Precise localization and trafficking of plant immune receptors are critical for their function. We identify the TNL-class nucleotide-binding leucine-rich repeat receptor (NLR) BURNOUT1 (BNT1) from Arabidopsis thaliana as localized to plastids, key organelles for plant immunity. Alternative transcription start site usage generates two isoforms of BNT1: BNT1.2, which is targeted to the plastid envelope via an N-terminal signal-anchored mechanism, and BNT1.1, which resides in the cytoplasm. Moreover, BNT1.2 is predominantly expressed in epidermal cells, where it localizes to the so-called sensory plastids. Functional analysis revealed that bnt1 mutants exhibit compromised PAMP-triggered immunity (PTI) responses, including impaired callose deposition and reduced flg22-induced resistance to Pseudomonas syringae pv. tomato, while flg22-induced apoplastic reactive oxygen species production remains unaffected. Notably, only the plastid-localized BNT1.2 isoform is required for these PTI responses. Our findings reveal a role for NLRs in regulating PTI responses from plastids and highlight these organelles as key hubs for signal(s) integration during plant-pathogen interactions.

The comet assay is classically applied to study DNA damage at the single-cell level. In plants, it is commonly used for the analysis of seedlings, leaves, and seeds. The assay is time-consuming and runs continuously, making it difficult to process large volumes of samples or samples derived from kinetic studies in parallel. To overcome these limitations, we present an optimized protocol of neutral comet assay for analysis of Arabidopsis seeds, seedlings and leaves. It includes the cryopreservation of nuclei under conditions that do not affect DNA integrity. This option is not present in other available protocols for plants and allows samples collected at different times to be preserved and then processed simultaneously. We provide details on all experimental steps, including slide preparation, to obtain high-quality samples without the need of commercial kits. Moreover, we demonstrated its effectiveness in quantifying DNA damage induced by abiotic and biotic stresses. This versatile approach will help to optimize sample processing and will contribute to improving the studies of DNA damage in plants.

SUMMARYDNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl‐CpG‐binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5‐fluorouracil and 5‐bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non‐aged seeds. Seeds from mbd4l‐1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l‐1 and 35S:MBD4L.4/mbd4l‐1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L‐dependent manner at 24 h post‐imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l‐1 and 35S:MBD4L.4/mbd4l‐1 than in mbd4l‐1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l‐1, but not in 35S:MBD4L.3/mbd4l‐1 or 35S:MBD4L.4/mbd4l‐1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.

DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.

In plants, the establishment of broad and long-lasting immunity is based on programs that control systemic resistance and immunological memory or "priming". Despite not showing activated defenses, a primed plant induces a more efficient response to recurrent infections. Priming might involve chromatin modifications that allow a faster/stronger activation of defense genes. The Arabidopsis chromatin regulator "Morpheus Molecule 1" (MOM1) has been recently suggested as a priming factor affecting the expression of immune receptor genes. Here, we show that mom1 mutants exacerbate the root growth inhibition response triggered by the key defense priming inducers azelaic acid (AZA), β-aminobutyric acid (BABA) and pipecolic acid (PIP). Conversely, mom1 mutants complemented with a minimal version of MOM1 (miniMOM1 plants) are insensitive. Moreover, miniMOM1 is unable to induce systemic resistance against Pseudomonas sp. in response to these inducers. Importantly, AZA, BABA and PIP treatments reduce the MOM1 expression, but not miniMOM1 transcript levels, in systemic tissues. Consistently, several MOM1-regulated immune receptor genes are upregulated during the activation of systemic resistance in WT plants, while this effect is not observed in miniMOM1. Taken together, our results position MOM1 as a chromatin factor that negatively regulates the defense priming induced by AZA, BABA and PIP.

SUMMARYDNA glycosylases are critical enzymes that recognize small base lesions in DNA and trigger their repair to preserve genome integrity. The Arabidopsis MBD4‐like (MBD4L) DNA glycosylase improves tolerance to genotoxic stress. This enzyme is encoded by a single gene carrying an exonic intron (exitron) at its 5′ region. Although alternative splicing (AS) of exitrons (protein‐coding cryptic introns within exons) is suspected to increase protein diversity, phenotypes associated to exitron removal or retention are only known for a few genes. Here, we show that AS of the MBD4L exitron determines the generation of two enzyme isoforms with different subnuclear localization. Both isoforms conserve the catalytic domain but are directed to either the nucleoplasm or the nucleolus. Interestingly, heat stress regulates the AS of the MBD4L exitron and increases the relative abundance of the nucleolar variant. This process depends on the splicing factors NTR1 and RS31. Next, we generated transgenic plants expressing a mutated MBD4L‐GFP gene version that abolished exitron splicing and found that nucleolar protein targeting was impaired in these plants. Our findings suggest that AS of the MBD4L exitron can function as a mechanism to drive the enzyme to the nucleolus during heat stress. Several DNA repair enzymes reach the nucleolus under particular conditions although AS of exitrons has not been so far associated with this process. To our knowledge, this is the first example of an exitron mediating enzyme localization in eukaryotes.

Proline is a multifunctional amino acid that is accumulated in high concentrations in plants under various stress conditions. Proline accumulation is intimately connected to many cellular processes, such as osmotic pressure, energy status, nutrient availability, changes in redox balance, and defenses against pathogens. Proline biosynthesis and catabolism is linked to photosynthesis and mitochondrial respiration, respectively. Proline can function as a signal, modulating gene expression and certain metabolic processes. We review important findings on proline metabolism and function of the last decade, giving a more informative picture about the function of this unusual amino acid in maintaining cellular homeostasis, modulating plant development, and promoting stress acclimation.

The gene pool encoding PRR and NLR immune receptors determines the ability of a plant to resist microbial infections. Basal expression of these genes is prevented by diverse mechanisms since their hyperactivity can be harmful. To approach the study of epigenetic control of PRR / NLR genes we here analyzed their expression in mutants carrying abnormal repressive 5-methyl cytosine (5-mC) and histone 3 lysine 9 dimethylation (H3K9me2) marks, due to lack of MET1, CMT3, MOM1, SUVH4/5/6, or DDM1. At optimal growth conditions, none of the mutants showed basal expression of the defense gene marker PR1 , but all of them had greater resistance to Pseudomonas syringae pv. tomato than wild type plants, suggesting they are primed to stimulate immune cascades. Consistently, analysis of available transcriptomes indicated that all mutants showed activation of particular PRR/NLR genes under some growth conditions. Under low defense activation, 37 PRR / NLR genes were expressed in these plants, but 29 of them were exclusively activated in specific mutants, indicating that MET1, CMT3, MOM1, SUVH4/5/6, and DDM1 mediate basal repression of different subsets of genes. Some epigenetic marks present at promoters, but not gene bodies, could explain the activation of these genes in the mutants. As expected, suvh4/5/6 and ddm1 activated genes carrying 5-mC and H3K9me2 marks in wild type plants. Surprisingly, all mutants expressed genes harboring promoter H2A.Z/H3K27me3 marks likely affected by the chromatin remodeler PIE1 and the histone demethylase REF6, respectively. Therefore, MET1, CMT3, MOM1, SUVH4/5/6, and DDM1, together with REF6, seemingly contribute to the establishment of chromatin states that prevent constitutive PRR/NLR gene activation, but facilitate their priming by modulating epigenetic marks at their promoters.

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.

The genus Phycomyces belongs to the class Zygomycetes, characterized by fusing gametangia which form thick-walled zygospores, by nonflagellated, nonmotile vegetative spores and by a mycelium that is almost completely nonseptate. The sporangiophores of Phycomyces show growth response to several stimuli including light, gravity, wind, chemicals, and the presence of objects in the proximity of the growing zone. The existence of mutants has facilitated the study of sensory transduction processes, as well as carotenogenesis, differentiation, and sexual development. Mutants with defective phototropism are designated mad. The isolation of mutants that carry recessive mutations has relied so far on the killing of most of the nuclei by chemical or physical agents, generally the same agents used to induce a high frequency of mutations. The values of biochemical variables or behavioral traits in heterokaryons containing a known proportion of nuclei presenting differences for these variables are obtained as a function of the nuclear proportion.

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 (βα) 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.

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Pattern recognition receptors (PRR) and nucleotide-binding leucine-rich repeat proteins (NLR) are major components of the plant immune system responsible for pathogen detection. To date, the transcriptional regulation of PRR/NLR genes is poorly understood. Some PRR/NLR genes are affected by epigenetic changes of neighboring transposable elements (TEs) (cis regulation). We analyzed whether these genes can also respond to changes in the epigenetic marks of distal pericentromeric TEs (trans regulation). We found that Arabidopsis tissues infected with Pseudomonas syringae pv. tomato (Pst) initially induced the expression of pericentromeric TEs, and then repressed it by RNA-directed DNA methylation (RdDM). The latter response was accompanied by the accumulation of small RNAs (sRNAs) mapping to the TEs. Curiously these sRNAs also mapped to distal PRR/NLR genes, which were controlled by RdDM but remained induced in the infected tissues. Then, we used non-infected mom1 (Morpheus' molecule 1) mutants that expressed pericentromeric TEs to test if they lose repression of PRR/NLR genes. mom1 plants activated several PRR/NLR genes that were unlinked to MOM1-targeted TEs, and showed enhanced resistance to Pst. Remarkably, the increased defenses of mom1 were abolished when MOM1/RdDM-mediated pericentromeric TEs silencing was re-established. Therefore, common sRNAs could control PRR/NLR genes and distal pericentromeric TEs and preferentially silence TEs when they are activated.

An increase in crop yield is essential to reassure food security to meet the accelerating global demand. Several genetic modifications can increase organ size, which in turn might boost crop yield. Still, only in a few cases their performance has been evaluated under stress conditions. MicroRNA miR396 repress the expression of GROWTH-REGULATING FACTOR (GRF) genes that codes for transcription factors that promote organ growth. Here, we show that both Arabidopsis thaliana At-GRF2 and At-GRF3 genes resistant to miR396 activity (rGRF2 and rGRF3) increased organ size, but only rGRF3 can produce this effect without causing morphological defects. Furthermore, introduction of At-rGRF3 in Brassica oleracea can increase organ size, and when At-rGRF3 homologs from soybean and rice are introduced in Arabidopsis, leaf size is also increased. This suggests that regulation of GRF3 activity by miR396 is important for organ growth in a broad range of species. Plants harboring rGRF3 have larger leaves also under drought stress, a condition that stimulates miR396 accumulation. These plants also showed an increase in the resistance to virulent bacteria, suggesting that the size increment promoted by rGRF3 occurs without an obvious cost on plant defenses. Our findings indicate that rGRF3 can increase plant organ size under both normal and stress conditions and is a valuable tool for biotechnological applications.

Resumen: Se presenta el caso de un hombre de 79 anos con miocardiopatia dilatada severa, disfuncion ventricular izquierda, fibrilacion auricular permanente y portador de un resincronizador ventricular. Al efectuar un recambio del resincronizador se implanto un electrodo adicional para estimulacion multisitio del ventriculo izquierdo. Ello condujo a significativa mejoria clinica y de la fraccion de eyeccion del ventriculo izquierdo.

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.

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.

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.

Natural and synthetic elicitors have contributed significantly to the study of plant immunity. Pathogen-derived proteins and carbohydrates that bind to immune receptors, allow the fine dissection of certain defence pathways. Lipids of a different nature that act as defence elicitors, have also been studied, but their specific effects have been less well characterized, and their receptors have not been identified. In animal cells, nanoliposomes of the synthetic cationic lipid 3-tetradecylamino-tert-butyl-N-tetradecylpropionamidine (diC14) activate the TLR4-dependent immune cascade. Here, we have investigated whether this lipid induces Arabidopsis defence responses. At the local level, diC14 activated early and late defence gene markers (FRK1, WRKY29, ICS1 and PR1), acting in a dose-dependent manner. This lipid induced the salicylic acid (SA)-dependent, but not jasmonic acid (JA)-dependent, pathway and protected plants against Pseudomonas syringae pv. tomato (Pst), but not Botrytis cinerea. diC14 was not toxic to plant or pathogen, and potentiated pathogen-induced callose deposition. At the systemic level, diC14 induced PR1 expression and conferred resistance against Pst. diC14-induced defence responses required the signalling protein EDS1, but not NDR1. Curiously, the lipid-induced defence gene expression was lower in the fls2/efr/cerk1 triple mutant, but still unchanged in the single mutants. The amidine headgroup and chain length were important for its activity. Given the robustness of the responses triggered by diC14, its specific action on a defence pathway and the requirement for well-known defence components, this synthetic lipid is emerging as a useful tool to investigate the initial events involved in plant innate immunity.