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.

A receptor-like kinase SlFERL mediates immune responses of tomato to Botrytis cinerea by recognizing BcPG1 and fine-tuning MAPK signaling.

FERONIA (FER) is a receptor-like kinase showing versatile functions during plant growth, development, and responses to environmental stimuli. However, its functions during the interaction between fruit and necrotrophic fungal pathogens are still unclear. Combining reverse genetic approaches, physiological assays, co-immunoprecipitation, protein phosphorylation identification, and site-directed mutagenesis, we reported a tomato FER homolog SlFERL (Solanum lycopersicum FERONIA Like) involved in the immune responses to Botrytis cinerea invasion. The results indicated that SlFERL extracellular domain recognized and interacted with the secreted virulence protein BcPG1 from B. cinerea, further revealed that SlFERL triggered downstream signaling by phosphorylating SlMAP3K18 at Thr45, Ser49, Ser76, and Ser135. Moreover, we verified that SlMAP2K2 and SlMAP2K4 synergistically contributed to immune response of tomato to B. cinerea, in which SlFERL-SlMAP3K18 module substantially modulated protein level and/or kinase activity of SlMAP2K2/SlMAP2K4. These findings reveal a new pattern-triggered immune pathway, indicating that SlFERL participates in the immune responses to B. cinerea invasion via recognizing BcPG1 and fine-tuning MAPK signaling.

Simultaneous determination of dissolved organic nitrogen, nitrite, nitrate and ammonia using size exclusion chromatography coupled with nitrogen detector.

Accurate quantification of dissolved organic nitrogen (DON) has been a challenge due to the cumulative analytical errors in the conventional method via subtracting dissolved inorganic nitrogen species (DIN) from total dissolved nitrogen (TDN). Size exclusion chromatography coupled with an organic nitrogen detector (SEC-OND) has been developed as a direct method for quantification and characterization of DON. However, the applications of SEC-OND method still subject to poor separations between DON and DIN species and unsatisfied N recoveries of macromolecules. In this study, we packed a series of SEC columns with different lengths and resin materials for separation of different N species and designed an independent vacuum ultraviolet (VUV) oxidation device for complete oxidation converting N species to nitrate. To guarantee sufficient N recoveries, the operation conditions were optimized as oxidation time ≥ 30 min, injection mass (sample concentration × injection volume) < 1000 µL × mg-N/L for macromolecular proteins, and neutral pH mobile eluent. The dissolved O2 concentration in SEC mobile phase determined the upper limit of VUV oxidation at a specific oxidation time. Compared to conventional HW50S column (20 × 250 mm), HW40S column (20 × 350 mm) with mobile phase comprising of 1.5 g/L Na2HPO4·2H2O + 2.5 g/L KH2PO4 (pH = 6.85) could achieve a better separation of DON, nitrite, nitrate, and ammonia. When applied to river water, lake water, wastewater effluent, groundwater, and landfill leachate, the SEC-OND method could quantify DON as well as DIN species accurately and conveniently even the DIN/TDN ratio reached 0.98.

Overlooked Formation of H2O2 during the Hydroxyl Radical-Scavenging Process When Using Alcohols as Scavengers.

Hydroxyl radical (•OH) is an active species widely reported in studies across many scientific fields, and hence, its reliable analysis is vitally important. Currently, alcohols are commonly used as scavengers for •OH determination. However, the impacts of alcohols on the reliability of •OH detection remain unknown. In this study, we found that adding different types and different amounts of alcohols in water samples treated with ultraviolet irradiation undesirably produced substantial amounts of hydrogen peroxide (H2O2), which is a known •OH precursor. This means that the conventional •OH determination method using alcohols is likely unreliable or even misleading. Through careful investigation, we revealed an overlooked reaction pathway during H2O2 and •OH transformations. Varying oxygen concentrations, pHs, alcohol dosages, and types altered H2O2 formation, which can affect •OH determination accuracy. Among alcohols, n-butanol is the best scavenger because it quenches •OH rapidly but re-forms little H2O2.

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.

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.

Function of small GTPase Rho3 in regulating growth, conidiation and virulence of Botrytis cinerea.

Small GTPases of the Rho family play an important role in regulating biological processes in fungi. In this study, we mainly investigated the biological functions of Rho3 in Botrytis cinerea, and found that deletion of the rho3 from B. cinerea significantly suppressed vegetative growth and conidiation, reduced appressorium formation and decreased virulence. Microscopy analysis revealed that the distance between septa was increased in the Δrho3 mutant. In addition, mitochondria were suggested to be the main sources of intracellular reactive oxygen species (ROS) in B. cinerea based on dual staining with 2',7'-dichlorodihydrofluorescein diacetate and MitoTracker orange. The Δrho3 mutant showed less accumulation of ROS in the hyphae tips compared to the WT strain of B. cinerea. These results provide the novel evidence to ascertain the function of small GTPase Rho3 in regulating growth, conidiation and virulence of B. cinerea.

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.

Challenging established norms: The unanticipated role of alcohols in UV/PDS radical quenching.

UV/peroxydisulfate (UV/PDS) process is known to be highly efficient for degrading micropollutants from water by generating sulfate (SO4•-) and hydroxyl radicals (HO•). Reliable analyses of short-lived SO4•- and HO• are therefore critical for understanding reaction mechanisms and optimizing operating conditions. Currently, alcohols are commonly used as quenchers to distinguish radicals based on the assumption that they exclusively react with target radicals without other influences. However, this study for the first time reveals a series of unexpected effects that challenge this conventional wisdom because: 1) adding alcohols altered the decomposition rates of PDS by replacing the reactions between SO4•- and HO• with PDS by the reactions between secondary reactive species and PDS; and 2) SO4•- preferably reacted with alcohols to generate nonnegligible level of hydrogen peroxide (H2O2) under oxygen-rich conditions, which subsequently led to indirect formation of HO•. Additionally, the formation of H2O2 was substantially impacted by the types of alcohols, dosages, dissolved oxygen, and solution pH. Using probe tests as tools, we found that the actual SO4•- levels after dosing alcohols were only slightly different from assumed/expected levels, whereas the actually HO• levels were 43.7, 3364.9, and 12.5 times higher than assumed/expected conditions for samples dosed with methanol, iso-propanol, and tert-butanol, respectively. These unanticipated effects thus suggest that cautions are needed when using alcohols to qualitative and quantitative determine HO• and SO4•- in UV/PDS process.

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.

Reactive oxygen species involved in regulating fruit senescence and fungal pathogenicity.

Senescence is a vital aspect of fruit life cycles, and directly affects fruit quality and resistance to pathogens. Reactive oxygen species (ROS), as the primary mediators of oxidative damage in plants, are involved in senescence. Mitochondria are the main ROS and free radical source. Oxidative damage to mitochondrial proteins caused by ROS is implicated in the process of senescence, and a number of senescence-related disorders in a variety of organisms. However, the specific sites of ROS generation in mitochondria remain largely unknown. Recent discoveries have ascertained that fruit senescence is greatly related to ROS and incidental oxidative damage of mitochondrial protein. Special mitochondrial proteins involved in fruit senescence have been identified as the targets of ROS. We focus in discussion on our recent advances in exploring the mechanisms of how ROS regulate fruit senescence and fungal pathogenicity.

A review of factors affecting the formation and roles of primary and secondary reactive species in UV254-based advanced treatment processes.

The presence of organic micropollutants (OMPs) in water has been threatening human health and aquatic ecosystems worldwide. Ultraviolet-based advanced treatment processes (UV-ATPs) are one of the most effective and promising technologies to transform OMPs in water; therefore, an increasing number of emerging UV-ATPs are proposed. However, appropriate selection of UV-ATPs for practical applications is challenging because each UV-ATP generates different types and concentrations of reactive species (RSs) that may not be sufficient to degrade specific types of OMPs. Furthermore, the concentrations and types of RSs are highly influenced by anions and dissolved organic matter (DOM) coexisting in real waters, making systematic understandings of their interfering mechanisms difficult. To identify and address the knowledge gaps, this review provides a comparison of the generations and variations of various types of RSs in different UV-ATPs. These analyses not only prove the importance of water matrices on formation and consumption of primary and secondary RSs under different conditions, but also highlight the non-negligible roles of optical properties and reactivities of DOM and anions. For example, different UV-ATPs may be applicable to different target OMPs under different conditions; and the concentrations and roles of secondary RSs may outperform those of primary RSs in OMP degradation for real applications. With continuous progress and outstanding achievements in the UV-ATPs, it is hoped that the findings and conclusions of this review could facilitate further research and application of UV-ATPs.