Fine-Tuning of Arabidopsis thaliana Response to Endophytic Colonization by Gluconacetobacter diazotrophicus PAL5 Revealed by Transcriptomic Analysis.

Gluconacetobacter diazotrophicus is a diazotrophic endophytic bacterium that promotes the growth and development of several plant species. However, the molecular mechanisms activated during plant response to this bacterium remain unclear. Here, we used the RNA-seq approach to understand better the effect of G. diazotrophicus PAL5 on the transcriptome of shoot and root tissues of Arabidopsis thaliana. G. diazotrophicus colonized A. thaliana roots and promoted growth, increasing leaf area and biomass. The transcriptomic analysis revealed several differentially expressed genes (DEGs) between inoculated and non-inoculated plants in the shoot and root tissues. A higher number of DEGs were up-regulated in roots compared to shoots. Genes up-regulated in both shoot and root tissues were associated with nitrogen metabolism, production of glucosinolates and flavonoids, receptor kinases, and transcription factors. In contrast, the main groups of down-regulated genes were associated with pathogenesis-related proteins and heat-shock proteins in both shoot and root tissues. Genes encoding enzymes involved in cell wall biogenesis and modification were down-regulated in shoots and up-regulated in roots. In contrast, genes associated with ROS detoxification were up-regulated in shoots and down-regulated in roots. These results highlight the fine-tuning of the transcriptional regulation of A. thaliana in response to colonization by G. diazotrophicus PAL5.

Decoding the effects of drought stress on popcorn (Zea mays var. everta) flowering combining proteomics and physiological analysis.

Under conditions of soil water limitation and adequate irrigation, we conducted an investigation into the growth dynamics, gas exchange performance, and proteomic profiles of two inbred popcorn lines-L71, characterized as drought-tolerant, and L61, identified as drought-sensitive. Our goal was to uncover the mechanisms associated with tolerance to soil water limitation during the flowering. The plants were cultivated until grain filling in a substrate composed of perlite and peat within 150cm long lysimeter, subjected to two water conditions (WC): i) irrigated (WW) at lysimeter capacity (LC - 100%), and ii) water-stressed (WS). Under WS conditions, the plants gradually reached 45% of LC and were maintained at this level for 10 days. Irrespective of the WC, L71 exhibited the highest values of dry biomass in both shoot and root systems, signifying its status as the most robust genotype. The imposed water limitation led to early senescence, chlorophyll degradation, and increased anthocyanin levels, with a more pronounced impact observed in L61. Traits related to gas exchange manifested differences between the lines only under WS conditions. A total of 1838 proteins were identified, with 169 differentially accumulated proteins (DAPs) in the tolerant line and 386 DAPs in the sensitive line. Notably, differences in energy metabolism, photosynthesis, oxidative stress response, and protein synthesis pathways were identified as the key distinctions between L71 and L61. Consequently, our findings offer valuable insights into the alterations in proteomic profiles associated with the adaptation to soil water limitation in popcorn.

Essential role of extracytoplasmic proteins in the resistance of Gluconacetobacter diazotrophicus to cadmium.

Cadmium (Cd) is a heavy metal used as raw material for several fertilizers and pesticides. The increase of Cd concentration in soils has been observed in cultivated areas, affecting animals, plants, and microorganisms. Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium able to survive under adverse environmental conditions. Here, we investigated key mechanisms involved with the resistance of G. diazotrophicus to Cd. Proteomic analyses revealed that the main pathways regulated in response to Cd are nutrient uptake, multidrug efflux pumps, response to oxidative stress, and protein quality control system. Extracytoplasmic proteins related to multidrug efflux pumps were up-accumulated, while several proteins related to nutrients uptake were down-accumulated. The relevance of these pathways for bacterial resistance to Cd was investigated by reverse genetic analysis using mutants defective for nutrient uptake (tdbr, ompW, and oprB), multidrug efflux (czcC), response to oxidative stress (ggt), and protein quality control system (clpX). Our data demonstrated the essential role of the tdbr and czcC genes for resistance to Cd in G. diazotrophicus. These results contribute to a better understanding of the resistance mechanisms to Cd in G. diazotrophicus, shedding light on responses associated with extracytoplasmic compartments.

Revealing the differential protein profiles behind the nitrogen use efficiency in popcorn (Zea mays var. everta).

We investigated the proteomic profiles of two popcorn inbred lines, P2 (N-efficient and N-responsive) and L80 (N-inefficient and nonresponsive to N), under low (10% of N supply) and high (100% of N supply) nitrogen environments, associated with agronomic- and physiological-related traits to NUE. The comparative proteomic analysis allowed the identification of 79 differentially accumulated proteins (DAPs) in the comparison of high/low N for P2 and 96 DAPs in the comparison of high/low N for L80. The NUE and N uptake efficiency (NUpE) presented high means in P2 in comparison to L80 at both N levels, but the NUE, NUpE, and N utilization efficiency (NUtE) rates decreased in P2 under a high N supply. DAPs involved in energy and carbohydrate metabolism suggested that N regulates enzymes of alternative pathways to adapt to energy shortages and that fructose-bisphosphate aldolase may act as one of the key primary nitrate responsive proteins in P2. Proteins related to ascorbate biosynthesis and nitrogen metabolism increased their regulation in P2, and the interaction of L-ascorbate peroxidase and Fd-NiR may play an important role in the NUE trait. Taken together, our results provide new insights into the proteomic changes taking place in contrasting inbred lines, providing useful information on the genetic improvement of NUE in popcorn.

Mitochondrial proteomics reveals new insights into embryogenic competence acquisition in Carica papaya L. callus.

Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (EC) and nonembryogenic calli (NEC) of using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide (H2O2) contents. EC and NEC presented remarkable differences in anatomical and histochemical characteristics, with EC showing a higher reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate that mitochondrial metabolism affects the embryogenic competence of C. papaya callus. The EC presented higher participation of alternative oxidase (AOX) enzymes, higher total cell respiration and presented a stronger accumulation of mitochondrial stress response proteins. Differential accumulation of auxin-responsive Gretchen Hagen 3 (GH3) family proteins in EC was related to a decrease in the content of free 2,4-dichlorophenoxyacetic acid (2,4-D). EC also showed higher endogenous H2O2 contents. H2O2 is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos. SIGNIFICANCE: To further advance the understanding of somatic embryogenesis, we performed a broad characterization of embryogenic and nonembryogenic callus, through global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. Based on these results, we propose a working model for the competence of papaya callus. This model suggests that GH3 proteins play an important role in the regulation of auxins. In addition, embryogenic callus showed a greater abundance of stress response proteins and folding proteins. Embryogenic callus respiration occurs predominantly via AOX, and the inhibition of its activity is capable of inhibiting callus differentiation. Although the embryogenic callus presented greater total respiration and a greater abundance of oxidative phosphorylation proteins, they had less COX participation and less coupling efficiency, indicating less ATP production.

Integrative proteomics and phosphoproteomics reveals phosphorylation networks involved in the maintenance and expression of embryogenic competence in sugarcane callus.

Plant embryogenic cell culture allows mass propagation and genetic manipulation, but the mechanisms that determine the fate of these totipotent cells in somatic embryos have not yet been elucidated. Here, we performed label-free quantitative proteomics and phosphoproteomics analyses to determine signaling events related to sugarcane somatic embryo differentiation, especially those related to protein phosphorylation. Embryogenic calli were compared at multiplication (EC0, dedifferentiated cells) and after 14 days of maturation (EC14, onset of embryo differentiation). Metabolic pathway analysis showed enriched lysine degradation and starch/sucrose metabolism proteins during multiplication, whereas the differentiation of somatic embryos was found to involve the enrichment of energy metabolism, including the TCA cycle and oxidative phosphorylation. Multiplication-related phosphoproteins were associated with transcriptional regulation, including SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH). The regulation of multiple light harvesting complex photosystem II proteins and phytochrome interacting factor 3-LIKE 5 were predicted to promote bioenergetic metabolism and carbon fixation during the maturation stage. A motif analysis revealed 15 phosphorylation motifs. The [D-pS/T-x-D] motif was overrepresented during somatic embryo differentiation. A protein-protein network analysis predicted interactions among SNF1-related protein kinase 2 (SnRK2), abscisic acid-responsive element-binding factor 2 (ABF2), and KIN10, which indicated the role of these proteins in embryogenic competence. The predicted interactions between TOPLESS (TPL) and histone deacetylase 19 (HD19) may be involved in posttranslational protein regulation during somatic embryo differentiation. These results reveal the protein regulation dynamics of somatic embryogenesis and new players in somatic embryo differentiation, including their predicted phosphorylation motifs and phosphosites.

Combination of osmotic stress and sugar stress response mechanisms is essential for Gluconacetobacter diazotrophicus tolerance to high-sucrose environments.

Sugar-rich environments represent an important challenge for microorganisms. The osmotic and molecular imbalances resulting from this condition severely limit microbial metabolism and growth. Gluconacetobacter diazotrophicus is one of the most sugar-tolerant prokaryotes, able to grow in the presence of sucrose concentrations up to 30%. However, the mechanisms that control its tolerance to such conditions remain poorly exploited. The present work investigated the key mechanisms of tolerance to high sugar in G. diazotrophicus. Comparative proteomics was applied to investigate the main functional pathways regulated in G. diazotrophicus when cultivated in the presence of high sucrose. Among 191 proteins regulated by high sucrose, regulatory pathways related to sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system were highlighted. The role of these pathways on high-sucrose tolerance was investigated by mutagenesis analysis, which revealed that the knockout mutants zwf::Tn5 (sugar metabolism), tbdr::Tn5 (nutrient uptake), mtlK::Tn5 (compatible solute synthesis), pepN::Tn5 (proteolytic system), metH::Tn5 (amino acid metabolism), and ilvD::Tn5 (amino acid metabolism) became more sensitive to high sucrose. Together, our results identified mechanisms involved in response to high sugar in G. diazotrophicus, shedding light on the combination of osmotolerance and sugar-tolerance mechanisms. KEY POINTS: • G. diazotrophicus intensifies glycolysis to metabolize the excess of sugar. • G. diazotrophicus turns down the uptake of nutrients in response to high sugar. • G. diazotrophicus requires amino acid availability to resist high sugar.

DegP protease is essential for tolerance to salt stress in the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.

Comparative proteomics reveals essential mechanisms for osmotolerance in Gluconacetobacter diazotrophicus.

Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.

Arabidopsis thaliana exudates induce growth and proteomic changes in Gluconacetobacter diazotrophicus.

BACKGROUND: Plants interact with a variety of microorganisms during their life cycle, among which beneficial bacteria deserve special attention. Gluconacetobacter diazotrophicus is a beneficial bacterium able to fix nitrogen and promote plant growth. Despite its biotechnological potential, the mechanisms regulating the interaction between G. diazotrophicus and host plants remain unclear. METHODS: We analyzed the response of G. diazotrophicus to cocultivation with Arabidopsis thaliana seedlings. Bacterial growth in response to cocultivation and plant exudates was analyzed. Through comparative proteomic analysis, G. diazotrophicus proteins regulated during cocultivation were investigated. Finally, the role of some up-accumulated proteins in the response G. diazotrophicus to cocultivation was analyzed by reverse genetics, using insertion mutants. RESULTS: Our results revealed the induction of bacterial growth in response to cocultivation. Comparative proteomic analysis identified 450 bacterial proteins, with 39 up-accumulated, and 12 down-accumulated in response to cocultivation. Among the up-accumulated pathways, the metabolism of pentoses and protein synthesis were highlighted. Proteins potentially relevant to bacterial growth response such as ABC-F-Etta, ClpX, Zwf, MetE, AcnA, IlvC, and AccC were also increased. Reverse genetics analysis, using insertion mutants, revealed that the lack of ABC-F-Etta and AccC proteins severely affects G. diazotrophicus response to cocultivation. Our data demonstrated that specific mechanisms are activated in the bacterial response to plant exudates, indicating the essential role of "ribosomal activity" and "fatty acid biosynthesis" in such a process. This is the first study to demonstrate the participation of EttA and AccC proteins in plant-bacteria interactions, and open new perspectives for understanding the initial steps of such associations.

Limited Nitrogen and Plant Growth Stages Discriminate Well Nitrogen Use, Uptake and Utilization Efficiency in Popcorn.

The extensive use of nitrogen (N) in agriculture has caused negative impacts on the environment and costs. In this context, two pot experiments were performed under different N levels and harvested at different vegetative stages to assess two popcorn inbred lines (P2 and L80) and their hybrid (F1 = P2 × L80) for the N use, uptake and utilization efficiency (with the inclusion and exclusion of root N content); to find the contrasting N levels and vegetative stages that effect nitrogen use efficiency (NUE) and to understand the relationship between the traits related to NUE. The hybrid and P2 were confirmed better than L80 for all the studied traits. NUE is mainly affected by the shoot dry weight, uptake and utilization efficiency. Extremely low and high N levels were found to be more discriminating for N use and dry weight, respectively. At the V6 (six fully expanded leaf) stage, root N content (RNC) should be considered; in contrast, at the VT (tasseling stage) stage, RNC should not be considered for the uptake and utilization efficiency. The genetic parameter performance for N use, uptake, shoot dry weight and N content could favor the achievement of the genetic gain in advanced segregating generations.

Essential role of K+ uptake permease (Kup) for resistance to sucrose-induced stress in Gluconacetobacter diazotrophicus PAl 5.

Microorganisms are constantly challenged by stressful conditions, such as sugar-rich environments. Such environments can cause an imbalance of biochemical activities and compromise cell multiplication. Gluconacetobacter diazotrophicus PAl 5 is among the most sugar-tolerant bacteria, capable of growing in the presence of up to 876 mM sucrose. However, the molecular mechanisms involved in its response to high sucrose remain unknown. The present work aimed to identify sucrose-induced stress resistance genes in G. diazotrophicus PAl 5. Screening of a Tn5 transposon insertion library identified a mutant that was severely compromised in its resistance to high sucrose concentrations. Molecular characterization revealed that the mutation affected the kupA gene, which encodes a K+ uptake transporter (KupA). Functional complementation of the mutant with the wild type kupA gene recovered the sucrose-induced stress resistance phenotype. High sucrose resistance assay, under different potassium concentrations, revealed that KupA acts as a high-affinity K+ transporter, which is essential for resistance to sucrose-induced stress, when extracellular potassium levels are low. This study is the first to show the essential role of the KupA protein for resistance to sucrose-induced stress in bacteria by acting as a high-affinity potassium transporter in G. diazotrophicus PAl 5.

Correction: Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses.

[This corrects the article DOI: 10.1371/journal.pgen.1004936.].

Specific control of Arabidopsis BAK1/SERK4-regulated cell death by protein glycosylation.

Precise control of cell death is essential for the survival of all organisms. Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate cell death through elusive mechanisms. By deploying a genetic screen for suppressors of cell death triggered by virus-induced gene silencing of BAK1/SERK4 on Arabidopsis knockout collections, we identified STT3a, a protein involved in N-glycosylation modification, as an important regulator of bak1/serk4 cell death. Systematic investigation of glycosylation pathway and endoplasmic reticulum (ER) quality control (ERQC) components revealed distinct and overlapping mechanisms of cell death regulated by BAK1/SERK4 and their interacting protein BIR1. Genome-wide transcriptional analysis revealed the activation of members of cysteine-rich receptor-like kinase (CRK) genes in the bak1/serk4 mutant. Ectopic expression of CRK4 induced STT3a/N-glycosylation-dependent cell death in Arabidopsis and Nicotiana benthamiana. Therefore, N-glycosylation and specific ERQC components are essential to activate bak1/serk4 cell death, and CRK4 is likely to be among client proteins of protein glycosylation involved in BAK1/SERK4-regulated cell death.

Differential effects of salinity and osmotic stress on the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

Plant growth-promoting bacteria (PGPB) represent a promising alternative to the massive use of industrial fertilizers in agriculture. Gluconacetobacter diazotrophicus is a PGPB that colonizes several plant species. Although this bacterium is able to grow at high sucrose concentrations, its response to environmental stresses is poorly understood. The present study evaluated G. diazotrophicus PAL5 response to stresses caused by sucrose, PEG 400, NaCl, KCl, Na2SO4 and K2SO4. Morphological, ultrastructural and cell growth analysis revealed that G. diazotrophicus PAL5 is more sensitive to salt than osmotic stress. Growth inhibition and strong morphological changes were caused by salinity, in consequence of Cl ion-specific toxic effect. Interestingly, low osmotic stress levels were beneficial for bacterial multiplication, which was able to tolerate high sucrose concentrations, Na2SO4 and K2SO4. Our data show that G. diazotrophicus PAL5 has differential response to osmotic and salinity stress, which may influence its use as inoculant in saline environments.

Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis.

The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L(-1)) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L(-1) AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.

Protein poly(ADP-ribosyl)ation regulates arabidopsis immune gene expression and defense responses.

Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks.

Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity.

Perception of microbe-associated molecular patterns (MAMPs) elicits host transcriptional reprogramming as part of the immune response. Although pathogen perception is well studied, the signaling networks orchestrating immune gene expression remain less clear. In a genetic screen for components involved in the early immune gene transcription reprogramming, we identified Arabidopsis RNA polymerase II C-terminal domain (CTD) phosphatase-like 3 (CPL3) as a negative regulator of immune gene expression. MAMP perception induced rapid and transient cyclin-dependent kinase C (CDKC)-mediated phosphorylation of Arabidopsis CTD. The CDKCs, which are in turn phosphorylated and activated by a canonical MAP kinase (MAPK) cascade, represent a point of signaling convergence downstream of multiple immune receptors. CPL3 directly dephosphorylated CTD to counteract MAPK-mediated CDKC regulation. Thus, modulation of the phosphorylation dynamics of eukaryotic RNA polymerase II transcription machinery by MAPKs, CTD kinases, and phosphatases constitutes an essential mechanism for rapid orchestration of host immune gene expression and defense upon pathogen attacks.

Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.

BACKGROUND: Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD1, from Phaseolus vulgaris L. (common bean) seeds. RESULTS: The aim of this study was to overexpress PvD1 in a prokaryotic system, verify the biologic function of recombinant PvD1 (PvD1r) by comparing the antimicrobial activity of PvD1r to that of the natural defensin, PvD1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD1r in C. albicans cells. The cDNA encoding PvD1, which was previously obtained, was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform bacterial cells (Rosetta Gami 2 (DE3) pLysS) leading to recombinant protein expression. After expression had been induced, PvD1r was purified, cleaved with enterokinase and repurified by chromatographic steps. N-terminal amino acid sequencing showed that the overall process of the recombinant production of PvD1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD1r had a structure that was similar to the defensin isolated from plants. Purified PvD1 and PvD1r possessed inhibitory activity against the growth of the wild-type pathogenic yeast strain C. albicans. Both defensins, however, did not present inhibitory activity against the mutant strain of C. albicans. Antifungal assays with the wild-type C. albicans strains showed morphological changes upon observation by light microscopy following growth assays. PvD1r was coupled to FITC, and the subsequent treatment of wild type C. albicans with DAPI revealed that the labeled peptide was intracellularly localized. In the mutant strain, no intracellular labeling was detected. CONCLUSION: Our results indicate that PvD1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD1r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD1 in C. albicans cells is the sphingolipid glucosylceramide.

Bacteria isolated from roots and rhizosphere of Vitis vinifera retard water losses, induce abscisic acid accumulation and synthesis of defense-related terpenes in in vitro cultured grapevine.

Eleven bacterial strains were isolated at different soil depths from roots and rhizosphere of grapevines from a commercial vineyard. By 16S rRNA gene sequencing 10 different genera and 8 possible at species level were identified. From them, Bacillus licheniformis Rt4M10 and Pseudomonas fluorescens Rt6M10 were selected according to their characteristics as plant growth promoting rhizobacteria (PGPR). Both produced abscisic acid (ABA), indole-3-acetic acid (IAA) and the gibberellins A1 and A3 in chemically-defined medium. They also colonized roots of in vitro grown Vitis vinifera cv. Malbec plants. As result of bacterization ABA levels in 45 days-old in vitro plants were increased 76-fold by B. licheniformis and 40-fold by P. fluorescens as compared to controls. Both bacteria diminished plant water loss rate in correlation with increments of ABA. Twenty and 30 days post bacterization the plants incremented terpenes. The monoterpenes α-pinene, terpinolene, 4-carene, limonene, eucalyptol and lilac aldehyde A, and the sesquiterpenes α-bergamotene, α-farnesene, nerolidol and farnesol were assessed by gas chromatography-electron impact mass spectrometry analysis. α-Pinene and nerolidol were the most abundant (µg per g of tissue in plants bacterized with P. fluorescens). Only α-pinene, eucalyptol and farnesol were identified at low concentration in non-bacterized plants treated with ABA, while no terpenes were detected in controls. The results obtained along with others from literature suggest that B. licheniformis and P. fluorescens act as stress alleviators by inducing ABA synthesis so diminishing water losses. These bacteria also elicit synthesis of compounds of plant defense via an ABA independent mechanism.