Transcriptional landscape of pathogen-responsive lncRNAs in tomato unveils the role of hydrolase encoding genes in response to Botrytis cinerea invasion.

LncRNAs have gained increasing attention owing to their important regulatory roles on growth and stress responses of plants. However, the mechanisms underlying the functions of lncRNAs in fruit-pathogen interaction are still largely unknown. In this study, a total of 273 lncRNAs responding to Botrytis cinerea infection were identified in tomato fruit, among which a higher percentage of antisense lncRNAs were targeted to the genes enriched in hydrolase activity. To ascertain the roles of these lncRNAs, seven hydrolase-related transcripts were transiently knocked-down by virus-induced gene silencing. Silencing of lncRNACXE20 reduced the expression level of a carboxylesterase gene, further enhancing the resistance of tomato to B. cinerea. In contrast, silencing of lncRNACHI, lncRNAMMP, lncRNASBT1.9 and lncRNAPME1.9 impaired the resistance to B. cinerea, respectively. Further RT-qPCR assay and enzymatic activity detection displayed that the attenuated resistance of lncRNAMMP and lncRNASBT1.9-silenced plants was associated with the inhibition on the expression of JA-related genes, while the decreased resistance of lncRNACHI-silenced plants resulted in reduced chitinase activity. Collectively, these results may provide references for deciphering the mechanisms underlying specific lncRNAs to interfere with B. cinerea infection by regulating the expression of defence-related genes or affecting hydrolase activity.

Bioactive sulforaphane from cruciferous vegetables: advances in biosynthesis, metabolism, bioavailability, delivery, health benefits, and applications.

Chronic inflammation-induced diseases (CID) are the dominant cause of death worldwide, contributing to over half of all global deaths. Sulforaphane (SFN) derived from cruciferous vegetables has been extensively studied for its multiple functional benefits in alleviating CID. This work comprehensively reviewed the biosynthesis, metabolism, bioavailability, delivery, health benefits, and applications of SFN and its potential mechanisms against CID (e.g., cancer, obesity, type 2 diabetes, et al.), and neurological disorders based on a decade of research. SFN exerts its biological functions through the hydrolysis of glucosinolates by gut microbiota, and exhibits rapid metabolism and excretion characteristics via metabolization of mercapturic acid pathway. Microencapsulation is an important way to improve the stability and targeted delivery of SFN. The health benefits of SNF against CID are attributed to the multiple regulatory mechanisms including modulating oxidative stress, inflammation, apoptosis, immune response, and intestinal homeostasis. The clinical applications of SFN and related formulations show promising potential; however, further exploration is required regarding the sources, dosages, toxicity profiles, and stability of SFN. Together, SFN is a natural product with great potential for development and application, which is crucial for the development of functional food and pharmaceutical industries.

PeMetR-mediated sulfur assimilation is essential for virulence and patulin biosynthesis in Penicillium expansum.

Penicillium expansum, as the causal agent of blue mould and a main producer of mycotoxin patulin, is a global concern for economic and food safety. To date, the nutritional requirements of the pathogen during infection and patulin biosynthesis are poorly understood. Here, we genetically characterized the role of the bZIP transcription factor PeMetR in sulfur metabolism, virulence and patulin biosynthesis of P. expansum. The PeMetR regulator is crucial for normal germination and growth on inorganic S-sources but dispensable for utilization of organic S-sources. Accordingly, it is involved in regulating the expression of genes in sulfur assimilation pathway rather than methionine metabolic processes. Disruption of PeMetR resulted in a complete loss of virulence on various fruits. Additionally, the mutant showed a remarkably reduced ability to produce patulin. Exogenous methionine could partially or completely rescue the impaired phenotypes of the mutant. Inactivation of the sulfur assimilation pathway genes, PesA, PesB, PesC, PesF, generated growth, virulence and patulin production defects similar to those of ΔPeMetR. Overall, our study provides evidence that PeMetR-mediated sulfur assimilation is essential for growth and infection and shows for the first time that regulation of sulfur assimilation affects biosynthesis of an important mycotoxin patulin in P. expansum.

Honokiol suppresses mycelial growth and reduces virulence of Botrytis cinerea by inducing autophagic activities and apoptosis.

Fungal pathogens lead to severe quality deterioration and yield loss, making it urgent to explore efficient measures to control fungal diseases at the preharvest and postharvest stages of plants. Therefore, studies on natural substances targeting alternative antimicrobial targets have become hot spots of research. Here, we show that honokiol, a polyphenolic compound obtained from Magnolia officinalis, significantly suppressed mycelial growth and reduced virulence of B. cinerea on harvested fruit by inducing autophagic activities and apoptosis. Moreover, honokiol was capable of abolishing the mitochondrial membrane potential and inducing the accumulation of reactive oxygen species. Some key genes involved in pathogenicity on fruit were also found significantly down-regulated. In summary, honokiol was effective as an alternative agent targeting autophagic and apoptotic machineries to control the incidence of gray mold, which may further enrich the toolkit of crop managers for fighting postharvest diseases caused by this and similar fungi.

Versatile Roles of the Receptor-Like Kinase Feronia in Plant Growth, Development and Host-Pathogen Interaction.

As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.

Molecular basis and regulation of pathogenicity and patulin biosynthesis in Penicillium expansum.

Penicillium expansum is a necrotrophic plant pathogen with a wide range of fruit hosts. It causes blue mold rot during fruit storage, transport, and sale, resulting in huge economic losses to the fruit industry. Moreover, this pathogen is also the main producer of patulin, a toxic secondary metabolite that contaminates fruit and fruit-derived products and impairs human health. Therefore, understanding molecular basis of the pathogenicity and patulin biosynthesis of the fungal pathogen has important scientific significance and also plays an important guiding role in the research and development of new control technologies. Here, we comprehensively summarize the recent research progress, particularly regarding the molecular aspects of pathogenicity, patulin biosynthesis, and the related regulatory mechanisms, as well as control technologies for blue mold rot in the fruit industry.

Dissection of patulin biosynthesis, spatial control and regulation mechanism in Penicillium expansum.

The patulin biosynthesis is one of model pathways in an understanding of secondary metabolite biology and network novelties in fungi. However, molecular regulation mechanism of patulin biosynthesis and contribution of each gene related to the different catalytic enzymes in the biochemical steps of the pathway remain largely unknown in fungi. In this study, the genetic components of patulin biosynthetic pathway were systematically dissected in Penicillium expansum, which is an important fungal pathogen and patulin producer in harvested fruits and vegetables. Our results revealed that all the 15 genes in the cluster are involved in patulin biosynthesis. Proteins encoded by those genes are compartmentalized in various subcellular locations, including cytosol, nucleus, vacuole, endoplasmic reticulum, plasma membrane and cell wall. The subcellular localizations of some proteins, such as PatE and PatH, are required for the patulin production. Further, the functions of eight enzymes in the 10-step patulin biosynthetic pathway were verified in P. expansum. Moreover, velvet family proteins, VeA, VelB and VelC, were proved to be involved in the regulation of patulin biosynthesis, but not VosA. These findings provide a thorough understanding of the biosynthesis pathway, spatial control and regulation mechanism of patulin in fungi.

Production, Signaling, and Scavenging Mechanisms of Reactive Oxygen Species in Fruit-Pathogen Interactions.

Reactive oxygen species (ROS) play a dual role in fruit-pathogen interaction, which largely depends on their different levels in cells. Fruit recognition of a pathogen immediately triggers an oxidative burst that is considered an integral part of the fruit defense response. ROS are also necessary for the virulence of pathogenic fungi. However, the accumulation of ROS in cells causes molecular damage and finally leads to cell death. In this review, on the basis of data regarding ROS production and the scavenging systems determining ROS homeostasis, we focus on the role of ROS in fruit defense reactions against pathogens and in fungi pathogenicity during fruit-pathogen interaction.

The pH-responsive PacC transcription factor plays pivotal roles in virulence and patulin biosynthesis in Penicillium expansum.

The PacC (loss or reduction in phosphatase activity at acid but not at alkaline pH [Pac]) transcription factor regulates environmental adaptation, secondary metabolism and virulence in many fungal pathogens. Here, we report the functions of PacC in Penicillium expansum, a postharvest pathogenic fungus in horticultural crops, and ascertain that the gene expression and proteolytic processing of PePacC are strictly pH-dependent. Loss of PePacC resulted in an obvious decrease in growth and conidiation of P. expansum cultured in both acidic and alkaline conditions. The ΔPePacC mutant lost the ability of patulin production at pH values above 6.0 because expressions of all the genes in patulin cluster were significantly down-regulated. Additionally, virulence of the ΔPePacC mutant was obviously reduced in pear and apple fruits. Proteome analysis revealed that PePacC could function as an activator or repressor for different target proteins, including calreticulin (PeCRT) and sulfate adenylyltransferase (PeSAT), which were further proved to be involved in virulence of P. expansum. Our results demonstrate important roles for PePacC in patulin biosynthesis via limiting expressions of the genes in the cluster, and in pathogenesis via mediating a known virulence factor glucose oxidase (PeGOD) and new virulence factors, such as PeCRT and PeSAT.

Comparative Proteomics Reveals the Potential Targets of BcNoxR, a Putative Regulatory Subunit of NADPH Oxidase of Botrytis cinerea.

The NADPH oxidase (NOX) complex has been shown to play a crucial role in stress response and in the virulence of various fungal pathogens. The underlying molecular mechanisms of NOX, however, remain largely unknown. In the present study, a comparative proteomic analysis compared changes in protein abundance in wild-type Botrytis cinerea and ΔbcnoxR mutants in which the regulatory subunit of NOX was deleted. The ΔbcnoxR mutants exhibited reduced growth, sporulation, and impaired virulence. A total of 60 proteins, representing 49 individual genes, were identified in ΔbcnoxR mutants that exhibited significant differences in abundance relative to wild-type. Reverse transcription-quantitative polymerase chain reaction analysis demonstrated that the differences in transcript levels for 36 of the genes encoding the identified proteins were in agreement with the proteomic analysis, while the remainder exhibited reverse levels. Functional analysis of four proteins that decreased abundance in the ΔbcnoxR mutants indicated that 6-phosphogluconate dehydrogenase (BcPGD) played a role in the growth and sporulation of B. cinerea. The Δbcpgd mutants also displayed impaired virulence on various hosts, such as apple, strawberry, and tomato fruit. These results suggest that NOX can influence the expression of BcPGD, which has an impact on growth, sporulation, and virulence of B. cinerea.

Aquaporin8 regulates cellular development and reactive oxygen species production, a critical component of virulence in Botrytis cinerea.

Aquaporins (AQPs) are ubiquitous in nearly all organisms, mediating selective and rapid flux of water across biological membranes. The role of AQPs in phytopathogenic fungi is poorly understood. Orthologs of AQP genes in Botrytis cinerea were identified and knocked out. The effects of AQPs on hyphal growth and conidiation, formation of infection structures and virulence on plant hosts were examined. The role of AQP8 in reactive oxygen species (ROS) production, distribution and transport were further determined. Among eight AQPs, only AQP8 was essential for the ability of B. cinerea to infect plants. AQP8 was demonstrated to be an intrinsic plasma membrane protein, which may function as a channel and mediate hydrogen peroxide uptake. Deletion of AQP8 in B. cinerea completely inhibited the development of conidia and infection structures, and significantly affected noxR expression. Further observations revealed that both AQP8 and noxR impacted ROS distribution in the hyphal tips of B. cinerea. Moreover, AQP8 affected the expression of a mitochondrial protein, NQO1. A knockout mutant of NQO1 was observed to display reduced virulence. These data lead to a better understanding of the important role of AQP8 in the development and pathogenesis of plant pathogens.

Genomic Characterization Reveals Insights Into Patulin Biosynthesis and Pathogenicity in Penicillium Species.

Penicillium species are fungal pathogens that infect crop plants worldwide. P. expansum differs from P. italicum and P. digitatum, all major postharvest pathogens of pome and citrus, in that the former is able to produce the mycotoxin patulin and has a broader host range. The molecular basis of host-specificity of fungal pathogens has now become the focus of recent research. The present report provides the whole genome sequence of P. expansum (33.52 Mb) and P. italicum (28.99 Mb) and identifies differences in genome structure, important pathogenic characters, and secondary metabolite (SM) gene clusters in Penicillium species. We identified a total of 55 gene clusters potentially related to secondary metabolism, including a cluster of 15 genes (named PePatA to PePatO), that may be involved in patulin biosynthesis in P. expansum. Functional studies confirmed that PePatL and PePatK play crucial roles in the biosynthesis of patulin and that patulin production is not related to virulence of P. expansum. Collectively, P. expansum contains more pathogenic genes and SM gene clusters, in particular, an intact patulin cluster, than P. italicum or P. digitatum. These findings provide important information relevant to understanding the molecular network of patulin biosynthesis and mechanisms of host-specificity in Penicillium species.

Effect of Cinnamic Acid for Controlling Gray Mold on Table Grape and Its Possible Mechanisms of Action.

Cinnamic acid (CA) is an organic acid and is widely used in food industry as a common food additive. Previous studies showed that CA has the antimicrobial activity in vitro, but little is known about the effect of CA on controlling the fruit decay in vivo. In present study, we showed that application of CA was significantly effective on controlling the gray mold of table grape caused by Botrytis cinerea. CA can directly inhibit the mycelial growth of B. cinerea on potato dextrose agar plates. The mechanisms by which CA inhibited fungal growth were assayed by staining the spores with fluorescent dyes propidium iodide and 7-dichlorodihydrofluorescein diacetate, respectively. The results indicated that CA can damage the integrity of plasma membrane and induce the intracellular reactive oxygen species level of B. cinerea which were responsible for the reduction of growth rate. Meanwhile, CA treatment significantly stimulated the activities of peroxidase and polyphenol oxidase which were closely related to the resistance of plant. Taken together, this study suggested that CA was effective on controlling the gray mold of table grape in postharvest period by inhibiting the growth of pathogen and inducing the resistance of host.

Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence.

Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

Characterization of two SGNH family cell death-inducing proteins from the horticulturally important fungal pathogen Botrytis cinerea based on the optimized prokaryotic expression system.

Botrytis cinerea is one of the most destructive phytopathogenic fungi, causing significant losses to horticultural crops. As a necrotrophic fungus, B. cinerea obtains nutrients by killing host cells. Secreted cell death-inducing proteins (CDIPs) play a crucial role in necrotrophic infection; however, only a limited number have been reported. For high-throughput CDIP screening, we optimized the prokaryotic expression system and compared its efficiency with other commonly used protein expression systems. The optimized prokaryotic expression system showed superior effectiveness and efficiency and was selected for subsequent CDIP screening. The screening system verified fifty-five candidate proteins and identified two novel SGNH family CDIPs: BcRAE and BcFAT. BcRAE and BcFAT exhibited high expression levels throughout the infection process. Site-directed mutagenesis targeting conserved Ser residues abolished the cell death-inducing activity of both BcRAE and BcFAT. Moreover, the transient expression of BcRAE and BcFAT in plants enhanced plant resistance against B. cinerea without inducing cell death, independent of their enzymatic activities. Our results suggest a high-efficiency screening system for high-throughput CDIP screening and provide new targets for further study of B. cinerea-plant interactions.

Research Progress on the Mechanism and Function of Histone Acetylation Regulating the Interaction between Pathogenic Fungi and Plant Hosts.

Histone acetylation is a crucial epigenetic modification, one that holds the key to regulating gene expression by meticulously modulating the conformation of chromatin. Most histone acetylation enzymes (HATs) and deacetylation enzymes (HDACs) in fungi were originally discovered in yeast. The functions and mechanisms of HATs and HDACs in yeast that have been documented offer us an excellent entry point for gaining insights into these two types of enzymes. In the interaction between plants and pathogenic fungi, histone acetylation assumes a critical role, governing fungal pathogenicity and plant immunity. This review paper delves deep into the recent advancements in understanding how histone acetylation shapes the interaction between plants and fungi. It explores how this epigenetic modification influences the intricate balance of power between these two kingdoms of life, highlighting the intricate network of interactions and the subtle shifts in these interactions that can lead to either mutual coexistence or hostile confrontation.

A comparative metabolomics analysis of phytochemcials and antioxidant activity between broccoli floret and by-products (leaves and stalks).

Leaves and stalks, which account for about 45% and 25% of broccoli biomass, respectively, are usually discarded during broccoli production, leading to the waste of green resources. In this study, the phytochemical composition and antioxidant capacity of broccoli florets and their by-products (leaves and stalks) were comprehensively analyzed. The metabolomics identified several unique metabolites (e.g., scopoletin, Harpagoside, and sinalbin) in the leaves and stalks compared to florets. Notably, the leaves were found to be a rich source of flavonoids and coumarins, with superior antioxidant capacity. The random forest model and correlation analysis indicated that flavonoids, coumarin, and indole compounds were the important factors contributing to the antioxidant activity. Moreover, the stalks contained higher levels of carbohydrates and exhibited better antioxidant enzyme activity. Together, these results provided valuable data to support the comprehensive utilization of broccoli waste, the development of new products, and the expansion of the broccoli industry chain.

Comparison analysis of bioactive metabolites in soybean, pea, mung bean, and common beans: reveal the potential variations of their antioxidant property.

This study showed the significantly differences of basic nutrients and metabolite compounds in nine types of beans involved in soybean, mung bean, pea, and common beans. The metabolomics results showed that serval metabolites such as histidine, proline, 3-alanine, and myricetin which could be used to identify different beans. The random forest model showed that amino acid and fatty acid could be used as special indexes to distinguish different types of beans in practice. The different expressed metabolites among different types of beans were involved in various pathways including alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and purine metabolism. The antioxidant activity was significantly different among different types of beans, and the contents of amino acid, coumarin, and polyphenol contributed the antioxidant activities of beans. Together, these results will provide a comprehensive understanding of metabolites in different types of beans and theoretical guideline for the future application of beans.

Ena Proteins Respond to PacC-Mediated pH Signaling Pathway and Play a Crucial Role in Patulin Biosynthesis.

Penicillium expansum is a main producer of patulin that causes severe postharvest decay and food safety issues in the fruit industry. Development, pathogenicity, and patulin production of P. expansum are strongly influenced by the PacC-pH signaling pathway. Global transcription factor PacC regulates various fungal biological processes through a complicated molecular network. In the present study, three Ena family genes (PeEnas), PeEnaA, PeEnaB, and PeEnaC, as important downstream targets of PePacC, were identified in P. expansum. Deletion of PeEnaA, PeEnaB, and PeEnaC showed little effect on mycelial growth under alkaline or high salinity conditions, but double and triple deletion of these genes impaired the virulence of P. expansum on apple fruit. Notably, patulin biosynthesis of P. expansum was distinctly inhibited in the deletion mutants of PeEnas. PeEnas regulated expressions of the patulin gene cluster, AP1, CreA, Sge1, and Hog1 at the transcriptional level and played roles in maintaining membrane potential. Overexpression of PeEnaC in ΔPePacC restored the patulin production defect of ΔPePacC. Our results indicated that, as downstream targets of PePacC, the PeEna family proteins play a crucial role in patulin biosynthesis in P. expansum.

PeAP1-mediated oxidative stress response plays an important role in the growth and pathogenicity of Penicillium expansum.

Penicillium expansum is the causal agent of post-harvest blue mold in various fruits and serves as a model for understanding fungal pathogenicity and mycotoxin production. The relevance of oxidative stress response in the growth and virulence of P. expansum has been largely unexplored. Here, we identify the transcriptional factor PeAP1 as a regulator of oxidative stress response in P. expansum. Gene expression and protein abundance of PeAP1, as well as its nuclear localization, are specifically induced by H2O2. Deletion of PeAP1 results in increased sensitivity to H2O2, and PeAP1 mutants exhibit a variety of defects in hyphal growth and virulence. PeAP1 prevents the accumulation of both intracellular H2O2 during vegetative growth and host-derived H2O2 during biotrophic growth. Application of an antioxidant glutathione and a NADPH oxidase inhibitor, diphenylene iodonium, to the PeAP1 mutant partially restored fungal growth and virulence. RNA sequencing analysis revealed 144 H2O2-induced PeAP1 target genes, including four antioxidant-related genes, PeGST1, PePrx1, PePrx2, and PeTRX2, that were also demonstrated to be involved in oxidative stress response and/or virulence. Collectively, our results demonstrate the global regulatory role of PeAP1 in response to oxidative stress and provide insights into the critical role of the PeAP1-mediated oxidative stress response to regulate growth and virulence of P. expansum. IMPORTANCE Reactive oxygen species are the core of host plant defense and also play a vital role in the successful invasion of host plants by pathogenic fungi. Despite its importance, the relevance of oxidative stress response in fungal growth and virulence is poorly understood in P. expansum. In this study, we reveal that the transcription factor PeAP1 acts as a central regulator of oxidative stress response in P. expansum and that there is a major link between PeAP1-mediated oxidative stress response and fungal growth and virulence. To explore the underlying mechanisms, we performed comparative transcriptomic studies and identified a number of H2O2-induced PeAP1 target genes, including four novel ones, PePrx1, PePrx2, PeGST1, and PeTRX2, whose functions were linked to PeAP1 and pathogenicity. These findings provide novel insights into the regulation mechanism of PeAP1 on growth and virulence, which might offer promising targets for control of blue mold and patulin contamination.