Generation of Transfer-DNA-Free Base-Edited Citrus Plants.

To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase (ALS) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.

Improvement of Antioxidant Properties in Fruit from Two Blood and Blond Orange Cultivars by Postharvest Storage at Low Temperature.

Numerous studies have revealed the remarkable health-promoting activities of citrus fruits, all of them related to the accumulation of bioactive compounds, including vitamins and phytonutrients. Anthocyanins are characteristic flavonoids present in blood orange, which require low-temperature for their production. Storage at low-temperature of blood oranges has been proven to be a feasible postharvest strategy to increase anthocyanins in those countries with warm climates. To our knowledge, no studies comparing the effect of postharvest storage effect on phenylpropanoid accumulation in cultivars with and without anthocyanins production have been published. We have investigated the effect of postharvest cold storage in flavonoid accumulation in juice from Citrus sinensis L. Osbeck in two different oranges: Pera, a blond cultivar, and Moro, a blood one. Our findings indicate a different response to low-temperature of fruit from both cultivars at biochemical and molecular levels. Little changes were observed in Pera before and after storage, while a higher production of phenylpropanoids (3.3-fold higher) and flavonoids (1.4-fold higher), including a rise in anthocyanins from 1.3 ± 0.7 mg/L to 60.0 ± 9.4 mg/L was observed in Moro concurrent with an upregulation of the biosynthetic genes across the biosynthetic pathway. We show that postharvest storage enhances not only anthocyanins but also other flavonoids accumulation in blood oranges (but not in blond ones), further stimulating the interest in blood orange types in antioxidant-rich diets.

Cultural Management of Huanglongbing: Current Status and Ongoing Research.

Huanglongbing (HLB), formerly known as greening, is a bacterial disease restricted to some Asian and African regions until two decades ago. Nowadays, associated bacteria and their vectors have spread to almost all citrus-producing regions, and it is currently considered the most devastating citrus disease. HLB management can be approached in terms of prevention, limiting or avoiding pathogen and associated vectors to reach an area, or in terms of control, trying to reduce the impact of the disease by adopting different cultural strategies depending on infestation/infection levels. In both cases, control of psyllid populations is currently the best way to stop HLB spread. Best cultural actions (CHMAs, TPS system) to attain this goal and, thus, able to limit HLB spread, and ongoing research in this regard is summarized in this review.

Engineering of citrus to obtain huanglongbing resistance.

Huanglongbing (HLB) disease is threatening the sustainability of citriculture in affected regions because of its rapid spread and the severity of the symptoms it induces. Herein, we summarise the main research findings that can be exploited to develop HLB-resistant cultivars. A major bottleneck has been the lack of a system for the ex vivo cultivation of HLB-associated bacteria (CLs) in true plant hosts, which precludes the evaluation of target genes/metabolites in reliable plant/pathogen/vector environments. With regard to HLB vectors, several biotechnologies which have been proven in laboratory settings to be effective for insect control are presented. Finally, new genotypes that are resistant to CLs or their insect vectors are described, and the most relevant strategies for fighting HLB are highlighted.

Curing and low-temperature combined post-harvest storage enhances anthocyanin biosynthesis in blood oranges.

Anthocyanins are pigments present in blood oranges which can be enriched by post-harvest cold storage. Additionally, citrus fruits contain appreciable levels of other flavonoids, whose content increases under post-harvest heat treatments. Here, we investigated the effects of curing (37 °C for 3 days) and storage at low-temperature (9 °C) during 15, 30 and 45 days on accumulation of anthocyanins and other flavonoids in Moro and Sanguinelli Polidori blood oranges (Citrus sinensis L. Osbeck). Cured fruits reached up to 191.4 ± 1.4 mg/L of anthocyanins in their juice after cold storage and a 3-fold enrichment of other flavonoids such as flavones and flavanones, compared to 85.7 ± 3.3 mg/L anthocyanins from fruits with cold storage alone. Concomitantly, qPCR analysis showed that curing enhanced upregulation of the main structural and transcription factor genes regulating the flavonoid pathway. GC-MS analysis showed that no unpleasant compounds were generated in the cured plus cold-stored juice volatilome.

[Reactivity to antigens of the microbiome of the respiratory tract in patients with respiratory allergic diseases]. / Reactividad a antígenos del microbioma de vías respiratorias en pacientes con enfermedad alérgica respiratoria.

BACKGROUND: The prevalence of allergic diseases has increased worldwide. Recent studies have informed that the dysbiosis of some specific members of the human microbiota may enhance the allergic response of the respiratory tract. OBJECTIVE: To retrospectively explore the role of some microorganisms of the human microbiota on the skin reactivity and their effect on the chronicity of allergic respiratory diseases in humans. METHODS: A retrospective analysis of a 5-year database of patients with allergic respiratory tract disease. The frequency and magnitude of the reactivity to 38 different allergens was determined. RESULTS: Dermatophagoides pteronyssinus had the highest frequency of reactivity (93.7 %), followed by the bacterial allergen (a mixture of Staphylococcus aureus and Staphylococcus epidermidis) with a frequency of reactivity of 91.82 %; whereas Candida albicans had a frequency of reactivity of only 79.32 %. The frequency of reactivity to the pollen of native Mexican weeds was even lower ~79 %. CONCLUSION: The microorganisms of the microbiota that were analyzed in this study seem to have an influence on the development of respiratory allergic inflammation, associated with long-term colonization of the pharynx, nasal mucosa, and sinuses because of these microorganisms.

Antecedentes: La prevalencia de las enfermedades alérgicas ha aumentado en todo el mundo. En estudios recientes se ha informado que la disbiosis de algunos miembros específicos de la microbiota humana puede potenciar la respuesta alérgica de las vías respiratorias. Objetivo: Explorar retrospectivamente el papel de algunos microorganismos de la microbiota humana en la reactividad cutánea y su efecto sobre la cronicidad de las enfermedades alérgicas respiratorias en el humano. Métodos: Análisis retrospectivo de la base de datos de un periodo de cinco años de pacientes con enfermedad alérgica de las vías respiratorias. Se determinó la frecuencia y magnitud de la reactividad a 38 alérgenos diferentes. Resultados: La mayor frecuencia de reactividad la presentó Dermatophagoides pteronyssinus (93.7 %), al que le siguió una combinación bacteriana de Staphylococcus aureus-Staphylococcus epidermidis (91.82 %) y Candida albicans (79.32 %). La reactividad a alérgenos de polen de malezas nativas de México fue aun menor, aproximadamente de 79 %. Conclusión: Los microorganismos de la microbiota analizados en este estudio parecen tener una influencia en el desarrollo de la inflamación alérgica respiratoria, asociada a la colonización a largo plazo de la faringe, la mucosa nasal y los senos paranasales.

Pasteurized Orange Juice Rich in Carotenoids Protects Caenorhabditis elegans against Oxidative Stress and ß-Amyloid Toxicity through Direct and Indirect Mechanisms.

'Cara Cara' is a red orange (Citrus sinensis (L.) Osbeck) variety originally from Venezuela characterized by a significantly higher and diversified carotenoid content including higher-concentration lycopene, all-E-ß-carotene, phytoene, and other carotenoids when compared with the carotenoid profile of its isogenic blond counterpart 'Bahia', also known as Washington navel. The exceptionally high carotenoid content of 'Cara Cara' is of special interest due to its neuroprotective potential. Here, we used the nematode Caenorhabditis elegans to analyze the antioxidant effect and the protection against ß-amyloid-induced toxicity of pasteurized orange juice (POJ) obtained from 'Cara Cara' and compare to that from 'Bahia'. POJ treatment reduced the endogenous ROS levels and increased the worm's survival rate under normal and oxidative stress conditions. POJ treatment also upregulated the expression of antioxidant (gcs-1, gst-4, and sod-3) and chaperonin (hsp-16.2) genes. Remarkably, ROS reduction, gene expression activation, oxidative stress resistance, and longevity extension were significantly increased in the animals treated with 'Cara Cara' orange juice compared to animals treated with 'Bahia' orange juice. Furthermore, the body paralysis induced by ß-amyloid peptide was delayed by both POJs but the mean paralysis time for the worms treated with 'Cara Cara' orange juice was significantly higher compared to 'Bahia' orange juice. Our mechanistic studies indicated that POJ-reduced ROS levels are primarily a result of the direct scavenging action of natural compounds available in the orange juice. Moreover, POJ-induced gst-4::GFP expression and -increased stress resistance was dependent of the SKN-1/Nrf2 transcription factor. Finally, the transcription factors SKN-1, DAF-16, and HSF-1 were required for the POJ-mediated protective effect against Aß toxicity. Collectively, these results suggest that orange juice from 'Cara Cara' induced a stronger response against oxidative stress and ß-amyloid toxicity compared to orange juice from 'Bahia' possibly due to its higher carotenoid content.

Structural characterisation of pectin obtained from cacao pod husk. Comparison of conventional and subcritical water extraction.

Pectin was obtained with citric acid and subcritical water extraction from cacao pod husk with or without a previous step consisting of a supercritical fluid extraction of phenols. By subcritical conditions a higher yield (10.9%) was attained in a time 3-fold shorter than that obtained by conventional extraction (˜8%) and a greater effectiveness in the recovery of pectin with higher molecular weight (750 kDa) was also found. Regarding pectin structure, galacturonic acid and degree of methyl esterification content were similar (˜55 and ˜36%, respectively) in both methods. Moreover, pectin recovered by citric acid presented 2-fold higher amount of impurities as compared to subcritical water extraction. Hardly any effects of a previous supercritical treatment were observed in the structure and composition of pectin, indicating the efficiency of the integrated supercritical carbon dioxide and subcritical water extraction as green processes for the obtainment of phenol and pectin from cacao pod husk.

Anthocyanin biosynthesis and accumulation in blood oranges during postharvest storage at different low temperatures.

Blood oranges require low temperature for anthocyanin production. We have investigated the activation of anthocyanin biosynthesis and accumulation in the pulp of Moro blood and Pera blond oranges (Citrus sinensis L. Osbeck) stored at either 4 or 9°C after harvesting. Both temperatures stimulated anthocyanin accumulation in blood but not in blond oranges. Nonetheless, blood orange fruits stored at 9°C reached a darker purple coloration, higher anthocyanin contents and enhanced upregulation of genes from the flavonoid pathway in the pulp and juice than those kept at 4°C. Our results indicated that dihydroflavonol channeling toward anthocyanin production was boosted during the storage at 9°C compared to 4°C, providing more leucoanthocyanidins to enzymes downstream in the pathway. Finally, despite both low temperatures stimulated the expression of key transcription factors likely regulating the pathway, their expression profiles could not explain the differences observed at 9 and 4°C.

Effect of microwave drying and oven drying on the water activity, color, phenolic compounds content and antioxidant activity of coconut husk (Cocos nucifera L.).

The coconut (Cocos nucifera L.) husk is basically composed by fiber and pith material and remained under-utilized. This is an important source of phenolic compounds that could be used as functional ingredients. The aim of this study was to determine the effect of: oven-drying (OD) and microwave drying (MD), on the water activity, color, phenolic compound content and antioxidant activity of coconut husk. The OD was performed at 60 °C for 12 h and MD was performed at 900 W for 10 min. The total phenolic content (TPC) in fresh coconut husk was 64.2 mg GAE/g dry wt and significant higher than observed after OD and MD of 35.8 and 45.5 mg GAE/g dry wt, respectively. Ten phenols were identified in fresh and dehydrated coconut husks. The husk MD showed an increase in the content of gallic, 4-hydroxybenzoic, ferulic and syringic acids and epicatechin compared with the fresh; while coconut husk OD and MD, showed a decrease in the content of vanillic acid, vanillin, catequin and kaempferol. The antioxidant activity decreased after both OD and MD. However, MD resulted in a better antioxidant activity in husk than OD. MD of husk resulted into better retention of preserved color, TPC and TFC than OD.

Comparative proteome analysis of Brettanomyces bruxellensisunder hydroxycinnamic acid growth

Background: Brettanomyces bruxellensis is an important spoilage yeast in the winemaking process. The capacity of this yeast to generate an undesired off-flavor constitutes a significant loss in the Chilean wine industry. Results: The proteomic profile of B. bruxellensis in the presence of p-coumaric acid was determined by 2D gel electrophoresis, gel image analysis and differential spot selection. A set of 41 proteins showed a differential accumulation of ±2 and a p-value <0.0001. The homology sequence analysis was performed using the databases available. Differential proteins belonged to the categories of 'energy production and conversion' and 'amino acid transport and metabolism'. Conclusions: The proteomic profile of B. bruxellensis cultivated in the presence of p-coumaric acid in synthetic wine, agrees with the hypothesis of metabolic flux regulation, allowing a better conditioning to an adverse environment. This study involved the translational level of B. bruxellensis in the production of ethylphenols and corroborated that this yeast presented an advantage in these stress conditions. Thus, this work will allow an understanding of the regulation and processes involved in the production of ethyl-derivate compounds by B. bruxellensis. Furthermore, it allows the development of newer and better techniques for spoilage yeast control.

Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus.

Nitric oxide (NO) is a signalling molecule involved in many biological processes in bacteria, plants and mammals. However, little is known about the role and biosynthesis of NO in fungi. Here we show that NO production is increased at the early stages of the transition from vegetative growth to development in Aspergillus nidulans. Full NO production requires a functional nitrate reductase (NR) gene (niaD) that is upregulated upon induction of conidiation, even under N-repressing conditions in the presence of ammonium. At this stage, NO homeostasis is achieved by balancing biosynthesis (NR) and catabolism (flavohaemoglobins). niaD and flavohaemoglobin fhbA are transiently upregulated upon induction of conidiation, and both regulators AreA and NirA are necessary for this transcriptional response. The second flavohaemoglobin gene fhbB shows a different expression profile being moderately expressed during the early stages of the transition phase from vegetative growth to conidiation, but it is strongly induced 24 h later. NO levels influence the balance between conidiation and sexual reproduction because artificial strong elevation of NO levels reduced conidiation and induced the formation of cleistothecia. The nitrate-independent and nitrogen metabolite repression-insensitive transcriptional upregulation of niaD during conidiation suggests a novel role for NR in linking metabolism and development.

Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides.

There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 µM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories.

The Penicillium digitatum protein O-mannosyltransferase Pmt2 is required for cell wall integrity, conidiogenesis, virulence and sensitivity to the antifungal peptide PAF26.

The activity of protein O-mannosyltransferases (Pmts) affects the morphogenesis and virulence of fungal pathogens. Recently, PMT genes have been shown to determine the sensitivity of Saccharomyces cerevisiae to the antifungal peptide PAF26. This study reports the identification and characterization of the three Pdpmt genes in the citrus post-harvest pathogen Penicillium digitatum. The Pdpmt genes are expressed during fungal growth and fruit infection, with the highest induction for Pdpmt2. Pdpmt2 complemented the growth defect of the S. cerevisiae Δpmt2 strain. The Pdpmt2 gene mutation in P. digitatum caused pleiotropic effects, including a reduction in fungal growth and virulence, whereas its constitutive expression had no phenotypic effect. The Pdpmt2 null mutants also showed a distinctive colourless phenotype with a strong reduction in the number of conidia, which was associated with severe alterations in the development of conidiophores. Additional effects of the Pdpmt2 mutation were hyphal morphological alterations, increased sensitivity to cell wall-interfering compounds and a blockage of invasive growth. In contrast, the Pdpmt2 mutation increased tolerance to oxidative stress and to the antifungal activity of PAF26. These data confirm the role of protein O-glycosylation in the PAF26-mediated antifungal mechanism present in distantly related fungal species. Important to future crop protection strategies, this study demonstrates that a mutation rendering fungi more resistant to an antifungal peptide results in severe deleterious effects on fungal growth and virulence.

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity.

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.

A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments.

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ß-citraurin (3-hydroxy-ß-apo-8'-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and Mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ß-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ß-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ß-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7',8' double bond in zeaxanthin and ß-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7',8' double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.

Genes involved in protein glycosylation determine the activity and cell internalization of the antifungal peptide PAF26 in Saccharomyces cerevisiae.

We have previously characterized the synthetic hexapeptide PAF26 as a cell-penetrating and non-lytic antifungal peptide that is active against Saccharomyces cerevisiae and filamentous fungi. Numerous cell wall (CW) proteins are glycosylated in fungi and many of these play important roles in fungal pathogenesis. In this study, we screened a collection of S. cerevisiae deletion mutants for protein glycosylation genes whose deletion altered the sensitivity to PAF26. Increased tolerance to PAF26 was observed in mutants with the following disrupted genes: PMT1-6, EOS1, ALG5, MNN1, MNN4 and MNN5. Significantly, genes coding for protein O-mannosyltransferase 2 (Pmt2p), which is responsible for the addition of the first mannosyl residue of O-linked carbohydrates, and for Eos1p, an enzyme involved in N-linked glycosylation of proteins, showed resistance to PAF26 and defects in CW integrity. Microscopic studies on the S. cerevisiae Δeos1 deletion mutant demonstrated a blockage of peptide internalization by cells. Protoplasts lacking CWs interacted with the peptide, but were more resistant to peptide killing than cells possessing CWs due to a blockage in PAF26 internalization. Interestingly, protoplasts obtained from Δeos1 behaved similarly to those of the parental strain. Collectively, these observations demonstrate that the CW is a positive factor that determines the internalization of the PAF26, and that Eos1p exerts its activity through the glycosylation of specific protein(s) involved in peptide internalization.

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide.

Saccharomyces cerevisiae "flor" yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends on FLO11 gene length and expression. FLO11 encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm of S. cerevisiae flor strains. We found that PAF26, a synthetic tryptophan-rich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functional FLO11 but was not accompanied by increased FLO11 expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that a flo11 gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that the FLO11 gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-to-cell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.

Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells.

The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW) is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW) and PAF96 (RKKAAA), were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i) its interaction with the cell envelope; (ii) its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii) its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the cytoplasm, which coincides with cell death. Peptide containment within vacuoles preserves fungal cells from peptide toxicity.

Sensitivity of Saccharomyces cerevisiae to the cell-penetrating antifungal peptide PAF26 correlates with endogenous nitric oxide (NO) production.

PAF26 is a synthetic fungicidal hexapeptide with cell-penetration properties and non-lytic mode of action. We demonstrate herein the endogenous accumulation of reactive oxygen species (ROS) and nitric oxide (NO) in the model fungus Saccharomyces cerevisiae treated with PAF26. However, the S. cerevisiae deletion mutant of YAP1 - the major inductor of defense to oxidative stress - did not show high sensitivity to PAF26 but rather increased resistance, and its ROS accumulation did not differ from that of the parental strain. Cross-protection experiments suggest that the oxidant H(2)O(2) and PAF26 kill yeast through different pathways. Overall, the data indicate that ROS are not the primary antifungal mechanism of the peptide. On the contrary, the PAF26-induced intracellular production of NO was blocked in two distinct resistant mutants: the above mentioned Δyap1, which had the induction of NO disrupted, and the previously reported Δarg1 from the biosynthetic pathway of arginine, which has reduced basal NO levels. The NO synthase inhibitor l-NAME partially restored yeast growth in the presence of PAF26. These findings correlate antifungal activity of PAF26 with NO production and provide a plausible explanation for the resistance phenotype of Δarg1 through its involvement in NO biosynthesis.