Identification of Three Type II Toxin-Antitoxin Systems in Model Bacterial Plant Pathogen Dickeya dadantii 3937

Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB_2Dda, phd-doc_Dda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.     

Multivariate Optimization of the FLC-dc-APGD-Based Reaction-Discharge System for Continuous Production of a Plasma-Activated Liquid of Defined Physicochemical and Anti-Phytopathogenic Properties

To the present day, no efficient plant protection method against economically important bacterial phytopathogens from the Pectobacteriaceae family has been implemented into agricultural practice. In this view, we have performed a multivariate optimization of the operating parameters of the reaction-discharge system, employing direct current atmospheric pressure glow discharge, generated in contact with a flowing liquid cathode (FLC-dc-APGD), for the production of a plasma-activated liquid (PAL) of defined physicochemical and anti-phytopathogenic properties. As a result, the effect of the operating parameters on the conductivity of PAL acquired under these conditions was assessed. The revealed optimal operating conditions, under which the PAL of the highest conductivity was obtained, were as follows: flow rate of the solution equaled 2.0 mL min⁻¹, the discharge current was 30 mA, and the inorganic salt concentration (ammonium nitrate, NH₄NO₃) in the solution turned out to be 0.50% (m/w). The developed PAL exhibited bacteriostatic and bactericidal properties toward Dickeya solani IFB0099 and Pectobacterium atrosepticum IFB5103 strains, with minimal inhibitory and minimal bactericidal concentrations equaling 25%. After 24 h exposure to 25% PAL, 100% (1−2 × 10⁶) of D. solani and P. atrosepticum cells lost viability. We attributed the antibacterial properties of PAL to the presence of deeply penetrating, reactive oxygen and nitrogen species (RONS), which were, in this case, OH, O, O₃, H₂O₂, HO₂, NH, N₂, N₂⁺, NO₂⁻, NO₃⁻, and NH₄⁺. Putatively, the generated low-cost, eco-friendly, easy-to-store, and transport PAL, exhibiting the required antibacterial and physicochemical properties, may find numerous applications in the plant protection sector.     

The Arabidopsis Iron-Sulfur (Fe-S) Cluster Gene MFDX1 Plays a Role in Host and Nonhost Disease Resistance by Accumulation of Defense-Related Metabolites

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.     

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.     

Induction of Nickel Accumulation in Response to Zinc Deficiency in Arabidopsis thaliana

Excessive accumulation of nickel (Ni) can be toxic to plants. In Arabidopsis thaliana, the Fe²⁺ transporter, iron (Fe)-regulated transporter1 (IRT1), mediates Fe uptake and also implicates in Ni²⁺ uptake at roots; however, the underlying mechanism of Ni²⁺ uptake and accumulation remains unelucidated. In the present study, we found that zinc (Zn) deficient conditions resulted in increased accumulation of Ni in plants, particularly in roots, in A. thaliana. In order to elucidate the underlying mechanisms of Ni uptake correlating zinc condition, we traced ⁶³Ni isotope in response to Zn and found that (i) Zn deficiency induces short-term Ni²⁺ absorption and (ii) Zn²⁺ inhibits Ni²⁺ uptake, suggesting competitive uptake between Ni and Zn. Furthermore, the Zrt/Irt-like protein 3 (ZIP3)-defective mutant with an elevated Zn-deficient response exhibited higher Ni accumulation than the wild type, further supporting that the response to Zn deficiency induces Ni accumulation. Previously, expression profile study demonstrated that IRT1 expression is not inducible by Zn deficiency. In the present study, we found increased Ni accumulation in IRT1-null mutant under Zn deficiency in agar culture. These suggest that Zn deficiency induces Ni accumulation in an IRT1-independen manner. The present study revealed that Ni accumulation is inducible in response to Zn deficiency, which may be attributable to a Zn uptake transporter induced by Zn deficiency.     

CAND2/PMTR1 Is Required for Melatonin-Conferred Osmotic Stress Tolerance in Arabidopsis

Osmotic stress severely inhibits plant growth and development, causing huge loss of crop quality and quantity worldwide. Melatonin is an important signaling molecule that generally confers plant increased tolerance to various environmental stresses, however, whether and how melatonin participates in plant osmotic stress response remain elusive. Here, we report that melatonin enhances plant osmotic stress tolerance through increasing ROS-scavenging ability, and melatonin receptor CAND2 plays a key role in melatonin-mediated plant response to osmotic stress. Upon osmotic stress treatment, the expression of melatonin biosynthetic genes including SNAT1, COMT1, and ASMT1 and the accumulation of melatonin are increased in the wild-type plants. The snat1 mutant is defective in osmotic stress-induced melatonin accumulation and thus sensitive to osmotic stress, while exogenous melatonin enhances the tolerance of the wild-type plant and rescues the sensitivity of the snat1 mutant to osmotic stress by upregulating the expression and activity of catalase and superoxide dismutase to repress H₂O₂ accumulation. Further study showed that the melatonin receptor mutant cand2 exhibits reduced osmotic stress tolerance with increased ROS accumulation, but exogenous melatonin cannot revert its osmotic stress phenotype. Together, our study reveals that CADN2 functions necessarily in melatonin-conferred osmotic stress tolerance by activating ROS-scavenging ability in Arabidopsis.     

CsSWEET2, a Hexose Transporter from Cucumber (Cucumis sativus L.), Affects Sugar Metabolism and Improves Cold Tolerance in Arabidopsis

Sugars, which are critical osmotic compounds and signalling molecules in plants, and Sugars Will Eventually be Exported Transporters (SWEETs), which constitute a novel family of sugar transporters, play central roles in plant responses to multiple abiotic stresses. In the present study, a member of the SWEET gene family from cucumber (Cucumis sativus L.), CsSWEET2, was identified and characterized. Histochemical analysis of β-glucuronidase expression in transgenic Arabidopsis plants showed that CsSWEET2 is highly expressed in the leaves; subcellular localization indicated that CsSWEET2 proteins are localized in the plasma membrane and endoplasmic reticulum. Heterologous expression assays in yeast demonstrated that CsSWEET2 encodes an energy-independent hexose/H⁺ uniporter that can complement both glucose and fructose transport deficiencies. Compared with wild-type Arabidopsis plants, transgenic Arabidopsis plants overexpressing CsSWEET2 had much lower relative electrolyte leakage levels and were much more resistant to cold stress. Sugar content analysis showed that glucose and fructose levels in the transgenic Arabidopsis plants were significantly higher than those in the wild-type plants. Taken together, our results suggest that, by mediating sugar metabolism and compartmentation, CsSWEET2 plays a vital role in improving plant cold tolerance.     

A Novel DUF569 Gene Is a Positive Regulator of the Drought Stress Response in Arabidopsis

In the last two decades, global environmental change has increased abiotic stress on plants and severely affected crops. For example, drought stress is a serious abiotic stress that rapidly and substantially alters the morphological, physiological, and molecular responses of plants. In Arabidopsis, several drought-responsive genes have been identified; however, the underlying molecular mechanism of drought tolerance in plants remains largely unclear. Here, we report that the “domain of unknown function” novel gene DUF569 (AT1G69890) positively regulates drought stress in Arabidopsis. The Arabidopsis loss-of-function mutant atduf569 showed significant sensitivity to drought stress, i.e., severe wilting at the rosette-leaf stage after water was withheld for 3 days. Importantly, the mutant plant did not recover after rewatering, unlike wild-type (WT) plants. In addition, atduf569 plants showed significantly lower abscisic acid accumulation under optimal and drought-stress conditions, as well as significantly higher electrolyte leakage when compared with WT Col-0 plants. Spectrophotometric analyses also indicated a significantly lower accumulation of polyphenols, flavonoids, carotenoids, and chlorophylls in atduf569 mutant plants. Overall, our results suggest that novel DUF569 is a positive regulator of the response to drought in Arabidopsis.     

Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.     

A Novel Peroxidase CanPOD Gene of Pepper Is Involved in Defense Responses to Phytophtora capsici Infection as well as Abiotic Stress Tolerance

Peroxidases are involved in many plant processes including plant defense responses to biotic and abiotic stresses. We isolated a novel peroxidase gene CanPOD from leaves of pepper cultivar A3. The full-length gene has a 1353-bp cDNA sequence and contains an open reading frame (ORF) of 975-bp, which encodes a putative polypeptide of 324 amino acids with a theoretical protein size of 34.93 kDa. CanPOD showed diverse expression levels in different tissues of pepper plants. To evaluate the role of CanPOD in plant stress responses, the expression patterns of CanPOD were examined using Real-Time RT-PCR. The results indicated that CanPOD was significantly induced by Phytophtora capsici. Moreover, CanPOD was also up-regulated in leaves after salt and drought stress treatments. In addition, CanPOD expression was strongly induced by signaling hormones salicylic acid (SA). In contrast, CanPOD was not highly expressed after treatment with cold. Meanwhile, in order to further assess the role of gene CanPOD in defense response to P. capsici attack, we performed a loss-of-function experiment using the virus-induced gene silencing (VIGS) technique in pepper plants. In comparison to the control plant, the expression levels of CanPOD were obviously decreased in CanPOD-silenced pepper plants. Furthermore, we analyzed the effect of P. capsici on detached-leaves and found that the CanPOD-silenced plant leaves were highly susceptible to P. capsici infection. Taken together, our results suggested that CanPOD is involved in defense responses to P. capsici infection as well as abiotic stresses in pepper plants.     

Geomagnetic Field (GMF)-Dependent Modulation of Iron-Sulfur Interplay in Arabidopsis thaliana

The geomagnetic field (GMF) is an environmental factor affecting the mineral nutrient uptake of plants and a contributing factor for efficient iron (Fe) uptake in Arabidopsis seedlings. Understanding the mechanisms underlining the impact of the environment on nutrient homeostasis in plants requires disentangling the complex interactions occurring among nutrients. In this study we investigated the effect of GMF on the interplay between iron (Fe) and sulfur (S) by exposing Arabidopsis thaliana plants grown under single or combined Fe and S deficiency, to near-null magnetic field (NNMF) conditions. Mineral analysis was performed by ICP-MS and capillary electrophoresis, whereas the expression of several genes involved in Fe and S metabolism and transport was assayed by qRT-PCR. The results show that NNMF differentially affects (i) the expression of some Fe- and S-responsive genes and (ii) the concentration of metals in plants, when compared with GMF. In particular, we observed that Cu content alteration in plant roots depends on the simultaneous variation of nutrient availability (Fe and S) and MF intensity (GMF and NNMF). Under S deficiency, NNMF-exposed plants displayed variations of Cu uptake, as revealed by the expression of the SPL7 and miR408 genes, indicating that S availability is an important factor in maintaining Cu homeostasis under different MF intensities. Overall, our work suggests that the alteration of metal homeostasis induced by Fe and/or S deficiency in reduced GMF conditions impacts the ability of plants to grow and develop.     

ZmCIPK21, A Maize CBL-Interacting Kinase,Enhances Salt Stress Tolerance in Arabidopsis thaliana

Salt stress represents an increasing threat to crop growth and yield in saline soil. In this study, we identified a maize calcineurin B-like protein-interacting protein kinase (CIPK), ZmCIPK21, which was primarily localized in the cytoplasm and the nucleus of cells and displayed enhanced expression under salt stress. Over-expression of ZmCIPK21 in wild-type Arabidopsis plants increased their tolerance to salt, as supported by the longer root lengths and improved growth. The downstream stress-response genes, including dehydration-responsive element-binding (DREB) genes were also activated in transgenic plants over-expressing ZmCIPK21. In addition, introduction of the transgenic ZmCIPK21 gene into the Arabidopsis mutant cipk1-2 rescued the salt-sensitive phenotype under high salt stress. Measurement of Na⁺ and K⁺ content in transgenic plants showed that over-expression of ZmCIPK21 decreased accumulation of Na⁺ and allowed retention of relatively high levels of K⁺, thereby enhancing plant tolerance to salt conditions.     

Tomato Root Colonization by Exogenously Inoculated Arbuscular Mycorrhizal Fungi Induces Resistance against Root-Knot Nematodes in a Dose-Dependent Manner

Arbuscular mycorrhizal fungi (AMF) are generally recognized to induce plant growth and prime plants against soil-borne parasites, such as plant parasitic nematodes. However, the effectiveness of commercial formulates containing AMF has been questioned. Increasing amounts per plant of one commercial AMF-containing formulate, reported in the text as Myco, were used to detect the effects on growth of tomato plants and the resistance induced against root-knot nematodes (RKNs) The doses used per plant (0.5, 1.0, 2.0 g, reported as Myco1, Myco2, Myco3, respectively) were soil-drenched to growing potted plants; the effects of such treatments were analyzed both in plants not inoculated or inoculated by Meloidogyne incognita juveniles. Consistent increases in plant weight were apparent as soon as 7 days only after Myco2 treatments. Moreover, only treatments with Myco2 induced a consistent repression of the nematode infection observed in untreated plants. Conversely, treatments with Myco1 and Myco3 did not produce such an early growth improvement; some plant weight increase was observable only at 28 dpt. Accordingly, such Myco doses did not restrict the level of infestation observed in untreated plants. Control of infection was dependent on the dose of Myco provided to plants five days before nematode inoculation. About one month after all Myco treatments, several areas of roots were found to be colonized by AMF, although in Myco2-treated plants, three genes involved in the AMF colonization process (SlCCaMK, SlLYK9, and SlLYK13) were found to be over-expressed already at 7 dpt; over-expression was generally less consistent at 14 and 21 dpt. The expressions of two key genes of plant defense, the hypersensitive cell death inducer PR4b gene and the glutathione peroxidase-encoding GPX gene, were monitored in roots of Myco2-treated plants 3 and 7 days after nematode inoculation. PR4b was over-expressed and GPX was silenced in treated plants with respect to untreated plants. The repressive effect of Myco2 treatment against RKN infection was completely abolished when Myco2 suspensions were autoclaved to sterilization or treated with the potent anti-fungal agent amphotericin B, thus indicating that the biological control agents contained in the commercial formulate were living fungi.     

Heterogenicity within the LPS Structure in Relation to the Chosen Genomic and Physiological Features of the Plant Pathogen Pectobacterium parmentieri

Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of accessory virulence factors such as motility, siderophores, biofilm and lipopolysaccharide (LPS). LPS belongs to pathogen-associated molecular patterns (PAMPs) and plays an important role in plant colonization and interaction with the defense systems of the host. Therefore, we decided to investigate the heterogeneity of O-polysaccharides (OPS) of LPS of different strains of P. parmentieri, in search of an association between the selected genomic and phenotypic features of the strains that share an identical structure of the OPS molecule. In the current study, OPS were isolated from the LPS of two P. parmentieri strains obtained either in Finland in the 1980s (SCC3193) or in Poland in 2013 (IFB5432). The purified polysaccharides were analyzed by utilizing 1D and 2D NMR spectroscopy (¹H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) in addition to chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides and NMR spectroscopy data revealed that these two P. parmentieri strains isolated in different countries possess the same structure of OPS with a very rare residue of 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (pseudaminic acid) substituted in the position C-8: →3)-β-d-Galf-(1→3)-α-d-Galp-(1→8)-β-Pse4Ac5Ac7Ac-(2→6)-α-d-Glcp-(1→6)-β-d-Glcp-(1→. The previous study indicated that three other P. parmentieri strains, namely IFB5427, IFB5408 and IFB5443, exhibit a different OPS molecule than SCC3193 and IFB5432. The conducted biodiversity-oriented assays revealed that the P. parmentieri IFB5427 and IFB5408 strains possessing the same OPS structure yielded the highest genome-wide similarity, according to average nucleotide identity analyses, in addition to the greatest ability to macerate chicory tissue among the studied P. parmentieri strains. The current research demonstrated a novel OPS structure, characteristic of at least two P. parmentieri strains (SCC3193 and IFB5432), and discussed the observed heterogenicity in the OPS of P. parmentieri in a broad genomic and phenotype-related context.