Polyamine-mediated mechanisms contribute to oxidative stress tolerance in Pseudomonas syringae.

Bacterial phytopathogens living on the surface or within plant tissues may experience oxidative stress because of the triggered plant defense responses. Although it has been suggested that polyamines can defend bacteria from this stress, the mechanism behind this action is not entirely understood. In this study, we investigated the effects of oxidative stress on the polyamine homeostasis of the plant pathogen Pseudomonas syringae and the functions of these compounds in bacterial stress tolerance. We demonstrated that bacteria respond to H2O2 by increasing the external levels of the polyamine putrescine while maintaining the inner concentrations of this compound as well as the analogue amine spermidine. In line with this, adding exogenous putrescine to media increased bacterial tolerance to H2O2. Deletion of arginine decarboxylase (speA) and ornithine decarboxylate (speC), prevented the synthesis of putrescine and augmented susceptibility to H2O2, whereas targeting spermidine synthesis alone through deletion of spermidine synthase (speE) increased the level of extracellular putrescine and enhanced H2O2 tolerance. Further research demonstrated that the increased tolerance of the ΔspeE mutant correlated with higher expression of H2O2-degrading catalases and enhanced outer cell membrane stability. Thus, this work demonstrates previously unrecognized connections between bacterial defense mechanisms against oxidative stress and the polyamine metabolism.

Inferring the Significance of the Polyamine Metabolism in the Phytopathogenic Bacteria Pseudomonas syringae: A Meta-Analysis Approach.

To succeed in plant invasion, phytopathogenic bacteria rely on virulence mechanisms to subvert plant immunity and create favorable conditions for growth. This process requires a precise regulation in the production of important proteins and metabolites. Among them, the family of compounds known as polyamines have attracted considerable attention as they are involved in important cellular processes, but it is not known yet how phytopathogenic bacteria regulate polyamine homeostasis in the plant environment. In the present study, we performed a meta-analysis of publicly available transcriptomic data from experiments conducted on bacteria to begin delving into this topic and better understand the regulation of polyamine metabolism and its links to pathogenicity. We focused our research on Pseudomonas syringae, an important phytopathogen that causes disease in many economically valuable plant species. Our analysis discovered that polyamine synthesis, as well as general gene expression activation and energy production are induced in the early stages of the disease. On the contrary, synthesis of these compounds is inhibited whereas its transport is upregulated later in the process, which correlates with the induction of virulence genes and the metabolism of nitrogen and carboxylic acids. We also found that activation of plant defense mechanisms affects bacterial polyamine synthesis to some extent, which could reduce bacterial cell fitness in the plant environment. Furthermore, data suggest that a proper bacterial response to oxidative conditions requires a decrease in polyamine production. The implications of these findings are discussed.

MAMP-elicited changes in amino acid transport activity contribute to restricting bacterial growth.

Plants live under the constant challenge of microbes that probe the environment in search of potential hosts. Plant cells perceive microbe-associated molecular patterns (MAMPs) from incoming microbes and activate defense responses that suppress attempted infections. Despite the substantial progress made in understanding MAMP-triggered signaling pathways, the downstream mechanisms that suppress bacterial growth and disease remain poorly understood. Here, we uncover how MAMP perception in Arabidopsis (Arabidopsis thaliana) elicits dynamic changes in extracellular concentrations of free L-amino acids (AA). Within the first 3 h of MAMP perception, a fast and transient inhibition of AA uptake produces a transient increase in extracellular AA concentrations. Within 4 and 12 h of MAMP perception, a sustained enhanced uptake activity decreases the extracellular concentrations of AA. Gene expression analysis showed that salicylic acid-mediated signaling contributes to inducing the expression of AA/H+ symporters responsible for the MAMP-induced enhanced uptake. A screening of loss-of-function mutants identified the AA/H+ symporter lysin/histidine transporter-1 as an important contributor to MAMP-induced enhanced uptake of AA. Infection assays in lht1-1 seedlings revealed that high concentrations of extracellular AA promote bacterial growth in the absence of induced defense elicitation but contribute to suppressing bacterial growth upon MAMP perception. Overall, the data presented in this study reveal a mechanistic connection between MAMP-induced plant defense and suppression of bacterial growth through the modulation of AA transport activity.

Modulation of polyamine metabolism in Arabidopsis thaliana by salicylic acid.

Polyamines (PAs) play important roles in plant defense against pathogens, but the regulation of PA metabolism by hormone-mediated defense signaling pathways has not been studied in depth. In this study, the modulation of PA metabolism by salicylic acid (SA) was analyzed in Arabidopsis by combining the exogenous application of this hormone with PA biosynthesis and SA synthesis/signaling mutants. SA induced notable modifications of PA metabolism, mainly consisting in putrescine (Put) accumulation both in whole-plant extracts and apoplastic fluids. Put was accumulated at the expense of increased biosynthesis by ARGININE DECARBOXYLASE 2 and decreased oxidation by copper amine oxidase. Enhancement of Put levels by SA was independent of the regulatory protein NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and the signaling kinases MKK4 and MPK3, but depended on MPK6. However, plant infection by Pseudomonas syringae pv. tomato DC3000 elicited Put accumulation in an SA-dependent way. The present study demonstrates a clear connection between SA signaling and plant PA metabolism in Arabidopsis and contributes to understanding the mechanisms by which SA modulates PA levels during plant-pathogen interactions.

Mechanisms of plant protection against two oxalate-producing fungal pathogens by oxalotrophic strains of Stenotrophomonas spp.

KEY MESSAGE: Oxalotrophic Stenotrophomonas isolated from tomato rhizosphere are able to protect plants against oxalate-producing pathogens by a combination of actions including induction of plant defence signalling callose deposition and the strengthening of plant cell walls and probably the degradation of oxalic acid. Oxalic acid plays a pivotal role in the virulence of the necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum. In this work, we isolated two oxalotrophic strains (OxA and OxB) belonging to the bacterial genus Stenotrophomonas from the rhizosphere of tomato plants. Both strains were capable to colonise endophytically Arabidopsis plants and protect them from the damage caused by high doses of oxalic acid. Furthermore, OxA and OxB protected Arabidopsis from S. sclerotiorum and B. cinerea infections. Bacterial inoculation induced the production of phenolic compounds and the expression of PR-1. Besides, both isolates exerted a protective effect against fungal pathogens in Arabidopsis mutants affected in the synthesis pathway of salicylic acid (sid2-2) and jasmonate perception (coi1). Callose deposition induced by OxA and OxB was required for protection against phytopathogens. Moreover, B. cinerea and S. sclerotiorum mycelial growth was reduced in culture media containing cell wall polysaccharides from leaves inoculated with each bacterial strain. These findings suggest that cell walls from Arabidopsis leaves colonised by these bacteria would be less susceptible to pathogen attack. Our results indicate that these oxalotrophic bacteria can protect plants against oxalate-producing pathogens by a combination of actions and show their potential for use as biological control agents against fungal diseases.

A Bacterial Endophyte from Apoplast Fluids Protects Canola Plants from Different Phytopathogens via Antibiosis and Induction of Host Resistance.

Endophytic bacteria colonize inner plant tissues and thrive at the apoplast, which is considered its main reservoir. Because this niche is the place where the main molecular events take place between beneficial and pathogenic microorganisms, the aim of this work was to characterize culturable endophytic bacteria from apoplastic fluids obtained from field-grown canola leaves and analyze their potential for biological control of diseases caused by Xanthomonas campestris, Sclerotinia sclerotiorum, and Leptosphaeria maculans. Dual-culture analysis indicated that three isolates (Apo8, Apo11, and Apo12) were able to inhibit the growth of all three phytopathogens. Sequencing of the 16S ribosomal RNA and rpoD genes of these isolates revealed that they are closely related to Pseudomonas viridiflava. One of the isolates, Apo11, was able to diminish the propagation of X. campestris in whole-plant assays. At the same time, Apo11 inoculation reduced the necrotic lesions provoked by S. sclerotiorum on canola leaves. This protective effect might be due to the induction of resistance in the host mediated by salicylic and jasmonic acid signaling pathways or the production of compounds with antimicrobial activity. At the same time, Apo11 inoculation promoted canola plant growth. Thus, the isolate characterized in this work has several desirable characteristics, which make it a potential candidate for the formulation of biotechnological products to control plant diseases or promote plant growth.

Polyamine Metabolism Responses to Biotic and Abiotic Stress.

Plants have developed different strategies to cope with the environmental stresses they face during their life cycle. The responses triggered under these conditions are usually characterized by significant modifications in the metabolism of polyamines such as putrescine, spermidine, and spermine. Several works have demonstrated that a fine-tuned regulation of the enzymes involved in the biosynthesis and catabolism of polyamines leads to the increment in the concentration of these compounds. Polyamines exert different effects that could help plants to deal with stressful conditions. For instance, they interact with negatively charged macromolecules and regulate their functions, they may act as compatible osmolytes, or present antimicrobial activity against plant pathogens. In addition, they have also been proven to act as regulators of gene expression during the elicitation of stress responses. In this chapter, we reviewed the information available till date in relation to the roles played by polyamines in the responses of plants during biotic and abiotic stress.

Phenotypic and Genotypic Characterization of Mutant Plants in Polyamine Metabolism Genes During Pathogenic Interactions.

Plants respond to pathogen attack by modifying defense gene expression and inducing the production of myriad proteins and metabolites. Among these responses, polyamine (PA) levels suffer remarkable modifications. Evidences demonstrate that plants make use of the polyamine biosynthetic pathway and the oxidative catabolism of these compounds in order to mount adequate defenses against pathogens. In Arabidopsis thaliana, putrescine is synthesized exclusively through the arginine decarboxylase (ADC) pathway, this enzyme exists as two isoforms named ADC1 and ADC2. Even though both isoforms participate in the response to pathogen attack, the mechanisms modulating ADC activity are not completely understood. Therefore, studies to clarify their roles are necessary. In this chapter, we describe the methods that can be applied for the study of plant-pathogen interactions using Arabidopsis adc mutant plants.

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea.

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense-related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid-targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild-type (WT) counterparts. ROS build-up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70-80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis-related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast-generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.

Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.

Overexpression of the carbohydrate binding module of strawberry expansin2 in Arabidopsis thaliana modifies plant growth and cell wall metabolism.

Several cell wall enzymes are carbohydrate active enzymes that contain a putative Carbohydrate Binding Module (CBM) in their structures. The main function of these non-catalitic modules is to facilitate the interaction between the enzyme and its substrate. Expansins are non-hydrolytic proteins present in the cell wall, and their structure includes a CBM in the C-terminal that bind to cell wall polymers such as cellulose, hemicelluloses and pectins. We studied the ability of the Expansin2 CBM (CBMFaEXP2) from strawberry (Fragaria x ananassa, Duch) to modify the cell wall of Arabidopsis thaliana. Plants overexpressing CBMFaEXP2 were characterized phenotypically and biochemically. Transgenic plants were taller than wild type, possibly owing to a faster growth of the main stem. Cell walls of CBMFaEXP2-expressing plants were thicker and contained higher amount of pectins. Lower activity of a set of enzymes involved in cell wall degradation (PG, ß-Gal, ß-Xyl) was found, and the expression of the corresponding genes (AtPG, Atß-Gal, Atß-Xyl5) was reduced also. In addition, a decrease in the expression of two A. thaliana Expansin genes (AtEXP5 and AtEXP8) was observed. Transgenic plants were more resistant to Botrytis cinerea infection than wild type, possibly as a consequence of higher cell wall integrity. Our results support the hypothesis that the overexpression of a putative CBM is able to modify plant cell wall structure leading to modulation of wall loosening and plant growth. These findings might offer a tool to controlling physiological processes where cell wall disassembly is relevant, such as fruit softening.

Physiological and molecular implications of plant polyamine metabolism during biotic interactions.

During ontogeny, plants interact with a wide variety of microorganisms. The association with mutualistic microbes results in benefits for the plant. By contrast, pathogens may cause a remarkable impairment of plant growth and development. Both types of plant-microbe interactions provoke notable changes in the polyamine (PA) metabolism of the host and/or the microbe, being each interaction a complex and dynamic process. It has been well documented that the levels of free and conjugated PAs undergo profound changes in plant tissues during the interaction with microorganisms. In general, this is correlated with a precise and coordinated regulation of PA biosynthetic and catabolic enzymes. Interestingly, some evidence suggests that the relative importance of these metabolic pathways may depend on the nature of the microorganism, a concept that stems from the fact that these amines mediate the activation of plant defense mechanisms. This effect is mediated mostly through PA oxidation, even though part of the response is activated by non-oxidized PAs. In the last years, a great deal of effort has been devoted to profile plant gene expression following microorganism recognition. In addition, the phenotypes of transgenic and mutant plants in PA metabolism genes have been assessed. In this review, we integrate the current knowledge on this field and analyze the possible roles of these amines during the interaction of plants with microbes.