Detoxifying bacterial genes for deoxynivalenol epimerization confer durable resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON-producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6-9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co-expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo-keto reductase AKR13B2 for 3-keto-DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes.

The Inhibitory Effect of Pseudomonas stutzeri YM6 on Aspergillus flavus Growth and Aflatoxins Production by the Production of Volatile Dimethyl Trisulfide.

Aspergillus flavus and the produced aflatoxins cause great hazards to food security and human health across all countries. The control of A. flavus and aflatoxins in grains during storage is of great significance to humans. In the current study, bacteria strain YM6 isolated from sea sediment was demonstrated effective in controlling A. flavus by the production of anti-fungal volatiles. According to morphological characteristics and phylogenetic analysis, strain YM6 was identified as Pseudomonas stutzeri. YM6 can produce abundant volatile compounds which could inhibit mycelial growth and conidial germination of A. flavus. Moreover, it greatly prevented fungal infection and aflatoxin production on maize and peanuts during storage. The inhibition rate was 100%. Scanning electron microscopy further supported that the volatiles could destroy the cell structure of A. flavus and prevent conidia germination on the grain surface. Gas chromatography/mass spectrometry revealed that dimethyl trisulfide (DMTS) with a relative abundance of 13% is the most abundant fraction in the volatiles from strain YM6. The minimal inhibitory concentration of DMTS to A. flavus conidia is 200 µL/L (compound volume/airspace volume). Thus, we concluded that Pseudomonas stutzeri YM6 and the produced DMTS showed great inhibition to A. flavus, which could be considered as effective biocontrol agents in further application.

Transcriptome-wide analyses of RNA m6A methylation in hexaploid wheat reveal its roles in mRNA translation regulation.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic messenger RNAs. m6A was discovered in wheat about 40 years ago; however, its potential roles in wheat remain unknown. In this study, we profiled m6As in spikelets transcriptome at the flowering stage of hexaploid wheat and found that m6As are evenly distributed across the A, B, and D subgenomes but their extents and locations vary across homeologous genes. m6As are enriched in homeologous genes with close expression levels and the m6A methylated genes are more conserved. The extent of m6A methylation is negatively correlated with mRNA expression levels and its presence on mRNAs has profound impacts on mRNA translation in a location-dependent manner. Specifically, m6As within coding sequences and 3'UTRs repress the translation of mRNAs while the m6As within 5'UTRs and start codons could promote it. The m6A-containing mRNAs are significantly enriched in processes and pathways of "translation" and "RNA transport," suggesting the potential role of m6As in regulating the translation of genes involved in translation regulation. Our data also show a stronger translation inhibition by small RNAs (miRNA and phasiRNA) than by m6A methylation, and no synergistical effect between the two was observed. We propose a secondary amplification machinery of translation regulation triggered by the changes in m6A methylation status. Taken together, our results suggest translation regulation as a key role played by m6As in hexaploid wheat.

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium-wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium-wheat interactions.

Application of Double-Strand RNAs Targeting Chitin Synthase, Glucan Synthase, and Protein Kinase Reduces Fusarium graminearum Spreading in Wheat.

Controlling the devastating fungal pathogen Fusarium graminearum (Fg) is a challenge due to inadequate resistance in nature. Here, we report on the identification of RNAi molecules and their applications for controlling Fg in wheat through silencing chitin synthase 7 (Chs7), glucan synthase (Gls) and protein kinase C (Pkc). From transgenic Fg strains four RNAi constructs from Chs7 (Chs7RNAi-1, -2, -3, and -4), three RNAi constructs from Gls (GlsRNAi-2, -3, and -6), and one RNAi construct from Pkc (PkcRNAi-5) were identified that displayed effective silencing effects on mycelium growth in medium and pathogenicity in wheat spikes. Transcript levels of Chs7, Gls and Pkc were markedly reduced in those strains. Double-strand RNAs (dsRNAs) of three selected RNAi constructs (Chs7RNAi-4, GlsRNAi-6 and PkcRNA-5) strongly inhibited mycelium growth in vitro. Spray of those dsRNAs on detached wheat leaves significantly reduced lesion sizes; the independent dsRNAs showed comparable effects on lesions with combination of two or three dsRNAs. Expression of three targets Chs7, Gls, and Pkc was substantially down-regulated in Fg-infected wheat leaves. Further application of dsRNAs on wheat spikes in greenhouse significantly reduced infected spikelets. The identified RNAi constructs may be directly used for spray-induced gene silencing and stable expression in plants to control Fusarium pathogens in agriculture.

Novel Soil Bacterium Strain Desulfitobacterium sp. PGC-3-9 Detoxifies Trichothecene Mycotoxins in Wheat via De-Epoxidation under Aerobic and Anaerobic Conditions.

Trichothecenes are the most common mycotoxins contaminating small grain cereals worldwide. The C12,13 epoxide group in the trichothecenes was identified as a toxic group posing harm to humans, farm animals, and plants. Aerobic biological de-epoxidation is considered the ideal method of controlling these types of mycotoxins. In this study, we isolated a novel trichothecene mycotoxin-de-epoxidating bacterium, Desulfitobacterium sp. PGC-3-9, from a consortium obtained from the soil of a wheat field known for the occurrence of frequent Fusarium head blight epidemics under aerobic conditions. Along with MMYPF media, a combination of two antibiotics (sulfadiazine and trimethoprim) substantially increased the relative abundance of Desulfitobacterium species from 1.55% (aerobic) to 29.11% (aerobic) and 28.63% (anaerobic). A single colony purified strain, PGC-3-9, was isolated and a 16S rRNA sequencing analysis determined that it was Desulfitobacterium. The PGC-3-9 strain completely de-epoxidated HT-2, deoxynivalenol (DON), nivalenol and 15-acetyl deoxynivalenol, and efficiently eliminated DON in wheat grains under aerobic and anaerobic conditions. The strain PGC-3-9 exhibited high DON de-epoxidation activity at a wide range of pH (6-10) and temperature (15-50 °C) values under both conditions. This strain may be used for the development of detoxification agents in the agriculture and feed industries and the isolation of de-epoxidation enzymes.

A quinone-dependent dehydrogenase and two NADPH-dependent aldo/keto reductases detoxify deoxynivalenol in wheat via epimerization in a Devosia strain.

The Fusarium mycotoxin deoxynivalenol (DON) is typically controlled by fungicides. Here, we report DON detoxification using enzymes from the highly active Devosia strain D6-9 which degraded DON at 2.5 µg/min/108 cells. Strain D6-9 catabolized DON to 3-keto-DON and 3-epi-DON, completely removing DON in wheat. Genome analysis of three Devosia strains (D6-9, D17, and D13584), with strain D6-9 transcriptomes, identified three genes responsible for DON epimerization. One gene encodes a quinone-dependent DON dehydrogenase QDDH which oxidized DON into 3-keto-DON. Two genes encode the NADPH-dependent aldo/keto reductases AKR13B2 and AKR6D1 that convert 3-keto-DON into 3-epi-DON. Recombinant proteins expressed in Escherichia coli efficiently degraded DON in wheat grains. Molecular docking and site-directed mutagenesis revealed that residues S497, E499, and E535 function in QDDH's DON-oxidizing activity. These results advance potential microbial and enzymatic elimination of DON in agricultural samples and lend insight into the underlying mechanisms and molecular evolution of DON detoxification.

Expression of microRNA-like RNA-2 (Fgmil-2) and bioH1 from a single transcript in Fusarium graminearum are inversely correlated to regulate biotin synthesis during vegetative growth and host infection.

MicroRNA-like RNAs (milRNAs) post-transcriptionally down-regulate target genes. We investigated Fusarium graminearum (Fg) milRNA expression during fungal vegetative growth and infection of wheat. Small RNA sequencing identified 36 milRNAs from Fg, one of which, Fgmil-2, had >100 transcripts per million in conidia, mycelia and infected wheat, with the highest expression in conidia and the lowest expression in colonized wheat tissue. Fgmil-2 displays perfect homology to the 3'-untranslated region (3'-UTR) of an FgbioH1 messenger RNA that is involved in biotin biosynthesis. Poly(A) polymerase-mediated rapid amplification of cDNA ends combined with sequencing analysis demonstrated that cleavage at a specific site by FgDicer2 in the 3'-UTR of FgbioH1 transcripts generated the Fgmil-2 precursor with a typical hairpin structure. Deletion of FgbioH1 or FgDicer2 genes abolished Fgmil-2 biogenesis. FgbioH1 had an inversely correlated pattern of expression to that of Fgmil-2 and FgDicer2. Deletion of FgbioH1 also showed that it is required for mycelial growth, virulence, mycotoxin biosynthesis and expression of biotin-dependent carboxylase genes. This study reveals in Fg a novel mode of inversely correlated post-transcriptional regulation in which Fgmil-2 originates from its own target transcript, FgbioH, to govern biotin biosynthesis.

Inhibitory Effect of Volatiles Emitted From Alcaligenes faecalis N1-4 on Aspergillus flavus and Aflatoxins in Storage.

Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 µL/L, respectively. The effective concentration for MI against A. flavus is 200 µL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.

Metabolic Changes of Fusarium graminearum Induced by TPS Gene Deletion.

Fusarium head blight (FHB) mainly resulting from Fusarium graminearum (Fg) Schwabe is a notorious wheat disease causing huge losses in wheat production globally. Fg also produces mycotoxins, which are harmful to human and domestic animals. In our previous study, we obtained two Fg mutants, TPS1- and TPS2-, respectively, with a single deletion of trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) compared with the wild type (WT). Both mutants were unable to synthesize trehalose and produced fewer mycotoxins. To understand the other biochemical changes induced by TPS gene deletion in Fg, we comprehensively analyzed the metabolomic differences between TPS- mutants and the WT using NMR together with gas chromatography-flame ionization detection/mass spectrometry. The expression of some relevant genes was also quantified. The results showed that TPS1- and TPS2- mutants shared some common metabolic feature such as decreased levels for trehalose, Val, Thr, Lys, Asp, His, Trp, malonate, citrate, uridine, guanosine, inosine, AMP, C10:0, and C16:1 compared with the WT. Both mutants also shared some common expressional patterns for most of the relevant genes. This suggests that apart from the reduced trehalose biosynthesis, both TPS1 and TPS2 have roles in inhibiting glycolysis and the tricarboxylic acid cycle but promoting the phosphopentose pathway and nucleotide synthesis; the depletion of either TPS gene reduces the acetyl-CoA-mediated mycotoxin biosynthesis. TPS2- mutants produced more fatty acids than TPS1- mutants, suggesting different roles for TPS1 and TPS2, with TPS2- mutants having impaired trehalose biosynthesis and trehalose 6-phosphate accumulation. This may offer opportunities for developing new fungicides targeting trehalose biosynthesis in Fg for FHB control and mycotoxin reduction in the FHB-affected cereals.

Variation in the Microbiome, Trichothecenes, and Aflatoxins in Stored Wheat Grains in Wuhan, China.

Contamination by fungal and bacterial species and their metabolites can affect grain quality and health of wheat consumers. In this study, sequence analyses of conserved DNA regions of fungi and bacteria combined with determination of trichothecenes and aflatoxins revealed the microbiome and mycotoxins of wheat from different silo positions (top, middle, and bottom) and storage times (3, 6, 9, and 12 months). The fungal community in wheat on the first day of storage (T0) included 105 classified species (81 genera) and 41 unclassified species. Four species had over 10% of the relative abundance: Alternaria alternata (12%), Filobasidium floriforme (27%), Fusarium graminearum (12%), and Wallemia sebi (12%). Fungal diversity and relative abundance of Fusarium in wheat from top silo positions were significantly lower than at other silo positions during storage. Nivalenol and deoxynivalenol in wheat were 13⁻34% higher in all positions at 3 months compared to T0, and mycotoxins in wheat from middle and bottom positions at 6 to 12 months were 24⁻57% higher than at T0. The relative abundance of toxigenic Aspergillus and aflatoxins were low at T0 and during storage. This study provides information on implementation and design of fungus and mycotoxin management strategies as well as prediction models.

MycoKey Round Table Discussions of Future Directions in Research on Chemical Detection Methods, Genetics and Biodiversity of Mycotoxins.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

An aldo-keto reductase is responsible for Fusarium toxin-degrading activity in a soil Sphingomonas strain.

Degradation of toxins by microorganisms is a promising approach for detoxification of agricultural products. Here, a bacterial strain, Sphingomonas S3-4, that has the ability to degrade the mycotoxin deoxynivalenol (DON) was isolated from wheat fields. Incubation of Fusarium-infected wheat grains with S3-4 completely eliminated DON. In S3-4 DON is catabolized into compounds with no detectable phytotoxicity, 3-oxo-DON and 3-epi-DON, via two sequential reactions. Comparative analysis of genome sequences from two DON-degrading strains, S3-4 and Devosia D17, and one non-DON-degrading strain, Sphingobium S26, combined with functional screening of a S3-4 genomic BAC library led to the discovery that a novel aldo/keto reductase superfamily member, AKR18A1, is responsible for oxidation of DON into 3-oxo-DON. DON-degrading activity is completely abolished in a mutant S3-4 strain where the AKR18A1 gene is disrupted. Recombinant AKR18A1 protein expressed in Escherichia coli catalyzed the reversible oxidation/reduction of DON at a wide range of pH values (7.5 to 11) and temperatures (10 to 50 °C). The S3-4 strain and recombinant AKR18A1 also catabolized zearalenone and the aldehydes glyoxal and methyglyoxal. The S3-4 strain and the AKR18A1 gene are promising agents for the control of Fusarium pathogens and detoxification of mycotoxins in plants and in food/feed products.

Combined Metabonomic and Quantitative RT-PCR Analyses Revealed Metabolic Reprogramming Associated with Fusarium graminearum Resistance in Transgenic Arabidopsis thaliana.

Fusarium head blight disease resulting from Fusarium graminearum (FG) infection causes huge losses in global production of cereals and development of FG-resistant plants is urgently needed. To understand biochemistry mechanisms for FG resistance, here, we have systematically investigated the plant metabolomic phenotypes associated with FG resistance for transgenic Arabidopsis thaliana expressing a class-I chitinase (Chi), a Fusarium-specific recombinant antibody gene (CWP2) and fused Chi-CWP2. Plant disease indices, mycotoxin levels, metabonomic characteristics, and expression levels of several key genes were measured together with their correlations. We found that A. thaliana expressing Chi-CWP2 showed higher FG resistance with much lower disease indices and mycotoxin levels than the wild-type and the plants expressing Chi or CWP2 alone. The combined metabonomic and quantitative RT-PCR analyses revealed that such FG-resistance was closely associated with the promoted biosynthesis of secondary metabolites (phenylpropanoids, alkanoids) and organic osmolytes (proline, betaine, glucose, myo-inositol) together with enhanced TCA cycle and GABA shunt. These suggest that the concurrently enhanced biosyntheses of the shikimate-mediated secondary metabolites and organic osmolytes be an important strategy for A. thaliana to develop and improve FG resistance. These findings provide essential biochemical information related to FG resistance which is important for developing FG-resistant cereals.

Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment.

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5-10) and temperatures (20-37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 µg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

An antibody that confers plant disease resistance targets a membrane-bound glyoxal oxidase in Fusarium.

Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species.

The Shewanella algae strain YM8 produces volatiles with strong inhibition activity against Aspergillus pathogens and aflatoxins.

Aflatoxigenic Aspergillus fungi and associated aflatoxins are ubiquitous in the production and storage of food/feed commodities. Controlling these microbes is a challenge. In this study, the Shewanella algae strain YM8 was found to produce volatiles that have strong antifungal activity against Aspergillus pathogens. Gas chromatography-mass spectrometry profiling revealed 15 volatile organic compounds (VOCs) emitted from YM8, of which dimethyl trisulfide was the most abundant. We obtained authentic reference standards for six of the VOCs; these all significantly reduced mycelial growth and conidial germination in Aspergillus; dimethyl trisulfide and 2,4-bis(1,1-dimethylethyl)-phenol showed the strongest inhibitory activity. YM8 completely inhibited Aspergillus growth and aflatoxin biosynthesis in maize and peanut samples stored at different water activity levels, and scanning electron microscopy revealed severely damaged conidia and a complete lack of mycelium development and conidiogenesis. YM8 also completely inhibited the growth of eight other agronomically important species of phytopathogenic fungi: A. parasiticus, A. niger, Alternaria alternate, Botrytis cinerea, Fusarium graminearum, Fusarium oxysporum, Monilinia fructicola, and Sclerotinia sclerotiorum. This study demonstrates the susceptibility of Aspergillus and other fungi to VOCs from marine bacteria and indicates a new strategy for effectively controlling these pathogens and the associated mycotoxin production during storage and possibly in the field.

Induction of Oral Tolerance with Transgenic Plants Expressing Antigens for Prevention/Treatment of Autoimmune, Allergic and Inflammatory Diseases.

The prevalence and incidence of autoimmune and allergic diseases have increased dramatically over the last several decades, especially in the developed world. The treatment of autoimmune and allergic diseases is typically with the use of non-specific immunosuppressive agents that compromise the integrity of the host immune system and therefore, increase the risk of infections. Antigenspecific immunotherapy by reinstating immunological tolerance towards self antigens without compromising immune functions is a much desired goal for the treatment of autoimmune and allergic diseases. Mucosal administration of antigen is a long-recognized method of inducing antigen-specific immune tolerance known as oral tolerance, which is viewed as having promising potential in the treatment of autoimmune and allergic diseases. Plant-based expression and delivery of recombinant antigens provide a promising new platform to induce oral tolerance, having considerable advantages including reduced cost and increased safety. Indeed, in recent years the use of tolerogenic plants for oral tolerance induction has attracted increasing attention, and considerable progress has been made. This review summarizes recent advances in using plants to deliver tolerogens for induction of oral tolerance in the treatment of autoimmune, allergic and inflammatory diseases.

Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat.

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co-expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T3 to T5 generations. Confocal microscopy revealed profoundly restricted mycelia in Fg-infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down-regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host-induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.