A biostimulant yeast, Hanseniaspora opuntiae, modifies Arabidopsis thaliana root architecture and improves the plant defense response against Botrytis cinerea.

MAIN CONCLUSION: The biostimulant Hanseniaspora opuntiae regulates Arabidopsis thaliana root development and resistance to Botrytis cinerea. Beneficial microbes can increase plant nutrient accessibility and uptake, promote abiotic stress tolerance, and enhance disease resistance, while pathogenic microorganisms cause plant disease, affecting cellular homeostasis and leading to cell death in the most critical cases. Commonly, plants use specialized pattern recognition receptors to perceive beneficial or pathogen microorganisms. Although bacteria have been the most studied plant-associated beneficial microbes, the analysis of yeasts is receiving less attention. This study assessed the role of Hanseniaspora opuntiae, a fermentative yeast isolated from cacao musts, during Arabidopsis thaliana growth, development, and defense response to fungal pathogens. We evaluated the A. thaliana-H. opuntiae interaction using direct and indirect in vitro systems. Arabidopsis growth was significantly increased seven days post-inoculation with H. opuntiae during indirect interaction. Moreover, we observed that H. opuntiae cells had a strong auxin-like effect in A. thaliana root development during in vitro interaction. We show that 3-methyl-1-butanol and ethanol are the main volatile compounds produced by H. opuntiae. Subsequently, it was determined that A. thaliana plants inoculated with H. opuntiae have a long-lasting and systemic effect against Botrytis cinerea infection, but independently of auxin, ethylene, salicylic acid, or jasmonic acid pathways. Our results demonstrate that H. opuntiae is an important biostimulant that acts by regulating plant development and pathogen resistance through different hormone-related responses.

AtRAC7/ROP9 Small GTPase Regulates A. thaliana Immune Systems in Response to B. cinerea Infection.

Botrytis cinerea is a necrotrophic fungus that can cause gray mold in over 1400 plant species. Once it is detected by Arabidopsis thaliana, several defense responses are activated against this fungus. The proper activation of these defenses determines plant susceptibility or resistance. It has been proposed that the RAC/ROP small GTPases might serve as a molecular link in this process. In this study, we investigate the potential role of the Arabidopsis RAC7 gene during infection with B. cinerea. For that, we evaluated A. thaliana RAC7-OX lines, characterized by the overexpression of the RAC7 gene. Our results reveal that these RAC7-OX lines displayed increased susceptibility to B. cinerea infection, with enhanced fungal colonization and earlier lesion development. Additionally, they exhibited heightened sensitivity to bacterial infections caused by Pseudomonas syringae and Pectobacterium brasiliense. By characterizing plant canonical defense mechanisms and performing transcriptomic profiling, we determined that RAC7-OX lines impaired the plant transcriptomic response before and during B. cinerea infection. Global pathway analysis of differentially expressed genes suggested that RAC7 influences pathogen perception, cell wall homeostasis, signal transduction, and biosynthesis and response to hormones and antimicrobial compounds through actin filament modulation. Herein, we pointed out, for first time, the negative role of RAC7 small GTPase during A. thaliana-B. cinerea interaction.

The serine-arginine (SR) protein UmRrm75 from Ustilago maydis is a functional ortholog of yeast ScHrb1.

The Basidiomycete fungus Ustilago maydis is a biotrophic pathogen of maize. The U. maydis UmRrm75 gene encodes an RNA-binding protein (RBP). In a previous study, we reported that ΔUmRrm75 null mutant strains accumulate H2O2, exhibit slow growth, and have decreased virulence in maize. Herein, we describe UmRrm75 as an ortholog of the ScHrb1, a serine-arginine (SR) protein identified in the yeast Saccharomyces cerevisiae, which plays a role in nuclear quality control, specifically in mRNA splicing and export processes. The yeast ScHrb1 mutant (ΔScHrb1) exhibits an increased sensitivity to elevated levels of boron. We noticed that the ΔScHrb1 displayed sensitivity to H2O2, which is consistent with previous findings in the ΔUmRrm75 mutant. We reversed the sensitivity phenotypes of boron and H2O2 by introducing the UmRrm75 gene into the ΔScHrb1 mutant. Furthermore, we generated complementary strains of U. maydis by expressing UmRrm75-GFP under its native promoter in the ∆UmRrm75 mutants. The UmRrm75-GFP/∆UmRrm75 complementary strains successfully recovered their growth capability under stressors, H2O2 and boron, resembling the parental strains FB2 and AB33. The subcellular localization experiments conducted in U. maydis revealed that the UmRrm75 protein is localized within the nucleus of both yeast and hyphae. The nuclear localization of the UmRrm75 protein remains unaltered even under conditions of heat or oxidative stress. This suggests that UmRrm75 might perform its RBP activity in the nucleus, as previously reported for ScHrb1. Our data contribute to understanding the role of the nuclear RBP UmRrm75 from the corn smut fungus U. maydis.

LEAfing through literature: late embryogenesis abundant proteins coming of age-achievements and perspectives.

To deal with increasingly severe periods of dehydration related to global climate change, it becomes increasingly important to understand the complex strategies many organisms have developed to cope with dehydration and desiccation. While it is undisputed that late embryogenesis abundant (LEA) proteins play a key role in the tolerance of plants and many anhydrobiotic organisms to water limitation, the molecular mechanisms are not well understood. In this review, we summarize current knowledge of the physiological roles of LEA proteins and discuss their potential molecular functions. As these are ultimately linked to conformational changes in the presence of binding partners, post-translational modifications, or water deprivation, we provide a detailed summary of current knowledge on the structure-function relationship of LEA proteins, including their disordered state in solution, coil to helix transitions, self-assembly, and their recently discovered ability to undergo liquid-liquid phase separation. We point out the promising potential of LEA proteins in biotechnological and agronomic applications, and summarize recent advances. We identify the most relevant open questions and discuss major challenges in establishing a solid understanding of how these intriguing molecules accomplish their tasks as cellular sentinels at the limits of surviving water scarcity.

Defining novel plant polyamine oxidase subfamilies through molecular modeling and sequence analysis.

BACKGROUND: The polyamine oxidases (PAOs) catabolize the oxidative deamination of the polyamines (PAs) spermine (Spm) and spermidine (Spd). Most of the phylogenetic studies performed to analyze the plant PAO family took into account only a limited number and/or taxonomic representation of plant PAOs sequences. RESULTS: Here, we constructed a plant PAO protein sequence database and identified four subfamilies. Subfamily PAO back conversion 1 (PAObc1) was present on every lineage included in these analyses, suggesting that BC-type PAOs might play an important role in plants, despite its precise function is unknown. Subfamily PAObc2 was exclusively present in vascular plants, suggesting that t-Spm oxidase activity might play an important role in the development of the vascular system. The only terminal catabolism (TC) PAO subfamily (subfamily PAOtc) was lost in Superasterids but it was present in all other land plants. This indicated that the TC-type reactions are fundamental for land plants and that their function could being taken over by other enzymes in Superasterids. Subfamily PAObc3 was the result of a gene duplication event preceding Angiosperm diversification, followed by a gene extinction in Monocots. Differential conserved protein motifs were found for each subfamily of plant PAOs. The automatic assignment using these motifs was found to be comparable to the assignment by rough clustering performed on this work. CONCLUSIONS: The results presented in this work revealed that plant PAO family is bigger than previously conceived. Also, they delineate important background information for future specific structure-function and evolutionary investigations and lay a foundation for the deeper characterization of each plant PAO subfamily.

The cysteine-rich receptor-like protein kinase CRK28 modulates Arabidopsis growth and development and influences abscisic acid responses.

MAIN CONCLUSION: CRK28, a cysteine-rich receptor-like kinase, plays a role in root organogenesis and overall growth of plants and antagonizes abscisic acid response in seed germination and primary root growth. Receptor-like kinases (RLK) orchestrate development and adaptation to environmental changes in plants. One of the largest RLK groups comprises cysteine-rich receptor-like kinases (CRKs), for which the function of most members remains unknown. In this report, we show that the loss of function of CRK28 led to the formation of roots that are longer and more branched than the parental (Col-0) plantlets, and this correlates with an enhanced domain of the mitotic reporter CycB1:uidA in primary root meristems, whereas CRK28 overexpressing lines had the opposite phenotype, including slow root growth and reduced lateral root formation. Epidermal cell analyses revealed that crk28 mutants had reduced root hair length and increased trichome number, whereas 35S::CRK28 lines present primary roots with longer root hairs but lesser trichomes in leaves. The overall growth in soil of crk28 mutant and CRK28 overexpressing lines was reduced or enhanced, respectively, when compared to the parental (Col-0) seedlings, while germination, root growth and expression analyses of ABI3 and ABI5 further showed that CRK28 modulates ABA responses, which may be important to fine-tune plant morphogenesis. Our study unravels the participation of RLK signaling in root growth and epidermal cell differentiation.

Modification of AtGRDP1 gene expression affects silique and seed development in Arabidopsis thaliana.

Glycine Rich Proteins (GRPs) are induced at different developmental stages and in specific plant tissues. Recently, we described a novel Arabidopsis gene encoding a short glycine-rich domain protein (AtGRDP1). This gene is involved in abiotic stress responsiveness; the Atgrdp1-null mutant seeds were more sensitive to stress, while the opposite phenotype was achieved by AtGRDP1 overexpression. In this study, we analyzed the phenotype of the fruits produced by Arabidopsis Atgrdp1 mutants and 35S::AtGRDP1 overexpression lines. Our analyses revealed important changes in silique length, seed number, seed weight and morphology in the analyzed lines. In particular, Atgrdp1 mutant lines exhibited several defects including short siliques, a diminished number of seeds per silique, and a reduction in seed size and weight as compared to Col-0. The overexpression of the AtGRDP1 gene also generated phenotypes with alterations in size of silique, number of seeds per silique, and size and weight of the seed. In addition, the expression analysis of AtGRDP1 gene showed that it was expressed in floral and fruit organs, with the highest expression level in mature siliques. The alterations in the siliques and seeds traits in the Atgrdp1 mutant line, as well as the phenotypes observed in AtGRDP1 overexpression lines, suggest a role of the AtGRDP1 gene in the Arabidopsis fruit development.

Cladosporium psychrotolerans strain T01 enhances plant biomass and also exhibits antifungal activity against pathogens.

An increasing number of microorganisms are being identified to enhance plant growth and inhibit phytopathogens. Some Cladosporium species form beneficial associations with plants, either as endophytes or by colonizing the rhizosphere. Herein, we evaluated the influence of the Cladosporium psychrotolerans (T01 strain) fungus on the in vitro growth of Arabidopsis thaliana plantlets through direct and split interactions. After 9 days post-inoculation with C. psychrotolerans, Arabidopsis plantlets exhibited a notable increase in fresh weight and lateral roots, particularly in split interactions. Chlorophyll content increased in both plant-fungus interaction conditions, whereas the primary root was inhibited during direct interaction. We observed an increase in the GUS signal from the Arabidopsis auxin-inducible DR5:uidA marker in lateral root tips in both contact and split fungal interactions, and primary root tips in a split interaction. Arabidopsis and tomato plants cultivated in soil pots and inoculated with C. psychrotolerans (T01 strain) showed a positive effect on biomass production. GC/MS analysis detected that the T01 strain emitted volatile organic compounds (VOCs), predominantly alcohols and aldehydes. These VOCs displayed potent inhibitory effects, with a 60% inhibition against Botrytis cinerea and a 50% inhibition against C. gloeosporioides. Our study demonstrates that C. psychrotolerans T01 has the potential to enhance biomass production and inhibit pathogens, making it a promising candidate for green technology applications.

Effect of fungi and light on seed germination of three Opuntia species from semiarid lands of central Mexico.

Fungal attack under light reduces mechanical resistance of the testa of Opuntia seeds, making it easier for the embryo to emerge. However, the effect of fungi on Opuntia seed germination in darkness is unknown. We evaluated the combined effects of light and inoculation with Phoma medicaginis, Trichoderma harzianum, Trichoderma koningii, and Penicillium chrysogenum on germination of O. streptacantha, O. leucotricha, and O. robusta seeds, from central Mexico. We also evaluated the combined effects of seed age (2-, 3-, and 12-year-old seeds) and presence of fungi on the testa on O. streptacantha germination. All fungal species eroded the funicular envelope and promoted seed germination for O. leucotricha and O. streptacantha, but did more so in light than in darkness. For the latter species, younger seeds inoculated with fungi had lower germination than older ones. For O. robusta, we found that seeds inoculated with P. medicaginis and T. harzianum had similar germination in light and in darkness. Our results strongly indicate that deterioration of the testa by fungi is higher in light than in darkness.

Expression of EPL1 from Trichoderma atroviride in Arabidopsis Confers Resistance to Bacterial and Fungal Pathogens.

During plant interaction with beneficial microorganisms, fungi secrete a battery of elicitors that trigger plant defenses against pathogenic microorganisms. Among the elicitor molecules secreted by Trichoderma are cerato-platanin proteins, such as EPL1, from Trichoderma atroviride. In this study, Arabidopsis thaliana plants that express the TaEPL1 gene were challenged with phytopathogens to evaluate whether expression of EPL1 confers increased resistance to the bacterial pathogen Pseudomonas syringae and the necrotrophic fungus Botrytis cinerea. Infection assays showed that Arabidopsis EPL1-2, EPL1-3, EPL1-4 expressing lines were more resistant to both pathogens in comparison to WT plants. After Pseudomonas syringae infection, there were reduced disease symptoms (e.g., small chlorotic spots) and low bacterial titers in the three 35S::TaEPL1 expression lines. Similarly; 35S::TaEPL1 expression lines were more resistant to Botrytis cinerea infection, showing smaller lesion size in comparison to WT. Interestingly, an increase in ROS levels was detected in 35S::TaEPL1 expression lines when compared to WT. A higher expression of SA- and JA-response genes occurred in the 35S::TaEPL1 lines, which could explain the resistance of these EPL1 expression lines to both pathogens. We propose that EPL1 is an excellent elicitor, which can be used to generate crops with improved resistance to broad-spectrum diseases.

HR4 gene is induced in the Arabidopsis-Trichoderma atroviride beneficial interaction.

Plants are constantly exposed to microbes, for this reason they have evolved sophisticated strategies to perceive and identify biotic interactions. Thus, plants have large collections of so-called resistance (R) proteins that recognize specific microbe factors as signals of invasion. One of these proteins is codified by the Arabidopsis thaliana HR4 gene in the Col-0 ecotype that is homologous to RPW8 genes present in the Ms-0 ecotype. In this study, we investigated the expression patterns of the HR4 gene in Arabidopsis seedlings interacting with the beneficial fungus Trichoderma atroviride. We observed the induction of the HR4 gene mainly at 96 hpi when the fungus interaction was established. Furthermore, we found that the HR4 gene was differentially regulated in interactions with the beneficial bacterium Pseudomonas fluorescens and the pathogenic bacterium P. syringae. When hormone treatments were applied to A. thaliana (Col-0), each hormone treatment induced changes in HR4 gene expression. On the other hand, the expression of the RPW8.1 and RPW8.2 genes of Arabidopsis ecotype Ms-0 in interaction with T. atroviride was assessed. Interestingly, these genes are interaction-responsive; in particular, the RPW8.1 gene shows a very high level of expression in the later stages of interaction. These results indicate that HR4 and RPW8 genes could play a role in the establishment of Arabidopsis interactions with beneficial microbes.

The Opuntia streptacantha OpsHSP18 gene confers salt and osmotic stress tolerance in Arabidopsis thaliana.

Abiotic stress limits seed germination, plant growth, flowering and fruit quality, causing economic decrease. Small Heat Shock Proteins (sHSPs) are chaperons with roles in stress tolerance. Herein, we report the functional characterization of a cytosolic class CI sHSP (OpsHSP18) from Opuntia streptacantha during seed germination in Arabidopsis thaliana transgenic lines subjected to different stress and hormone treatments. The over-expression of the OpsHSP18 gene in A. thaliana increased the seed germination rate under salt (NaCl) and osmotic (glucose and mannitol) stress, and in ABA treatments, compared with WT. On the other hand, the over-expression of the OpsHSP18 gene enhanced tolerance to salt (150 mM NaCl) and osmotic (274 mM mannitol) stress in Arabidopsis seedlings treated during 14 and 21 days, respectively. These plants showed increased survival rates (52.00 and 73.33%, respectively) with respect to the WT (18.75 and 53.75%, respectively). Thus, our results show that OpsHSP18 gene might have an important role in abiotic stress tolerance, in particular in seed germination and survival rate of Arabidopsis plants under unfavorable conditions.

The entomopathogenic fungus Metarhizium anisopliae enhances Arabidopsis, tomato, and maize plant growth.

Species of the entomopathogenic fungi Metarhizium are used worldwide as biocontrol agents. Recently, other lifestyles have been associated with some Metarhizium species, which include their role as saprophytes, endophytes, and plant growth promoters. Herein, the effect of three Metarhizium anisopliae strains on the growth of Arabidopsis thaliana plantlets was evaluated using an in vitro split system. Arabidopsis fresh weight and total chlorophyll content significantly increased 7 days post-inoculation with the three Metarhizium anisopliae strains evaluated. The primary root length was promoted by all fungal strains without physical contact, whereas in direct contact primary root growth was inhibited. Volatile organic compounds identification revealed that during the interaction of Arabidopsis with Ma-20 and Ma-25 strains only ß-caryophyllene was produced, whereas in the Arabidopsis-Ma-28 interaction o-cymene was mainly emitted. The plant growth promoting effect induced by Metarhizium anisopliae strains was also achieved in Arabidopsis, tomato and maize plants grown in soil pots. Our results showed that three Metarhizium anisopliae strains were able to increase plant fresh weight, opening promising perspectives for field production, with the advantages of insect biocontrol and plant growth promotion induced by this species of fungus.

Marker-Free Rice (Oryza sativa L. cv. IR 64) Overexpressing PDH45 Gene Confers Salinity Tolerance by Maintaining Photosynthesis and Antioxidant Machinery.

Helicases function as key enzymes in salinity stress tolerance, and the role and function of PDH45 (pea DNA helicase 45) in stress tolerance have been reported in different crops with selectable markers, raising public and regulatory concerns. In the present study, we developed five lines of marker-free PDH45-overexpressing transgenic lines of rice (Oryza sativa L. cv. IR64). The overexpression of PDH45 driven by CaMV35S promoter in transgenic rice conferred high salinity (200 mM NaCl) tolerance in the T1 generation. Molecular attributes such as PCR, RT-PCR, and Southern and Western blot analyses confirmed stable integration and expression of the PDH45 gene in the PDH45-overexpressing lines. We observed higher endogenous levels of sugars (glucose and fructose) and hormones (GA, zeatin, and IAA) in the transgenic lines in comparison to control plants (empty vector (VC) and wild type (WT)) under salt treatments. Furthermore, photosynthetic characteristics such as net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 (Ci), and chlorophyll (Chl) content were significantly higher in transgenic lines under salinity stress as compared to control plants. However, the maximum primary photochemical efficiency of PSII, as an estimated from variable to maximum chlorophyll a fluorescence (Fv/Fm), was identical in the transgenics to that in the control plants. The activities of antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (GPX), were significantly higher in transgenic lines in comparison to control plants, which helped in keeping the oxidative stress burden (MDA and H2O2) lesser on transgenic lines, thus protecting the growth and photosynthetic efficiency of the plants. Overall, the present research reports the development of marker-free PDH45-overexpressing transgenic lines for salt tolerance that can potentially avoid public and biosafety concerns and facilitate the commercialization of genetically engineered crop plants.

An interactome analysis reveals that Arabidopsis thaliana GRDP2 interacts with proteins involved in post-transcriptional processes.

The Arabidopsis thaliana glycine-rich domain protein 2 (AtGRDP2) gene encodes a protein of unknown function that is involved in plant growth and salt stress tolerance. The AtGRDP2 protein (787 aa, At4g37900) is constituted by three domains: a DUF1399 located at the N-terminus, a potential RNA Recognition Motif (RRM) in the central region, and a short glycine-rich domain at the C-terminus. Herein, we analyzed the subcellular localization of AtGRDP2 protein as a GFP translational fusion and found it was localized in the cytosol and the nucleus of tobacco leaf cells. Truncated versions of AtGRDP2 showed that the DUF1399 or the RRM domains were sufficient for nuclear localization. In addition, we performed a yeast two-hybrid split-ubiquitin assay (Y2H) to identify potential interactors for AtGRDP2 protein. The Y2H assay identified proteins associated with RNA binding functions such as PABN3 (At5g65260), EF-1α (At1g07920), and CL15 (At3g25920). Heterodimeric associations in planta between AtGRDP2 and its interactors were carried out by Bimolecular Fluorescence Complementation (BiFC) assays. The data revealed heterodimeric interactions between AtGRDP2 and PABN3 in the nucleus and AtGRDP2 with EF-1α in the cytosol, while AtGRDP2-CL15 associations occurred only in the chloroplasts. Finally, functional characterization of the protein-protein interaction regions revealed that both DUF1399 and RRM domains were key for heterodimerization with its interactors. The AtGRDP2 interaction with these proteins in different compartments suggests that this glycine-rich domain protein is involved in post-transcriptional processes.

Arabidopsis adc-silenced line exhibits differential defense responses to Botrytis cinerea and Pseudomonas syringae infection.

During plant-microbe interactions, polyamines participate in the plant defense response. Previously, we reported that silencing of ADC genes in Arabidopsis thaliana causes a drastic reduction of polyamine levels as well as increments in reactive oxygen species content. In this study, we examined the response of the adc-silenced line to Botrytis cinerea and Pseudomonas syringae infection. The adc-silenced line was more susceptible to Botrytis cinerea, showing larger lesion length and a higher incidence of fungal infection. Pre-treatments with putrescine reestablished the response of the adc-silenced line to Botrytis cinerea, resulting in a similar phenotype to the parental plant. Expression levels of defense-related genes were analyzed during fungal infection showing that the salicylic acid-induced gene PR1 was up-regulated, while the jasmonic acid-related genes LOX3 and PDF1.2, as well as, the camalexin biosynthetic gene PAD3 were down-regulated in the adc-silenced line. Furthermore, methyl jasmonate pre-treatments reduced Botrytis cinerea infection in the adc-silenced line. On the other hand, the adc-silenced line showed an increased resistance to Pseudomonas syringae infection. SA-related genes such as PR1, ZAT1.2, WRKY54 and WRKY70 were highly expressed in the adc-silenced line upon bacterial interaction. Our data show that the adc-silenced line has altered the defense-response against Botrytis cinerea and Pseudomonas syringae, that is consistent with deregulation of SA- and JA-mediated response pathways.

Decrease of Arabidopsis PAO activity entails increased RBOH activity, ROS content and altered responses to Pseudomonas.

Polyamines (PAs) are small aliphatic amines with important regulatory activities in plants. Biotic stress results in changes in PA levels due to de novo synthesis and PA oxidation. In Arabidopsis thaliana five FAD-dependent polyamine oxidase enzymes (AtPAO1-5) participate in PA back-conversion and degradation. PAO activity generates H2O2, an important molecule involved in cell signaling, elongation, programmed cell death, and defense responses. In this work we analyzed the role of AtPAO genes in the Arabidopsis thaliana-Pseudomonas syringae pathosystem. AtPAO1 and AtPAO2 genes were transcriptionally up-regulated in infected plants. Atpao1-1 and Atpao2-1 single mutant lines displayed altered responses to Pseudomonas, and an increased susceptibility was found in the double mutant Atpao1-1 x Atpao2-1. These polyamine oxidases mutant lines showed disturbed contents of ROS (H2O2 and O2-) and altered activities of RBOH, CAT and SOD enzymes both in infected and control plants. In addition, changes in the expression levels of AtRBOHD, AtRBOHF, AtPRX33, and AtPRX34 genes were also noticed. Our data indicate an important role for polyamine oxidases in plant defense and ROS homeostasis.

PEST sequences from a cactus dehydrin regulate its proteolytic degradation.

Dehydrins (DHNs) are intrinsically disordered proteins expressed under cellular dehydration-related stresses. In this study, we identified potential proteolytic PEST sequences located at the central and C-terminal regions from the Opuntia streptacantha OpsDHN1 protein. In order to evaluate these PEST sequences as proteolytic tags, we generated a translational fusion with the GUS reporter protein and OpsDHN1 coding sequence. We found a GUS degradation effect in tobacco agro-infiltrated leaves and Arabidopsis transgenic lines that expressed the fusion GUS::OpsDHN1 full-length. Also, two additional translational fusions between OpsDHN1 protein fragments that include the central (GUS::PEST-1) or the C-terminal (GUS::PEST-2) PEST sequences were able to decrease the GUS activity, with PEST-2 showing the greatest reduction in GUS activity. GUS signal was abated when the OpsDHN1 fragment that includes both PEST sequences (GUS::PEST-1-2) were fused to GUS. Treatment with the MG132 proteasome inhibitor attenuated the PEST-mediated GUS degradation. Point mutations of phosphorylatable residues in PEST sequences reestablished GUS signal, hence these sequences are important during protein degradation. Finally, in silico analysis identified potential PEST sequences in other plant DHNs. This is the first study reporting presence of PEST motifs in dehydrins.

The Ustilago maydis null mutant strains of the RNA-binding protein UmRrm75 accumulate hydrogen peroxide and melanin.

Ustilago maydis is a dimorphic fungus that has emerged as a model organism for the study of fungal phytopathogenicity and RNA biology. In a previous study, we isolated the U. maydis UmRrm75 gene. The deletion of the UmRrm75 gene affected morphogenesis and pathogenicity. UmRrm75 gene encodes a protein containing three RNA recognition motifs. Here we determined that UmRrm75 has chaperone activity in Escherichia coli using the transcription anti-termination assay. Subsequently, we analyzed the growth of ΔUmRrm75 mutants at 15 °C and 37 °C, observing that mutant strains had reduced growth in comparison to parental strains. UmRrm75 gene expression was induced under these non-optimal temperatures. ΔUmRrm75 mutant colonies displayed a dark-brown color at 28 °C, which was confirmed to be melanin based on spectroscopic analysis and spectrometric data. Furthermore, ΔUmRrm75 mutant strains showed the presence of peroxisomes, and increased H2O2 levels, even at 28 °C. The ΔUmRrm75 mutant strains displayed a higher expression of redox-sensor UmYap1 gene and increased catalase activity than the parental strains. Our data show that deletion of the UmRrm75 gene results in higher levels of H2O2, increased melanin content, and abiotic stress sensitivity.

Polyamine metabolism in maize tumors induced by Ustilago maydis.

Alterations occurring in polyamine metabolism of maize in tumors formed during the interaction with the biotrophic pathogenic fungus Ustilago maydis were analyzed. During the process, a striking increase in maize polyamine biosynthesis, mainly free and conjugated putrescine occurred in the tumors induced by the fungus, and in the neighbor plant tissues. This increase correlated with an activation mainly of Adc, Samdc1, Zmsamdc2 and Zmsamdc3, but not of Zmodc, Zmspds1 and Zmspds2 genes, and an elevation in arginine decarboxylase activity, confirming a predominant role of this enzyme in the process. Evidences for a possible contribution of spermidine and spermine degradation by polyamine oxidase activity, probably related to cell wall stiffening or lignification during tumor growth, were also obtained. It is suggested that polyamines, mainly putrescine, might play an active role in the pathosystem maize-U. maydis.