Beneficial soil-borne bacteria and fungi: a promising way to improve plant nitrogen acquisition.

Nitrogen (N) is an essential element for plant productivity, thus, it is abundantly applied to the soil in the form of organic or chemical fertilizers that have negative impacts on the environment. Exploiting the potential of beneficial microbes and identifying crop genotypes that can capitalize on symbiotic associations may be possible ways to significantly reduce the use of N fertilizers. The best-known example of symbiotic association that can reduce the use of N fertilizers is the N2-fixing rhizobial bacteria and legumes. Bacterial taxa other than rhizobial species can develop associative symbiotic interactions with plants and also fix N. These include bacteria of the genera Azospirillum, Azotobacter, and Bacillus, some of which are commercialized as bio-inoculants. Arbuscular mycorrhizal fungi are other microorganisms that can develop symbiotic associations with most terrestrial plants, favoring access to nutrients in a larger soil volume through their extraradical mycelium. Using combinations of different beneficial microbial species is a promising strategy to boost plant N acquisition and foster a synergistic beneficial effect between symbiotic microorganisms. Complex biological mechanisms including molecular, metabolic, and physiological processes dictate the establishment and efficiency of such multipartite symbiotic associations. In this review, we present an overview of the current knowledge and future prospects regarding plant N nutrition improvement through the use of beneficial bacteria and fungi associated with plants, individually or in combination.

Scavenging iron: a novel mechanism of plant immunity activation by microbial siderophores.

Siderophores are specific ferric iron chelators synthesized by virtually all microorganisms in response to iron deficiency. We have previously shown that they promote infection by the phytopathogenic enterobacteria Dickeya dadantii and Erwinia amylovora. Siderophores also have the ability to activate plant immunity. We have used complete Arabidopsis transcriptome microarrays to investigate the global transcriptional modifications in roots and leaves of Arabidopsis (Arabidopsis thaliana) plants after leaf treatment with the siderophore deferrioxamine (DFO). Physiological relevance of these transcriptional modifications was validated experimentally. Immunity and heavy-metal homeostasis were the major processes affected by DFO. These two physiological responses could be activated by a synthetic iron chelator ethylenediamine-di(o-hydroxyphenylacetic) acid, indicating that siderophores eliciting activities rely on their strong iron-chelating capacity. DFO was able to protect Arabidopsis against the pathogenic bacterium Pseudomonas syringae pv tomato DC3000. Siderophore treatment caused local modifications of iron distribution in leaf cells visible by ferrocyanide and diaminobenzidine-H2O2 staining. Metal quantifications showed that DFO causes a transient iron and zinc uptake at the root level, which is presumably mediated by the metal transporter iron regulated transporter1 (IRT1). Defense gene expression and callose deposition in response to DFO were compromised in an irt1 mutant. Consistently, plant susceptibility to D. dadantii was increased in the irt1 mutant. Our work shows that iron scavenging is a unique mechanism of immunity activation in plants. It highlights the strong relationship between heavy-metal homeostasis and immunity.

New insights into the role of siderophores as triggers of plant immunity: what can we learn from animals?

Microorganisms use siderophores to obtain iron from the environment. In pathogenic interactions, siderophores are involved in iron acquisition from the host and are sometimes necessary for the expression of full virulence. This review summarizes the main data describing the role of these iron scavengers in animal and plant defence systems. To protect themselves against iron theft, mammalian hosts have developed a hypoferremia strategy that includes siderophore-binding molecules called siderocalins. In addition to microbial ferri-siderophore sequestration, siderocalins are involved in triggering immunity. In plants, no similar mechanisms have been described and many fewer data are available, although recent advances have shed light on the role of siderophores in plant-pathogen interactions. Siderophores can trigger immunity in plants in several contexts. The most frequently described situation involving siderophores is induced systemic resistance (ISR) triggered by plant-growth-promoting rhizobacteria. Although ISR responses have been observed after treating roots with certain siderophores, the underlying mechanisms are poorly understood. Immunity can also be triggered by siderophores in leaves. Siderophore perception in plants appears to be different from the well-known perception mechanisms of other microbial compounds, known as microbe-associated molecular patterns. Scavenging iron per se appears to be a novel mechanism of immunity activation, involving complex disturbance of metal homeostasis. Receptor-specific recognition of siderophores has been described in animals, but not in plants. The review closes with an overview of the possible mechanisms of defence activation, via iron scavenging by siderophores or specific siderophore recognition by the plant host.

Nitrogen metabolism meets phytopathology.

Nitrogen (N) is essential for life and is a major limiting factor of plant growth. Because soils frequently lack sufficient N, large quantities of inorganic N fertilizers are added to soils for crop production. However, nitrate, urea, and ammonium are a major source of global pollution, because much of the N that is not taken up by plants enters streams, groundwater, and lakes, where it affects algal production and causes an imbalance in aquatic food webs. Many agronomical data indicate that the higher use of N fertilizers during the green revolution had an impact on the incidence of crop diseases. In contrast, examples in which a decrease in N fertilization increases disease severity are also reported, indicating that there is a complex relationship linking N uptake and metabolism and the disease infection processes. Thus, although it is clear that N availability affects disease, the underlying mechanisms remain unclear. The aim of this review is to describe current knowledge of the mechanisms that link plant N status to the plant's response to pathogen infection and to the virulence and nutritional status of phytopathogens.

A multi-organ maize metabolic model connects temperature stress with energy production and reducing power generation.

Climate change has adversely affected maize productivity. Thereby, a holistic understanding of metabolic crosstalk among its organs is important to address this issue. Thus, we reconstructed the first multi-organ maize metabolic model, iZMA6517, and contextualized it with heat and cold stress transcriptomics data using expression distributed reaction flux measurement (EXTREAM) algorithm. Furthermore, implementing metabolic bottleneck analysis on contextualized models revealed differences between these stresses. While both stresses had reducing power bottlenecks, heat stress had additional energy generation bottlenecks. We also performed thermodynamic driving force analysis, revealing thermodynamics-reducing power-energy generation axis dictating the nature of temperature stress responses. Thus, a temperature-tolerant maize ideotype can be engineered by leveraging the proposed thermodynamics-reducing power-energy generation axis. We experimentally inoculated maize root with a beneficial mycorrhizal fungus, Rhizophagus irregularis, and as a proof-of-concept demonstrated its efficacy in alleviating temperature stress. Overall, this study will guide the engineering effort of temperature stress-tolerant maize ideotypes.

Corrigendum: Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

[This corrects the article DOI: 10.3389/fpls.2021.807798.].

Salicylic acid is a key player of Arabidopsis autophagy mutant susceptibility to the necrotrophic bacterium Dickeya dadantii.

Autophagy is a ubiquitous vesicular process for protein and organelle recycling in eukaryotes. In plant, autophagy is reported to play pivotal roles in nutrient recycling, adaptation to biotic and abiotic stresses. The role of autophagy in plant immunity remains poorly understood. Several reports showed enhanced susceptibility of different Arabidopsis autophagy mutants (atg) to necrotrophic fungal pathogens. Interaction of necrotrophic bacterial pathogens with autophagy is overlooked. We then investigated such interaction by inoculating the necrotrophic enterobacterium Dickeya dadantii in leaves of the atg2 and atg5 mutants and an ATG8a overexpressing line. Overexpressing ATG8a enhances plant tolerance to D. dadantii. While atg5 mutant displayed similar susceptibility to the WT, the atg2 mutant exhibited accelerated leaf senescence and enhanced susceptibility upon infection. Both phenotypes were reversed when the sid2 mutation, abolishing SA signaling, was introduced in the atg2 mutant. High levels of SA signaling in atg2 mutant resulted in repression of the jasmonic acid (JA) defense pathway known to limit D. dadantii progression in A. thaliana. We provide evidence that in atg2 mutant, the disturbed hormonal balance leading to higher SA signaling is the main factor causing increased susceptibility to the D. dadantii necrotroph by repressing the JA pathway and accelerating developmental senescence.

Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

Owing to the large genetic diversity of barley and its resilience under harsh environments, this crop is of great value for agroecological transition and the need for reduction of nitrogen (N) fertilizers inputs. In the present work, we investigated the diversity of a North African barley genotype collection in terms of growth under limiting N (LN) or ample N (HN) supply and in terms of physiological traits including amino acid content in young seedlings. We identified a Moroccan variety, Laanaceur, accumulating five times more lysine in its leaves than the others under both N nutritional regimes. Physiological characterization of the barley collection showed the genetic diversity of barley adaptation strategies to LN and highlighted a genotype x environment interaction. In all genotypes, N limitation resulted in global biomass reduction, an increase in C concentration, and a higher resource allocation to the roots, indicating that this organ undergoes important adaptive metabolic activity. The most important diversity concerned leaf nitrogen use efficiency (LNUE), root nitrogen use efficiency (RNUE), root nitrogen uptake efficiency (RNUpE), and leaf nitrogen uptake efficiency (LNUpE). Using LNUE as a target trait reflecting barley capacity to deal with N limitation, this trait was positively correlated with plant nitrogen uptake efficiency (PNUpE) and RNUpE. Based on the LNUE trait, we determined three classes showing high, moderate, or low tolerance to N limitation. The transcriptomic approach showed that signaling, ionic transport, immunity, and stress response were the major functions affected by N supply. A candidate gene encoding the HvNRT2.10 transporter was commonly up-regulated under LN in the three barley genotypes investigated. Genes encoding key enzymes required for lysine biosynthesis in plants, dihydrodipicolinate synthase (DHPS) and the catabolic enzyme, the bifunctional Lys-ketoglutarate reductase/saccharopine dehydrogenase are up-regulated in Laanaceur and likely account for a hyperaccumulation of lysine in this genotype. Our work provides key physiological markers of North African barley response to low N availability in the early developmental stages.

NRAMP genes function in Arabidopsis thaliana resistance to Erwinia chrysanthemi infection.

AtNRAMP3 and AtNRAMP4 are two Arabidopsis metal transporters sharing about 50% sequence identity with mouse NRAMP1. The NRAMP1/Slc11A1 metal ion transporter plays a crucial role in the innate immunity of animal macrophages targeted by intracellular bacterial pathogens. AtNRAMP3 and AtNRAMP4 localize to the vacuolar membrane. We found that AtNRAMP3 is upregulated in leaves challenged with the bacterial pathogens Pseudomonas syringae and Erwinia chrysanthemi, whereas AtNRAMP4 expression is not modified. Using single and double nramp3 and nramp4 mutants, as well as lines ectopically expressing either of these genes, we show that AtNRAMP3 and, to a lesser extent, AtNRAMP4 are involved in Arabidopsis thaliana resistance against the bacterial pathogen E. chrysanthemi. The susceptibility of the double nramp3 nramp4 mutant is associated with the reduced accumulation of reactive oxygen species and ferritin (AtFER1), an iron storage protein known to participate in A. thaliana defense. Interestingly, roots from infected plants accumulated transcripts of AtNRAMP3 as well as the iron-deficiency markers IRT1 and FRO2. This finding suggests the existence of a shoot-to-root signal reminiscent of an iron-deficiency signal activated by pathogen infection. Our data indicate that the functions of NRAMP proteins in innate immunity have been conserved between animals and plants.

Plant Defense Stimulator Mediated Defense Activation Is Affected by Nitrate Fertilization and Developmental Stage in Arabidopsis thaliana.

Plant defense stimulators, used in crop protection, are an attractive option to reduce the use of conventional crop protection products and optimize biocontrol strategies. These products are able to activate plant defenses and thus limit infection by pathogens. However, the effectiveness of these plant defense stimulators remains erratic and is potentially dependent on many agronomic and environmental parameters still unknown or poorly controlled. The developmental stage of the plant as well as its fertilization, and essentially nitrogen nutrition, play major roles in defense establishment in the presence of pathogens or plant defense stimulators. The major nitrogen source used by plants is nitrate. In this study, we investigated the impact of Arabidopsis thaliana plant developmental stage and nitrate nutrition on its capacity to mount immune reactions in response to two plant defense stimulators triggering two major defense pathways, the salicylic acid and the jasmonic acid pathways. We show that optimal nitrate nutrition is needed for effective defense activation and protection against the pathogenic bacteria Dickeya dadantii and Pseudomonas syringae pv. tomato. Using an npr1 defense signaling mutant, we showed that nitrate dependent protection against D. dadantii requires a functional NPR1 gene. Our results indicate that the efficacy of plant defense stimulators is strongly affected by nitrate nutrition and the developmental stage. The nitrate dependent efficacy of plant defense stimulators is not only due to a metabolic effect but also invloves NPR1 mediated defense signaling. Plant defense stimulators may have opposite effects on plant resistance to a pathogen. Together, our results indicate that agronomic use of plant defense stimulators must be optimized according to nitrate fertilization and developmental stage.

A secreted metal-binding protein protects necrotrophic phytopathogens from reactive oxygen species.

Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H2O2-induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.

Arabidopsis thaliana expresses multiple lines of defense to counterattack Erwinia chrysanthemi.

Many taxonomically diverse plant species are attacked by Erwinia chrysanthemi, a member of the causal agents of soft-rotting diseases. Symptom development is due to the collective action of pectin-degrading enzymes secreted by the bacterium through a type II secretion system (T2SS). Using Arabidopsis thaliana as a susceptible host, we show that plants respond to E. chrysanthemi 3937 by expressing cell-wall reactions, production of an oxidative burst, and activation of salicylic acid (SA) and jasmonic acid (JA) or ethylene (ET) signaling pathways. We found that the oxidative burst is mainly generated via the expression of the AtrbohD gene, constitutes a barrier of resistance to bacterial attack, and acts independently of the SA-mediated response. To determine the importance of T2SS-secreted proteins in elicitation of these defenses, we used a T2SS deficient mutant and purified enzymatic preparations of representative members of strain 3937 pectate lyase activity. The T2SS-secreted proteins were responsible only partially for the activation of SA and JA or ET signaling pathways observed after infection with the wild-type bacterium and were not involved in the expression of other identified defense reactions. Our study shows the differential role played by pectate lyases isoenzymes in this process and highlights the complexity of the host immune network, which is finely controlled by the bacterium.

Quantitative Methods to Assess Differential Susceptibility of Arabidopsis thaliana Natural Accessions to Dickeya dadantii.

Among the most devastating bacterial diseases of plants, soft rot provoked by Dickeya spp. cause crop yield losses on a large range of species with potato being the most economically important. The use of antibiotics being prohibited in most countries in the field, identifying tolerance genes is expected to be one of the most effective alternate disease control approaches. A prerequisite for the identification of tolerance genes is to develop robust disease quantification methods and to identify tolerant plant genotypes. In this work, we investigate the feasibility of the exploitation of Arabidopsis thaliana natural variation to find tolerant genotypes and to develop robust quantification methods. We compared different quantification methods that score either symptom development or bacterial populations in planta. An easy to set up and reliable bacterial quantification method based on qPCR amplification of bacterial DNA was validated. This study demonstrates that it is possible to conduct a robust phenotyping of soft rot disease, and that Arabidopsis natural accessions are a relevant source of tolerance genes.

Alterations of iron distribution in Arabidopsis tissues infected by Dickeya dadantii.

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for plant soft rot disease in a wide range of hosts, including the model plant Arabidopsis thaliana. Iron distribution in infected A. thaliana was investigated at the cellular scale using the Perls'-diaminobenzidine-H2 O2 (PDH) method. Iron visualization during infection reveals a loss of iron from cellular compartments and plant cell walls. During symptom progression, two distinct zones are clearly visible: a macerated zone displaying weak iron content and a healthy zone displaying strong iron content. Immunolabelling of cell wall methylated pectin shows that pectin degradation is correlated with iron release from cell walls, indicating a strong relationship between cell wall integrity and iron in plant tissues. Using a D. dadantii lipopolysaccharide antibody, we show that bacteria are restricted to the infected tissue, and that they accumulate iron in planta. In conclusion, weak iron content is strictly correlated with bacterial cell localization in the infected tissues, indicating a crucial role of this element during the interaction. This is the first report of iron localization at the cellular level during a plant-microbe interaction and shows that PDH is a method of choice in this type of investigation.

Immunity to plant pathogens and iron homeostasis.

Iron is essential for metabolic processes in most living organisms. Pathogens and their hosts often compete for the acquisition of this nutrient. However, iron can catalyze the formation of deleterious reactive oxygen species. Hosts may use iron to increase local oxidative stress in defense responses against pathogens. Due to this duality, iron plays a complex role in plant-pathogen interactions. Plant defenses against pathogens and plant response to iron deficiency share several features, such as secretion of phenolic compounds, and use common hormone signaling pathways. Moreover, fine tuning of iron localization during infection involves genes coding iron transport and iron storage proteins, which have been shown to contribute to immunity. The influence of the plant iron status on the outcome of a given pathogen attack is strongly dependent on the nature of the pathogen infection strategy and on the host species. Microbial siderophores emerged as important factors as they have the ability to trigger plant defense responses. Depending on the plant species, siderophore perception can be mediated by their strong iron scavenging capacity or possibly via specific recognition as pathogen associated molecular patterns. This review highlights that iron has a key role in several plant-pathogen interactions by modulating immunity.

Evolutionary tinkering of the expression of PDF1s suggests their joint effect on zinc tolerance and the response to pathogen attack.

Multigenic families of Plant Defensin type 1 (PDF1) have been described in several species, including the model plant Arabidopsis thaliana as well as zinc tolerant and hyperaccumulator A. halleri. In A. thaliana, PDF1 transcripts (AtPDF1) accumulate in response to pathogen attack following synergic activation of ethylene/jasmonate pathways. However, in A. halleri, PDF1 transcripts (AhPDF1) are constitutively highly accumulated. Through an evolutionary approach, we investigated the possibility of A. halleri or A. thaliana species specialization in different PDF1s in conveying zinc tolerance and/or the response to pathogen attack via activation of the jasmonate (JA) signaling pathway. The accumulation of each PDF1 from both A. halleri and A. thaliana was thus compared in response to zinc excess and MeJA application. In both species, PDF1 paralogues were barely or not at all responsive to zinc. However, regarding the PDF1 response to JA signaling activation, A. thaliana had a higher number of PDF1s responding to JA signaling activation. Remarkably, in A. thaliana, a slight but significant increase in zinc tolerance was correlated with activation of the JA signaling pathway. In addition, A. halleri was found to be more tolerant to the necrotrophic pathogen Botrytis cinerea than A. thaliana. Since PDF1s are known to be promiscuous antifungal proteins able to convey zinc tolerance, we propose, on the basis of the findings of this study, that high constitutive PDF1 transcript accumulation in A. halleri is a potential way to skip the JA signaling activation step required to increase the PDF1 transcript level in the A. thaliana model species. This could ultimately represent an adaptive evolutionary process that would promote a PDF1 joint effect on both zinc tolerance and the response to pathogens in the A. halleri extremophile species.

Iron deficiency affects plant defence responses and confers resistance to Dickeya dadantii and Botrytis cinerea.

Iron is an essential element for most living organisms, and pathogens are likely to compete with their hosts for the acquisition of this element. The bacterial plant pathogen Dickeya dadantii has been shown to require its siderophore-mediated iron uptake system for systemic disease progression on several host plants, including Arabidopsis thaliana. In this study, we investigated the effect of the iron status of Arabidopsis on the severity of disease caused by D. dadantii. We showed that symptom severity, bacterial fitness and the expression of bacterial pectate lyase-encoding genes were reduced in iron-deficient plants. Reduced symptoms correlated with enhanced expression of the salicylic acid defence plant marker gene PR1. However, levels of the ferritin coding transcript AtFER1, callose deposition and production of reactive oxygen species were reduced in iron-deficient infected plants, ruling out the involvement of these defences in the limitation of disease caused by D. dadantii. Disease reduction in iron-starved plants was also observed with the necrotrophic fungus Botrytis cinerea. Our data demonstrate that the plant nutritional iron status can control the outcome of an infection by acting on both the pathogen's virulence and the host's defence. In addition, iron nutrition strongly affects the disease caused by two soft rot-causing plant pathogens with a large host range. Thus, it may be of interest to take into account the plant iron status when there is a need to control disease without compromising crop quality and yield in economically important plant species.

Microbial siderophores exert a subtle role in Arabidopsis during infection by manipulating the immune response and the iron status.

Siderophores (ferric ion chelators) are secreted by organisms in response to iron deficiency. The pathogenic enterobacterium Erwinia chrysanthemi produces two siderophores, achromobactin and chrysobactin (CB), which are required for systemic dissemination in host plants. Previous studies have shown that CB is produced in planta and can trigger the up-regulation of the plant ferritin gene AtFER1. To further investigate the function of CB during pathogenesis, we analyzed its effect in Arabidopsis (Arabidopsis thaliana) plants following leaf infiltration. CB activates the salicylic acid (SA)-mediated signaling pathway, while the CB ferric complex is ineffective, suggesting that the elicitor activity of this siderophore is due to its iron-binding property. We confirmed this hypothesis by testing the effect of siderophores structurally unrelated to CB, including deferrioxamine. There was no activation of SA-dependent defense in plants grown under iron deficiency before CB treatment. Transcriptional analysis of the genes encoding the root ferrous ion transporter and ferric chelate reductase, and determination of the activity of this enzyme in response to CB or deferrioxamine, showed that these compounds induce a leaf-to-root iron deficiency signal. This root response as well as ferritin gene up-regulation in the leaf were not compromised in a SA-deficient mutant line. Using the Arabidopsis-E. chrysanthemi pathosystem, we have shown that CB promotes bacterial growth in planta and can modulate plant defenses through an antagonistic mechanism between SA and jasmonic acid signaling cascades. Collectively, these data reveal a new link between two processes mediated by SA and iron in response to microbial siderophores.

Ferritins, bacterial virulence and plant defence.

The enterobacterial pathogen Erwinia chrysanthemi causes soft rot diseases on a wide range of plants, including the model plant Arabidopsis thaliana. This bacterium proliferates in the host by secreting a set of pectin degrading enzymes responsible for symptom development. In addition, survival of this bacterium in planta requires two high-affinity iron acquisition systems mediated by siderophores and protective systems against oxidative damages, suggesting the implication by both partners of accurate mechanisms controlling their iron homeostasis under conditions of infection. In this review, we address this question and we show that ferritins both from the pathogen and the host are subtly implicated in the control of this interplay.

Siderophore-mediated upregulation of Arabidopsis ferritin expression in response to Erwinia chrysanthemi infection.

Ferritins are multimeric iron storage proteins encoded by a four-member gene family in Arabidopsis (AtFer1-4). To investigate whether iron sequestration in ferritins is a part of an iron-withholding defense system induced in response to bacterial invasion, we used Arabidopsis thaliana as a susceptible host for the pathogenic bacterium Erwinia chrysanthemi. In this study, we used a T-DNA insertion mutant line to show that the lack of a functional AtFer1 gene resulted in an enhanced susceptibility of Arabidopsis plants to E. chrysanthemi. We found that the AtFer1 gene is upregulated during infection, with a biphasic accumulation of the transcript at critical time points 0.5 and 24 h post-infection (p.i.). The activation of AtFer1 expression observed at 24 h p.i. was independent of the iron-dependent regulatory sequence (IDRS) known to mediate the transcriptional response of the AtFer1 gene to iron excess and to nitric oxide. Upregulation of AtFer1 gene expression was compromised after inoculation with an E. chrysanthemi siderophore null mutant. Infiltration of the purified siderophores chrysobactin and desferrioxamine strongly increased AtFer1 transcript abundance and it did not occur with the iron-loaded forms of these siderophores. We found that neither oxidative stress nor nitric oxide was involved in the plant response to chrysobactin. Our data show that ferritin accumulation during infection of Arabidopsis by E. chrysanthemi is a basal defense mechanism which is mainly activated by bacterial siderophores. The potential role of siderophores in this process is discussed.