Inhibition of AcrAB-TolC enhances antimicrobial activity of phytochemicals in Pectobacterium brasiliense.

Introduction: The eons-long co-evolvement of plants and bacteria led to a plethora of interactions between the two kingdoms, in which bacterial pathogenicity is counteracted by plant-derived antimicrobial defense molecules. In return, efflux pumps (EP) form part of the resistance mechanism employed by bacteria to permit their survival in this hostile chemical environment. In this work we study the effect of combinations of efflux pump inhibitors (EPIs) and plant-derived phytochemicals on bacterial activity using Pectobacteriun brasiliense 1692 (Pb1692) as a model system. Methods: We measured the minimal inhibitory concentration (MIC) of two phytochemicals, phloretin (Pht) and naringenin (Nar), and of one common antibiotic ciprofloxacin (Cip), either alone or in combinations with two known inhibitors of the AcrB EP of Escherichia coli, a close homolog of the AcrAB-TolC EP of Pb1692. In addition, we also measured the expression of genes encoding for the EP, under similar conditions. Results: Using the FICI equation, we observed synergism between the EPIs and the phytochemicals, but not between the EPIs and the antibiotic, suggesting that EP inhibition potentiated the antimicrobial activity of the plant derived compounds, but not of Cip. Docking simulations were successfully used to rationalize these experimental results. Discussion: Our findings suggest that AcrAB-TolC plays an important role in survival and fitness of Pb1692 in the plant environment and that its inhibition is a viable strategy for controlling bacterial pathogenicity.

Extract from the Macroalgae Ulva rigida Induces Table Grapes Resistance to Botrytis cinerea.

Fungal pathogens are a central cause of the high wastage rates of harvested fruit and vegetables. Seaweeds from the genus Ulva are fast-growing edible green macroalgae whose species can be found on the shore of every continent, and therefore present a resource that can be utilized on a global scale. In this study, we found that the application of ulvan extract, a sulfated polysaccharide extracted from Ulva rigida (1000 mg/L), elicited table grapes defense and reduced the incidence and decay area of Botrytis cinerea by 43% and 41%, respectively. In addition, compared to the control group at two days post-treatment, ulvan extract elicited a variety of defense-related biomarkers such as a 43% increase in the activity of reactive oxygen species, 4-fold increase in the activity of catalase, 2-fold increase in the activity of superoxide dismutase and 1.4-fold increase in the activity of chitinase. No increase was observed in phenylalanine ammonia-lyase activity, and the treatment did not affect fruit quality parameters such as the pH levels, sugar levels, and titratable acidity of grapes. These results illustrate the potential of ulvan extract to naturally induce the plant defense response and to reduce postharvest decay.

Double-stranded RNA targeting fungal ergosterol biosynthesis pathway controls Botrytis cinerea and postharvest grey mould.

Pathogenic fungi cause major postharvest losses. During storage and ripening, fruit becomes highly susceptible to fungi that cause postharvest disease. Fungicides are effective treatments to limit disease. However, due to increased public concern for their possible side effects, there is a need to develop new strategies to control postharvest fungal pathogens. Botrytis cinerea, a common postharvest pathogen, was shown to uptake small double-stranded RNA (dsRNA) molecules from the host plant. Such dsRNA can regulate gene expression through the RNA interference system. This work aimed to develop a synthetic dsRNA simultaneously targeting three essential transcripts active in the fungal ergosterol biosynthesis pathway (dsRNA-ERG). Our results show initial uptake of dsRNA in the emergence zone of the germination tube that spreads throughout the fungus and results in down-regulation of all three targeted transcripts. Application of dsRNA-ERG decreased B. cinerea germination and growth in in vitro conditions and various fruits, leading to reduce grey-mould decay. The inhibition of growth or decay was reversed by the addition of ergosterol. While dual treatment with dsRNA-ERG and ergosterol-inhibitor fungicide reduced by 100-fold the required amount of fungicide to achieve the same protection rate. The application of dsRNA-ERG induced systemic protection as shown by decreased decay development at inoculation points distant from the treatment point in tomato and pepper fruits. Overall, this study suggests that dsRNA-ERG can effectively control B. cinerea growth and grey-mould development suggesting its efficacy as a future method for postharvest control of fungal pathogens.

CRISPR/Cas9 mutants of tomato MICRORNA164 genes uncover their functional specialization in development.

Plant MICRORNA164 (miR164) plays diverse regulatory functions by post-transcriptional repression of certain NAM/ATAF/CUC-domain transcription factors. However, the involvement of miR164 in fleshy fruit development and ripening remains poorly understood. Here, de novo prediction of tomato (Solanum lycopersicum) MIR164 genes identified four genes (SlMIR164a-d), of which SlMIR164d has an atypically long pre-miRNA. The roles of the fruit expressed SlMIR164a, b, and d were studied by analysis of their Clustered Regularly Interspaced Short Palindromic Repeats mutants. The slmir164bCR mutant plants exhibited shoot and flower abnormalities characteristic of ectopic boundary specification, whereas the shoot and flower development of slmir164aCR and slmir164dCR mutants were indistinguishable from wild-type. Strikingly, the knockout of SlMIR164a practically eliminated sly-miR164 from the developing and ripening fruit pericarp. The sly-miR164-deficient slmir164aCR fruits were smaller than the wild-type, due to reduced pericarp cell division and expansion, and displayed intense red color and matte, instead of glossy appearance, upon ripening. We found that the fruit skin phenotypes were associated with morphologically abnormal outer epidermis and thicker cuticle. Quantitation of sly-miR164 target transcripts in slmir164aCR ripening fruits demonstrated the upregulation of SlNAM3 and SlNAM2. Specific expression of their miR164-resistant versions in the pericarp resulted in the formation of extremely small fruits with abnormal epidermis, highlighting the importance of their negative regulation by sly-miR164a. Taken together, our results demonstrate that SlMIR164a and SlMIR164b play specialized roles in development: SlMIR164b is required for shoot and flower boundary specification, and SlMIR164a is required for fruit growth including the expansion of its outer epidermis, which determines the properties of the fruit skin.

Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing.

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl-homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound's inhibition of virulence.

Detection of quiescent fungi in harvested fruit using CMOS biosensor: A proof of concept study.

Postharvest fruit decay is caused by fungal pathogens and leads to major losses. In this study, specific mRNA sequences that are upregulated in the fungus Colletotrichum gloeosporioides during its quiescent stage in fruits, were identified using a CMOS sensor. The identification process was based on sandwich approach, where strands complementary to the C. gloeosporioides mRNA sequences (quiescent stage-specific) were immobilized on the CMOS surface, and exposed to the target complementary reporter strands. In the presence of a target sequence, the reporter strand (linked to the enzyme horseradish peroxidase (HRP)) was left in the system and a measurable light signal was produced. The complementary strands specifically anneal to the mRNA in the sample. The sensitivity of the technology was assessed by mRNA sequences isolated from C. gloeosporioides, and identified as 10 nM RNA. The effect of the pathogenicity state on the sensor performance was also evaluated. The CMOS sensor could detect quiescent fungi, which are barely detectable by other means. The unique capability of the proposed system to detect and recognize the fungus during both pathogenic and quiescent stages, will allow the development of new sensors that can monitor the amount of undetectable quiescent fungi in harvested fruit, enabling improved food management.

Harvesting Mango Fruit with a Short Stem-End Altered Endophytic Microbiome and Reduce Stem-End Rot.

Stem-end rot (SER) is a serious postharvest disease of mango fruit grown in semi-dry area. Pathogenic and non-pathogenic microorganisms endophytically colonize fruit stem-end. As fruit ripens, some pathogenic fungi switch from endophytic colonization to necrotrophic stage and cause SER. Various pre/post-treatments may alter the stem-end community and modify SER incidence. This study investigates the effects of harvesting mango with or without short stem-end on fruit antifungal and antioxidant activities, the endophytic microbiome, and SER during fruit storage. Our results show that harvesting mango with short stem significantly reduced SER during storage. At harvest, fruit harvested with or without stem exhibit a similar microorganisms community profile. However, after storage and shelf life, the community of fruit without stem shifted toward more SER-causing-pathogens, such as Lasiodiplodia, Dothiorella, and Alternaria, and separated from the community of fruit with stem. This change correlated to the high antifungal activity of stem extract that strongly inhibited both germination and growth of Lasiodiplodia theobromae and Alternaria alternata. Additionally, fruit that was harvested with stem displayed more antioxidant activity and less ROS. Altogether, these findings indicate that harvesting mango with short stem leads to higher antifungal and antioxidant activity, retaining a healthier microbial community and leading to reduced postharvest SER.

Increased phenylalanine levels in plant leaves reduces susceptibility to Botrytis cinerea.

Botrytis cinerea is a major plant pathogen, causing losses in crops during growth and storage. Here we show that increased accumulation of phenylalanine (Phe) and Phe-derived metabolites in plant leaves significantly reduces their susceptibility to B. cinerea. Arabidopsis, petunia and tomato plants were enriched with Phe by either overexpressing a feedback-insensitive E.coli DAHP synthase (AroG*), or by spraying or drenching detached leaves or whole plants with external Phe, prior to infection with B. cinerea. Metabolic analysis of Arabidopsis and petunia plants overexpressing AroG* as well as wt petunia plants treated externally with Phe, revealed an increase in Phe-derived phenylpropanoids accumulated in their leaves, and specifically in those inhibiting B. cinerea germination and growth, suggesting that different compounds reduce susceptibility to B. cinerea in different plants. Phe itself had no inhibitory effect on germination or growth of B. cinerea, and inhibition of Phe metabolism in petunia plants treated with external Phe prevented decreased susceptibility to the fungus. Thus, Phe metabolism into an array of metabolites, unique to each plant and plant organ, is the most probable cause for increased resistance to Botrytis. This mechanism may provide a basis for ecologically friendly control of a wide range of plant pathogens.

Differential response to heat stress in outer and inner onion bulb scales.

The formation of brown protective skin in onion bulbs can be induced by rapid post-harvest heat treatment. Onions that are peeled to different depths and are exposed to heat stress show that only the outer scales form the dry brown skin, whereas the inner scales maintain high water content and do not change color. Our study demonstrates that browning of the outer scale during heat treatment is due to an enzymatic process that is associated with high levels of oxidation components, such as peroxidase and quercetin glucoside. De novo transcriptome analysis revealed differential molecular responses of the outer and inner scales to heat stress. Genes involved in lipid metabolism, oxidation pathways, and cell-wall modification were highly expressed in the outer scale during heating. Defense response-related genes such as those encoding heat-shock proteins, antioxidative stress defense, or production of osmoprotectant metabolites were mostly induced in the inner scale in response to heat exposure. These transcriptomic data led to a conceptual model that suggests sequential processes for the development of browning and desiccation of the outer scale versus processes associated with defense response and heat tolerance in the inner scales.

Cellular and Molecular Changes Associated with Onion Skin Formation Suggest Involvement of Programmed Cell Death.

Skin formation of onion (Allium cepa L.) bulb involves scale desiccation accompanied by scale senescence, resulting in cell death and tissue browning. Understanding the mechanism of skin formation is essential to improving onion skin and bulb qualities. Although onion skin plays a crucial role in postharvest onion storage and shelf life, its formation is poorly understood. We investigated the mode of cell death in the outermost scales that are destined to form the onion skin. Surprisingly, fluorescein diacetate staining and scanning electron microscopy indicated that the outer scale desiccates from the inside out. This striking observation suggests that cell death in the outer scales, during skin formation, is an internal and organized process that does not derive only from air desiccation. DNA fragmentation, a known hallmark of programmed cell death (PCD), was revealed in the outer scales and gradually decreased toward the inner scales of the bulb. Transmission electron microscopy further revealed PCD-related structural alterations in the outer scales which were absent from the inner scales. De novo transcriptome assembly for three different scales: 1st (outer), 5th (intermediate) and 8th (inner) fleshy scales identified 2,542 differentially expressed genes among them. GO enrichment for cluster analysis revealed increasing metabolic processes in the outer senescent scale related to defense response, PCD processes, carbohydrate metabolism and flavonoid biosynthesis, whereas increased metabolism and developmental growth processes were identified in the inner scales. High expression levels of PCD-related genes were identified in the outer scale compared to the inner ones, highlighting the involvement of PCD in outer-skin development. These findings suggest that a program to form the dry protective skin exists and functions only in the outer scales of onion.