The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Rapid Detection and Quantification of Patulin and Citrinin Contamination in Fruits.

Patulin (PAT) and citrinin (CTN) are the most common mycotoxins produced by Penicillium and Aspergillus species and are often associated with fruits and fruit by-products. Hence, simple and reliable methods for monitoring these toxins in foodstuffs are required for regular quality assessment. In this study, we aimed to establish a cost-effective method for detection and quantification of PAT and CTN in pome fruits, such as apples and pears, using high-performance liquid chromatography (HPLC) coupled with spectroscopic detectors without the need for any clean-up steps. The method showed good performance in the analysis of these mycotoxins in apple and pear fruit samples with recovery ranges of 55-97% for PAT and 84-101% for CTN, respectively. The limits of detection (LOD) of PAT and CTN in fruits were 0.006 µg/g and 0.001 µg/g, while their limits of quantification (LOQ) were 0.018 µg/g and 0.003 µg/g, respectively. The present findings indicate that the newly developed HPLC method provides rapid and accurate detection of PAT and CTN in fruits.

Pseudozyma aphidis Induces Salicylic-Acid-Independent Resistance to Clavibacter michiganensis in Tomato Plants.

The ability of plant pathogens to rapidly develop resistance to commonly used pesticides challenges efforts to maximize crop production. Fungal biocontrol agents have become an important alternative to chemical fungicides as a result of environmental concerns regarding conventional pesticides, including resistance issues. The complex mode of action of biocontrol agents reduces the likelihood that pathogens will develop resistance to them. We recently isolated a unique, biologically active isolate of the epiphytic fungus Pseudozyma aphidis. We show that the extracellular metabolites secreted by our P. aphidis isolate can inhibit Xanthomonas campestris pv. vesicatoria, X. campestris pv. campestris, Pseudomonas syringae pv. tomato, Erwinia amylovora, Clavibacter michiganensis, and Agrobacterium tumefaciens in vitro. Moreover, application of Pseudozyma aphidis spores on tomato plants in the greenhouse significantly reduced (by 60%) the incidence of bacterial wilt and canker disease caused by C. michiganensis subsp. michiganensis on those plants as well as disease severity by 35%. Furthermore, infected plants treated with P. aphidis were 25% taller than control infected plants. We found that P. aphidis activates PR1a-and other pathogenesis-related genes in tomato plants-and can trigger an induced-resistance response against C. michiganensis that proceeds in a salicylic-acid-independent manner, as shown using NahG-transgenic tomato plants.

The transcription factor SlSHINE3 modulates defense responses in tomato plants.

The cuticle plays an important role in plant interactions with pathogens and with their surroundings. The cuticle acts as both a physical barrier against physical stresses and pathogens and a chemical deterrent and activator of the plant defense response. Cuticle production in tomato plants is regulated by several transcription factors, including SlSHINE3, an ortholog of the Arabidopsis WIN/SHN3. Here we used a SlSHINE3-overexpressing (SlSHN3-OE) and silenced (Slshn3-RNAi) lines and a mutant in SlCYP86A69 (Slcyp86A69)--a direct target of SlSHN3--to analyze the roles of the leaf cuticle and cutin content and composition in the tomato plant's defense response to the necrotrophic foliar pathogen Botrytis cinerea and the biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria. We showed that SlSHN3, which is predominantly expressed in tomato fruit epidermis, also affects tomato leaf cuticle, as morphological alterations in the SlSHN3-OE leaf tissue resulted in shiny, stunted and permeable leaves. SlSHN3-OE leaves accumulated 38% more cutin monomers than wild-type leaves, while Slshn3-RNAi and Slcyp86A69 plants showed a 40 and 70% decrease in leaf cutin monomers, respectively. Overexpression of SlSHN3 resulted in resistance to B. cinerea infection and to X. campestris pv. vesicatoria, correlated with cuticle permeability and elevated expression of pathogenesis-related genes PR1a and AOS. Further analysis revealed that B. cinerea-infected Slshn3-RNAi plants are more sensitive to B. cinerea and produce more hydrogen peroxide than wild-type plants. Cutin monomer content and composition differed between SlSHN3-OE, Slcyp86A69, Slshn3-RNAi and wild-type plants, and cutin monomer extracted from SlSHN3-OE plants altered the expression of pathogenesis-related genes in wild-type plants.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA.

In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT-PCR and quantitative real-time PCR assays were established. The system was used to examine the structural requirements for SL RNA as a substrate in the reaction. Interestingly, synthetic SL RNA assembled poorly to its cognate particle and was not utilized in the reaction. However, SL RNA synthesized in cells lacking Sm proteins, which is defective in cap-4 modification, was active in the reaction. This study is the first step towards further elucidating the mechanism of trans-splicing, an essential reaction which determines the trypanosome transcriptome.

IQD1 Involvement in Hormonal Signaling and General Defense Responses Against Botrytis cinerea.

IQ Domain 1 (IQD1) is a novel Arabidopsis thaliana calmodulin-binding protein, which was found to be a positive regulator of glucosinolate (GS) accumulation and plant defense responses against insects. We demonstrate here that the IQD1 overexpressing line (IQD1 OXP ) was also more resistant also to the necrotrophic fungus Botrytis cinerea, whereas an IQD1 knockout line (iqd1-1) was much more sensitive. Furthermore, we showed that IQD1 is up-regulated by jasmonic acid (JA) and downregulated by salicylic acid (SA). A comparison of whole transcriptome expression between iqd1-1 and wild type plants revealed a substantial downregulation of genes involved in plant defense and hormone regulation. Further examination revealed a marked reduction of SA and increases in the levels of ethylene, JA and abscisic acid response genes in the iqd1-1 line. Moreover, quantification of SA, JA, and abscisic acids in IQD1 OXP and iqd1-1 lines relative to the wild type, showed a significant reduction in endogenous JA levels in the knockout line, simultaneously with increased SA levels. Relations between IQD1 OXP and mutants defective in plant-hormone response indicated that IQD1 cannot rescue the absence of NPR1 or impaired SA accumulation in the NahG line. IQD1 cannot rescue ein2 or eto1 mutations connected to the ethylene pathway involved in both defense responses against B. cinerea and in regulating GS accumulation. Furthermore, IQD1cannot rescue the aos, coi1 or jar1mutations, all involved in the defense response against B. cinerea and it depends on JAR1 to control indole glucosinolate accumulation. We also found that in the B. cinerea, which infected the iqd1-1 mutant, the most abundant upregulated group of proteins is involved in the degradation of complex carbohydrates, as correlated with the sensitivity of this mutant. In summary, our results suggest that IQD1 is an important A. thaliana defensive protein against B. cinerea that is integrated into several important pathways, such as those involved in plant defense and hormone responses.

Ensiling process and pomegranate peel extract as a natural additive in potential prevention of fungal and mycotoxin contamination in silage.

A study was conducted on six animal feed centers in Israel where fungal and mycotoxin presence was examined in maize and wheat silages. Fumonisin mycotoxins FB1 and FB2 were present in every maize silage sample analyzed. Interestingly, no correlation was found between the occurrence of specific mycotoxins and the presence of the fungal species that might produce them in maize and wheat silages. We further investigated the effect of pomegranate peel extract (PPE) on Fusarium infection and fumonisin biosynthesis in laboratory-prepared maize silage. PPE had an inhibitory effect on FB1 and FB2 biosynthesis by Fusarium proliferatum, which resulted in up to 90 % reduction of fumonisin production in silage samples compared to untreated controls. This finding was supported by qRT-PCR analysis, showing downregulation of key genes involved in the fumonisin-biosynthesis pathway under PPE treatment. Our results present promising new options for the use of natural compounds that may help reduce fungal and mycotoxin contamination in agricultural foodstuff, and potentially replace traditionally used synthetic chemicals.

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism.

Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 5' and 3' flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. PTB1, but not PTB2, also affects cis-splicing. The specificity of binding of PTB1 was established in vivo and in vitro using a model substrate. This study demonstrates for the first time that trans-splicing of only certain substrates requires specific factors such as PTB proteins for their splicing. The trypanosome PTB proteins, like their mammalian homologs, represent multivalent RNA binding proteins that regulate mRNAs from their synthesis to degradation.

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in Aspergillus carbonarius.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC resulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB.

Penicillium expansum is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, P. expansum produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in Aspergillus species, and impact virulence of plant pathogenic fungi. Here we show that deletion of sntB in P. expansum results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis in vitro and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔsntB strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO2 levels) to which P. expansum is commonly exposed during fruit storage, resulted in a significant reduction of sntB expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.

Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1.

Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF, consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 were affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing one of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or not detectable.

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some Fusarium and Aspergillus spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi Aspergillus flavus and Fusarium proliferatum, both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by A. flavus up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.

MFS transporter from Botrytis cinerea provides tolerance to glucosinolate-breakdown products and is required for pathogenicity.

Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.

The effects of glucosinolates and their breakdown products on necrotrophic fungi.

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.