices. The expression of two types of N-terminal deletion proteins indicated that this domain affected enzyme activity. Full-length enzymes had Km values similar to those of native proteins, whereas truncated proteins were inactive. Moreover, the first α helix proved to be necessary for AtProDH1 and AtProDH2 activities. Interestingly, both isoenzymes were able to oligomerize and this also required the first N-terminal α helix. Thus, we report the first insights into structure-function relationship of plant ProDHs demonstrating that the N-terminus, although not directly involved in catalysis, controls enzyme arrangement and activity. The resources generated here could be useful to analyze other plant ProDH features, such as its coordination with other enzymes, and differences between ProDH1 and ProDH2, providing new information on its effects on stress tolerance.

Characterization of Arabidopsis mur3 mutations that result in constitutive activation of defence in petioles, but not leaves.

A screen was established for mutants in which the plant defence response is de-repressed. The pathogen-inducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1:luc fusion is co-regulated with ICS1. This line was mutagenized and M(2) seedlings screened for constitutive ICS1:luc expression (cie). The cie mutants fall into distinct phenotypic classes based on tissue-specific localization of luciferase activity. One mutant, cie1, that shows constitutive luciferase activity specifically in petioles, was chosen for further analysis. In addition to ICS1, PR and other defence-related genes are constitutively expressed in cie1 plants. The cie1 mutant is also characterized by an increased production of conjugated salicylic acid and reactive oxygen intermediates, as well as spontaneous lesion formation, all confined to petiole tissue. Significantly, defences activated in cie1 are sufficient to prevent infection by a virulent isolate of Hyaloperonospora parasitica, and this enhanced resistance response protects petiole tissue alone. Furthermore, cie1-mediated resistance, along with PR gene expression, is abolished in a sid2-1 mutant background, consistent with a requirement for salicylic acid. A positional cloning approach was used to identify cie1, which carries two point mutations in a gene required for cell wall biosynthesis and actin organization, MUR3. A mur3 knockout mutant also resists infection by H. parasitica in its petioles and this phenotype is complemented by transformation with wild-type MUR3. We propose that perturbed cell wall biosynthesis may activate plant defence and provide a rationale for the cie1 and the mur3 knockout phenotypes.

Differential control and function of Arabidopsis ProDH1 and ProDH2 genes on infection with biotrophic and necrotrophic pathogens.

Arabidopsis contains two proline dehydrogenase (ProDH) genes, ProDH1 and ProDH2, encoding for homologous and functional isoenzymes. Although ProDH1 has been studied extensively, especially under abiotic stress, ProDH2 has only started to be analysed in recent years. These genes display distinctive expression patterns and show weak transcriptional co-regulation, but are both activated in pathogen-infected tissues. We have demonstrated previously that Arabidopsis plants with silenced ProDH1/2 expression fail to trigger defences against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato AvrRpm1 (Pst-AvrRpm1), and that ProDH1 and ProDH2 are differentially regulated by salicylic acid (SA). In the current work, we used prodh1 and prodh2 single-mutant plants to assess the particular contribution of each gene to resistance against Pst-AvrRpm1 and the necrotrophic fungal pathogen Botrytis cinerea. In addition, we studied the sensitivity of ProDH1 and ProDH2 to the jasmonic acid (JA) defence pathway. We found that ProDH1 and ProDH2 are both necessary to achieve maximum resistance against Pst-AvrRpm1 and B. cinerea. However, ProDH2 has a major effect on early restriction of B. cinerea growth. Interestingly, ProDH1 is up-regulated by SA and JA, whereas ProDH2 is only activated by JA, and both genes display transcriptional inter-regulation at basal and infection conditions. These studies provide the first evidence of the contribution of ProDH2 to disease resistance, and describe the differential regulation and non-redundant but complementary function of both enzyme isoforms in infected tissues, providing support for a fundamental role of ProDH in the control of biotrophic and necrotrophic pathogens.

P5CDH affects the pathways contributing to Pro synthesis after ProDH activation by biotic and abiotic stress conditions.

Plants facing adverse conditions usually alter proline (Pro) metabolism, generating changes that help restore the cellular homeostasis. These organisms synthesize Pro from glutamate (Glu) or ornithine (Orn) by two-step reactions that share Δ(1) pyrroline-5-carboxylate (P5C) as intermediate. In the catabolic process, Pro is converted back to Glu using a different pathway that involves Pro dehydrogenase (ProDH), P5C dehydrogenase (P5CDH), and P5C as intermediate. Little is known about the coordination of the catabolic and biosynthetic routes under stress. To address this issue, we analyzed how P5CDH affects the activation of Pro synthesis, in Arabidopsis tissues that increase ProDH activity by transient exposure to exogenous Pro, or infection with Pseudomonas syringae pv. tomato. Wild-type (Col-0) and p5cdh mutant plants subjected to these treatments were used to monitor the Pro, Glu, and Orn levels, as well as the expression of genes from Pro metabolism. Col-0 and p5cdh tissues consecutively activated ProDH and Pro biosynthetic genes under both conditions. However, they manifested a different coordination between these routes. When external Pro supply was interrupted, wild-type leaves degraded Pro to basal levels at which point Pro synthesis, mainly via Glu, became activated. Under the same condition, p5cdh leaves sustained ProDH induction without reducing the Pro content but rather increasing it, apparently by stimulating the Orn pathway. In response to pathogen infection, both genotypes showed similar trends. While Col-0 plants seemed to induce both Pro biosynthetic routes, p5cdh mutant plants may primarily activate the Orn route. Our study contributes to the functional characterization of P5CDH in biotic and abiotic stress conditions, by revealing its capacity to modulate the fate of P5C, and prevalence of Orn or Glu as Pro precursors in tissues that initially consumed Pro.

Proline accumulation and AtP5CS2 gene activation are induced by plant-pathogen incompatible interactions in Arabidopsis.

Accumulation of free L-proline (Pro) is a typical stress response incited by osmotic injuries in plants and microorganisms. Although the protective role of Pro in osmotic stress is not well understood, it is thought to function as compatible osmolyte or as a scavenger of reactive oxygen species (ROS). Here we show that, in Arabidopsis thaliana, Pro biosynthesis can be activated by incompatible plant-pathogen interactions triggering a hypersensitive response (HR). Pro accumulates in leaf tissues treated with Pseudomonas syringae pv. tomato avirulent strains (avrRpt2 and avrRpm1) but remains unchanged in leaves infected with isogenic virulent bacteria. Incompatible interactions lead to transcriptional activation of AtP5CS2, but not AtP5CS1, encoding the rate limiting enzyme in Pro biosynthesis pyrroline-5-carboxylate synthase (P5CS). AtP5CS2:GUS and AtP5CS2:LUC transgenes were induced inside and around the HR lesions produced by avirulent Pseudomonas spp. in transgenic plants. Pro accumulation was faster and stronger when stimulated by avrRpm1 than by avrRpt2, and was compromised in the low-salicylic acid plants NahG and eds5 when signaled through the RPS2-dependent pathway. In addition, Pro content and AtP5CS2 expression were enhanced by ROS in wild-type plants, suggesting that ROS may function as an intermediate signal in AtP5CS2-mediated Pro accumulation.

Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract.

The time course of the level of A23187-induced acrosome reaction between human and rabbit spermatozoa was compared. It was extended in the former (a periodic ovulator) and short in the latter (an induced ovulator). This finding suggests that the capacitated state is programmed to maximize the prospects that an ovulated egg will meet spermatozoa in the best functional state.