Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit.

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

Avirulent Isolates of Penicillium chrysogenum to Control the Blue Mold of Apple Caused by P. expansum.

Blue mold is an economically significant postharvest disease of pome fruit that is primarily caused by Penicillium expansum. To manage this disease and sustain product quality, novel decay intervention strategies are needed that also maintain long-term efficacy. Biocontrol organisms and natural products are promising tools for managing postharvest diseases. Here, two Penicillium chrysogenum isolates, 404 and 413, were investigated as potential biocontrol agents against P. expansum in apple. Notably, 404 and 413 were non-pathogenic in apple, yet they grew vigorously in vitro when compared to the highly aggressive P. expansum R19 and Pe21 isolates. Whole-genome sequencing and species-specific barcoding identified both strains as P. chrysogenum. Each P. chrysogenum strain was inoculated in apple with the subsequent co-inoculation of R19 or Pe21 simultaneously, 3, or 7 days after prior inoculation with 404 or 413. The co-inoculation of these isolates showed reduced decay incidence and severity, with the most significant reduction from the longer establishment of P. chrysogenum. In vitro growth showed no antagonism between species, further suggesting competitive niche colonization as the mode of action for decay reduction. Both P. chrysogenum isolates had incomplete patulin gene clusters but tolerated patulin treatment. Finally, hygromycin resistance was observed for both P. chrysogenum isolates, yet they are not multiresistant to apple postharvest fungicides. Overall, we demonstrate the translative potential of P. chrysogenum to serve as an effective biocontrol agent against blue mold decay in apples, pending practical optimization and formulation.

A Bitter, Complex Problem: Causal Colletotrichum Species in Virginia Orchards and Apple Fruit Susceptibility.

Bitter rot, caused by Colletotrichum species, is one of the most devastating summer rot diseases affecting apple production in the Eastern United States. Given the differences in virulence and fungicide sensitivity levels between organisms belonging to the acutatum species complex (CASC) and the gloeosporioides species complex (CGSC), monitoring their diversity, geographic distribution, and frequency are essential for successful bitter rot management. In a 662-isolate collection from apple orchards in Virginia, isolates from CGSC were dominant (65.5%) in comparison to the CASC (34.5%). In a subsample of 82 representative isolates, using morphological and multilocus phylogenetic analyses, we identified C. fructicola (26.2%), C. chrysophilum (15.6%), C. siamense (0.8%), and C. theobromicola (0.8%) from CGSC and C. fioriniae (22.1%) and C. nymphaeae (1.6%) from CASC. The dominant species were C. fructicola, followed by C. fioriniae and C. chrysophilum. C. siamense followed by C. theobromicola developed the largest and deepest rot lesions on Honeycrisp fruit in our virulence tests. Detached fruit of nine apple cultivars and one wild accession (Malus sylvestris) were harvested early and late season and tested in controlled conditions for their susceptibility to C. fioriniae and C. chrysophilum. All cultivars were susceptible to both representative bitter rot species, with Honeycrisp fruit being the most susceptible and M. sylvestris, accession PI 369855, being the most resistant. We demonstrate that the frequency and prevalence of species in Colletotrichum complexes are highly variable in the Mid-Atlantic and provide region-specific data on apple cultivar susceptibility. Our findings are necessary for the successful management of bitter rot as an emerging and persistent problem in apple production both pre- and postharvest.

Genomic Resources of Four Colletotrichum Species (C. fioriniae, C. chrysophilum, C. noveboracense, and C. nupharicola) Threatening Commercial Apple Production in the Eastern United States.

The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic time traveling: sequencing old herbarium specimens, including the oldest herbarium specimen sequenced from kingdom Fungi, reveals the population structure of an agriculturally significant rust.

Sequencing herbarium specimens can be instrumental in answering ecological, evolutionary, and taxonomic inquiries. We developed a protocol for sequencing herbarium specimens of rust fungi (Pucciniales) and proceeded to sequence specimens ranging from 4 to 211 yr old from five different genera. We then obtained sequences from an economically important biological control agent, Puccinia suaveolens, to highlight the potential of sequencing herbarium specimens in an ecological sense and to evaluate the following hypotheses: (1) The population structure of a plant pathogen changes over time, and (2) introduced pathogens are more diverse in their native range. Our efforts resulted in sequences from 87 herbarium specimens that revealed a high level of diversity with a population structure that exhibited spatial-temporal patterns. The specimens sequenced from Europe showed more diversity than the ones from North America, uncovering an invasion pattern likely related to its European native host in North America. Additionally, to the best of our knowledge, the specimen from France collected in c. 1811 is the oldest herbarium specimen sequenced from kingdom Fungi. In conclusion, sequencing old herbarium specimens is an important tool that can be extrapolated to better understand plant-microbe evolution and to evaluate old type specimens to solidify the taxonomy of plant pathogenic fungi.

Would You Like Wood or Plastic? Bin Material, Sanitation Treatments, and Bin Inoculum Levels Impact Blue Mold Decay of Stored Apple Fruit.

Blue mold, caused primarily by Penicillium expansum, is a significant postharvest disease of apples. It not only causes economic losses but also produces mycotoxins that contaminate processed fruit products, which contributes to food waste and loss. Previous research has shown that packing and storage bins harbor Penicillium spores and that steam and hot water efficiently reduce spore inoculum levels. However, studies using wooden and plastic bins regarding their ability to harbor spores, the effect of chemical sanitation treatments on spore levels, and the impact of rinsate from treated bins on apple fruit decay have not been investigated for the Mid-Atlantic area (Okull et al. 2006; Rosenberger 2009). We evaluated different sanitation treatments (chemical and physical) to reduce P. expansum inoculum levels on wooden and plastic bins. We determined that wooden bins bound P. expansum spores four orders of magnitude higher than plastic. When both bin types were treated with steam (wooden) or sterile hot water (plastic), Thyme Guard, or Academy, all treatments resulted in significantly (P < 0.05) lower spore levels compared to untreated controls. Although, plastic bins retained lower numbers of spores after inoculation with contaminated spore rinsate and required much higher concentrations of P. expansum spores in rinsate to retain spores at levels that would lead to decay on apple fruit. Overall, we demonstrated that plastic bins retain fewer spores than wooden bins and that both can be sanitized by various physical or chemical treatments. We envision that our findings will be applicable in the future as the techniques implemented in this study were used to investigate industry-relevant questions. Our goal is that the research techniques and findings become feasible with advancements in technology and/or accompany other shifts in existing processes in commercial pome fruit packing and storage facilities.

Comparative Penicillium spp. Transcriptomics: Conserved Pathways and Processes Revealed in Ungerminated Conidia and during Postharvest Apple Fruit Decay.

Blue mold, caused by Penicillium spp., is an impactful postharvest disease resulting in significant economic losses due to reduced pome fruit quality and mycotoxin contamination. Using two Penicillium species with different levels of aggressiveness, transcriptomics were implemented in order to identify genes expressed during apple fruit decay and loci expressed in ungerminated conidia. Total RNA was isolated from ungerminated conidia and decayed apple fruit infected with P. expansum R19 or P. polonicum RS1. There were 2442 differentially expressed genes (DEGs) between the R19 and RS1 in apple. Comparisons within species between apple and conidia revealed 4404 DEGs for R19 and 2935 for RS1, respectively. Gene ontology (GO) analysis revealed differential regulation in fungal transport and metabolism genes during decay, suggesting a flux in nutrient acquisition and detoxification strategies. In R19, the oxidoreductase GO category comprised 20% of all DEG groups in apple verses conidia. Ungerminated conidia from both species showed DEGs encoding the glyoxylate shunt and beta-oxidation, specifying the earliest metabolic requirements for germination. This is the first study to identify pre-loaded transcripts in conidia from blue mold fungi, reveal unique genes between species expressed during apple decay, and show the expression dynamics of known fungal virulence factors. These findings will enable development of targeted approaches for blue mold abatement strategies.

More than a Virulence Factor: Patulin Is a Non-Host-Specific Toxin that Inhibits Postharvest Phytopathogens and Requires Efflux for Penicillium Tolerance.

Mycotoxin contamination is a leading cause of food spoilage and waste on a global scale. Patulin, a mycotoxin produced by Penicillium spp. during postharvest pome fruit decay, causes acute and chronic effects in humans, withstands pasteurization, and is not eliminated by fermentation. While much is known about the impact of patulin on human health, there are significant knowledge gaps concerning the effect of patulin during postharvest fruit-pathogen interactions. Application of patulin on six apple cultivars reproduced some blue mold symptoms that were cultivar-independent and dose-dependent. Identical symptoms were also observed in pear and mandarin orange. Six Penicillium isolates exposed to exogenous patulin exhibited delayed germination after 24 h, yet all produced viable colonies in 7 days. However, four common postharvest phytopathogenic fungi were completely inhibited by patulin during conidial germination and growth, suggesting the toxin is important for Penicillium to dominate the postharvest niche. Using clorgyline, a broad-spectrum efflux pump inhibitor, we demonstrated that efflux plays a role in Penicillium auto-resistance to patulin during conidial germination. The work presented here contributes new knowledge of patulin auto-resistance, its mode of action, and inhibitory role in fungal-fungal interactions. Our findings provide a solid foundation to develop toxin and decay mitigation approaches.

Incidence, Speciation, and Morpho-Genetic Diversity of Penicillium spp. Causing Blue Mold of Stored Pome Fruits in Serbia.

Blue mold, caused by Penicillium spp., is one of the most economically important postharvest diseases of pome fruits, globally. Pome fruits, in particular apple, is the most widely grown pome fruit in Serbia, and the distribution of Penicillium spp. responsible for postharvest decay is unknown. A two-year survey was conducted in 2014 and 2015, where four pome fruits (apple, pear, quince, and medlar) with blue mold symptoms were collected from 20 storage locations throughout Serbia. Detailed morphological characterization, analysis of virulence in three apple cultivars, and multilocus phylogeny revealed three main Penicillium spp. in order of abundance: P. expansum, P. crustosum, and P. solitum. Interestingly, P. expansum split into two distinct clades with strong statistical support that coincided with several morphological observations. Findings from this study are significant and showed previously undocumented diversity in blue mold fungi responsible for postharvest decay including the first finding of P. crustosum, and P. solitum as postharvest pathogens of quince and P. crustosum of medlar fruit in the world, and P. expansum of quince in Serbia. Data from this study provide timely information regarding phenotypic, morphological and genotypic plasticity in P. expansum that will impact the design of species-specific detection tools and guide the development of blue mold management strategies.

Genomic Analyses of Penicillium Species Have Revealed Patulin and Citrinin Gene Clusters and Novel Loci Involved in Oxylipin Production.

Blue mold of apple is caused by several different Penicillium species, among which P. expansum and P. solitum are the most frequently isolated. P. expansum is the most aggressive species, and P. solitum is very weak when infecting apple fruit during storage. In this study, we report complete genomic analyses of three different Penicillium species: P. expansum R21 and P. crustosum NJ1, isolated from stored apple fruit; and P. maximae 113, isolated in 2013 from a flooded home in New Jersey, USA, in the aftermath of Hurricane Sandy. Patulin and citrinin gene cluster analyses explained the lack of patulin production in NJ1 compared to R21 and lack of citrinin production in all three strains. A Drosophila bioassay demonstrated that volatiles emitted by P. solitum SA and P. polonicum RS1 were more toxic than those from P. expansum and P. crustosum strains (R27, R11, R21, G10, and R19). The toxicity was hypothesized to be related to production of eight-carbon oxylipins. Putative lipoxygenase genes were identified in P. expansum and P. maximae strains, but not in P. crustosum. Our data will provide a better understanding of Penicillium spp. complex secondary metabolic capabilities, especially concerning the genetic bases of mycotoxins and toxic VOCs.

An Analysis of Postharvest Fungal Pathogens Reveals Temporal-Spatial and Host-Pathogen Associations with Fungicide Resistance-Related Mutations.

Fungicides are the primary tools to control a wide range of postharvest fungal pathogens. Fungicide resistance is a widespread problem that has reduced the efficacy of fungicides. Resistance to FRAC-1 (Fungicide Resistance Action Committee-1) chemistries is associated with mutations in amino acid position 198 in the ß-tubulin gene. In our study, we conducted a meta-analysis of ß-tubulin sequences to infer temporal, spatial, plant host, and pathogen genus patterns of fungicide resistance in postharvest fungal pathogens. In total, data were acquired from 2,647 specimens from 12 genera of fungal phytopathogens residing in 53 countries on >200 hosts collected between 1926 and 2020. The specimens containing a position 198 mutation were globally distributed in a variety of pathosystems. Analyses showed that there are associations among the mutation and the year an isolate was collected, the pathogen genus, the pathogen host, and the collection region. Interestingly, fungicide-resistant ß-tubulin genotypes have been in a decline since their peak between 2005 and 2009. FRAC-1 fungicide usage data followed a similar pattern in that applications have been in a decline since their peak between 1997 and 2003. The data show that, with the reduction of selection pressure, FRAC-1 fungicide resistance in fungal populations will decline within 5 to 10 years. Based on this line of evidence, we contend that a ß-tubulin position 198 mutation has uncharacterized fitness cost(s) on fungi in nature. The compiled dataset can inform end users on the regions and hosts that are most prone to contain resistant pathogens and assist decisions concerning fungicide resistance management strategies.

The Good, the Bad, and the Ugly: Mycotoxin Production During Postharvest Decay and Their Influence on Tritrophic Host-Pathogen-Microbe Interactions.

Mycotoxins are a prevalent problem for stored fruits, grains, and vegetables. Alternariol, aflatoxin, and patulin, produced by Alternaria spp., Aspergillus spp., and Penicillium spp., are the major mycotoxins that negatively affect human and animal health and reduce fruit and produce quality. Control strategies for these toxins are varied, but one method that is increasing in interest is through host microbiome manipulation, mirroring a biocontrol approach. While the majority of mycotoxins and other secondary metabolites (SM) produced by fungi impact host-fungal interactions, there is also an interplay between the various organisms within the host microbiome. In addition to SMs, these interactions involve compounds such as signaling molecules, plant defense and growth hormones, and metabolites produced by both the plants and microbial community. Therefore, studies to understand the impact of the various toxins impacting the beneficial and harmful microorganisms that reside within the microbiome is warranted, and could lead to identification of safe analogs for antimicrobial activity to reduce fruit decay. Additionally, exploring the composition of the microbial carposphere of host plants is likely to shed light on developing a microbial consortium to maintain quality during storage and abate mycotoxin contamination.

Botrytis spp.: A Contemporary Perspective and Synthesis of Recent Scientific Developments of a Widespread Genus that Threatens Global Food Security.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

Penicillium expansum: biology, omics, and management tools for a global postharvest pathogen causing blue mould of pome fruit.

Blue mould, caused primarily by Penicillium expansum, is a major threat to the global pome fruit industry, causing multimillion-dollar losses annually. The blue mould fungus negatively affects fruit quality, thereby reducing fresh fruit consumption, and significantly contributes to food loss. P. expansum also produces an array of mycotoxins that are detrimental to human health. Management options are limited and the emergence of fungicide-resistant Penicillium spp. makes disease management difficult, therefore new approaches and tools are needed to combat blue mould in storage. This species profile comprises a comprehensive literature review of this aggressive pathogen associated with pomes (apple, pear, quince), focusing on biology, mechanisms of disease, control, genomics, and the newest developments in disease management. TAXONOMY: Penicillium expansum Link 1809. Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass: Eurotiomycetidae, Order Eurotiales; Family Trichocomaceae, Genus Penicillium, Species expansum. BIOLOGY: A wide host range necrotrophic postharvest pathogen that requires a wound (e.g., stem pull, punctures, bruises, shoulder cracks) or natural openings (e.g., lenticel, stem end, calyx sinus) to gain ingress and infect. TOXINS: Patulin, citrinin, chaetoglobosins, communesins, roquefortine C, expansolides A and B, ochratoxin A, penitrem A, rubratoxin B, and penicillic acid. HOST RANGE: Primarily apples, European pear, Asian pear, medlar, and quince. Blue mould has also been reported on stone fruits (cherry, plum, peach), small fruits (grape, strawberry, kiwi), and hazel nut. DISEASE SYMPTOMS: Blue mould initially appears as light tan to dark brown circular lesions with a defined margin between the decayed and healthy tissues. The decayed tissue is soft and watery, and blue-green spore masses appear on the decayed area, starting at the infection site and radiating outward as the decayed area ages. DISEASE CONTROL: Preharvest fungicides with postharvest activity and postharvest fungicides are primarily used to control decay. Orchard and packinghouse sanitation methods are also critical components of an integrated pest management strategy. USEFUL WEBSITES: Penn State Tree Fruit Production Guide (https://extension.psu.edu/forage-and-food-crops/fruit), Washington State Comprehensive Tree Fruit (http://treefruit.wsu.edu/crop-protection/disease-management/blue-mold/), The Apple Rot Doctor (https://waynejurick.wixsite.com/applerotdr), penicillium expansum genome sequences and resources (https://www.ncbi.nlm.nih.gov/genome/browse/#!/eukaryotes/11336/).

Global transcriptomic responses orchestrate difenoconazole resistance in Penicillium spp. causing blue mold of stored apple fruit.

BACKGROUND: Blue mold is a globally important and economically impactful postharvest disease of apples caused by multiple Penicillium spp. There are currently four postharvest fungicides registered for blue mold control, and some isolates have developed resistance manifesting in decay on fungicide-treated fruit during storage. To date, mechanisms of fungicide resistance have not been explored in this fungus using a transcriptomic approach. RESULTS: We have conducted a comparative transcriptomic study by exposing naturally-occurring difenoconazole (DIF) resistant (G10) and sensitive (P11) blue mold isolates to technical grade difenoconazole, an azole fungicide in the commercial postharvest product Academy (Syngenta Crop Protection, LLC). Dynamic changes in gene expression patterns were observed encompassing candidates involved in active efflux and transcriptional regulators between the resistant and sensitive isolates. Unlike other systems, 3 isoforms of cytochrome P450 monoxygenase (CYP51A-C) were discovered and expressed in both sensitive and resistant strains upon difenoconazole treatment. Active efflux pumps were coordinately regulated in the resistant isolate and were shown to mediate the global resistance response as their inhibition reversed the difenoconazole-resistant phenotype in vitro. CONCLUSIONS: Our data support the observation that global transcriptional changes modulate difenoconazole resistance in Penicillium spp. While the dogma of CYP51 overexpression is supported in the resistant isolate, our studies shed light on additional new mechanisms of difenoconazole resistance on a global scale in Penicillium spp. These new findings broaden our fundamental understanding of azole fungicide resistance in fungi, which has identified multiple genetic targets, that can be used for the detection, management, and abatement of difenoconazole-resistant blue mold isolates during long-term storage of apples.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

A Genome Resource for Several North American Venturia inaequalis Isolates with Multiple Fungicide Resistance Phenotypes.

The apple scab pathogen, Venturia inaequalis, is among the most economically important fungal pathogens that affects apples. Fungicide applications are an essential part of disease management. Implementation of cultural practices and genetic sources of resistance in the host are vital components of scab management. This is the first presentation of multiple, high quality, well-annotated genomes of four North American V. inaequalis isolates having both sensitive and multiple fungicide resistance phenotypes. We envision that these isolates will enable investigations into fungicide resistance mechanisms, exploring fungal virulence factors and delineating phylogenomic relationships among apple scab isolates from around the world.

A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants.

BACKGROUND: Hydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits. RESULTS: The putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine. CONCLUSION: SrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism.

Influence of R and S enantiomers of 1-octen-3-ol on gene expression of Penicillium chrysogenum.

Inhibition of spore germination offers an attractive and effective target for controlling fungal species involved in food spoilage. Mushroom alcohol (1-octen-3-ol) functions as a natural self-inhibitor of spore germination for many fungi and, therefore, provides a useful tool for probing the molecular events controlling the early stages of fungal growth. In Penicillium spp., the R and S enantiomers of 1-octen-3-ol delayed spore germination and sporulation in four species of Penicillium involved in soils of fruit and grains, but to different degrees. Because of its well-annotated genome, we used Penicillium chrysogenum to perform a comprehensive comparative transcriptomic analysis of cultures treated with the two enantiomers. Altogether, about 80% of the high-quality reads could be mapped to 11,396 genes in the reference genome. The top three active pathways were metabolic (978 transcripts), biosynthesis of secondary metabolites (420 transcripts), and microbial metabolism in diverse environments (318 transcripts). When compared to the control, treatment with (R)-(-)-1-octen-3-ol affected the transcription levels of 91 genes, while (S)-(+)-1-octen-3-ol affected only 41 genes. Most of the affected transcripts were annotated and predicted to be involved in transport, establishment of localization, and transmembrane transport. Alternative splicing and SNPs' analyses indicated that, compared to the control, the R enantiomer had greater effects on the gene expression pattern of Penicillium chrysogenum than the S enantiomer. A qRT-PCR analysis of 28 randomly selected differentially expressed genes confirmed the transcriptome data. The transcriptomic data have been deposited in NCBI SRA under the accession number SRX1065226.