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.

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.

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.

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.

Tissue-specific and pathogen-inducible expression of a fusion protein containing a Fusarium-specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins.

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.-specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea-specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea-specific in transgenic wheat. Single-floret inoculation of independent transgenic wheat lines of the T3 to T6 generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography-mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB-susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real-time PCR analysis revealed that the tissue-specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue-specific and pathogen-inducible expression of this Fusarium-specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.

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.

Recent developments in therapeutic protein expression technologies in plants.

Infectious diseases and cancers are some of the commonest causes of deaths throughout the world. The previous two decades have witnessed a combined endeavor across various biological sciences to address this issue in novel ways. The advent of recombinant DNA technologies has provided the tools for producing recombinant proteins that can be used as therapeutic agents. A number of expression systems have been developed for the production of pharmaceutical products. Recently, advances have been made using plants as bioreactors to produce therapeutic proteins directed against infectious diseases and cancers. This review highlights the recent progress in therapeutic protein expression in plants (stable and transient), the factors affecting heterologous protein expression, vector systems and recent developments in existing technologies and steps towards the industrial production of plant-made vaccines, antibodies, and biopharmaceuticals.

Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum.

Trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) are required for trehalose biosynthesis in yeast and filamentous fungi, including Fusarium graminearum. Three null mutants Δtps1, Δtps2 and Δtps1-Δtps2, each carrying either a single deletion of TPS1 or TPS2 or a double deletion of TPS1-TPS2, were generated from a toxigenic F. graminearum strain and were not able to synthesize trehalose. In contrast to its reported function in yeasts and filamentous fungi, TPS1 appeared dispensable for development and virulence. However, deletion of TPS2 abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. The cell polarity alteration was exhibited as reduced apical growth and increased lateral growth and branching with increased hyphal and cell wall widths. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution in its conidia and vegetative hyphae. The Δtps2 mutant also had 62% lower mycelial growth on potato dextrose agar and 99% lower virulence on wheat compared with the wild-type. The Δtps1, Δtps2 and Δtps1-Δtps2 mutants synthesized over 3.08-, 7.09- and 2.47-fold less mycotoxins, respectively, on rice culture compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively, with significantly changed expression profiles compared with the wild-type. Further dissection of this pathway will provide new insights into regulation of fungal development, virulence and trichothecene biosynthesis.

Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) µg/mL, 1000-fold more sensitive than that reported previously (1 µg/mL). The fusion protein was able to detect fungal concentrations below 1 µg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 µg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity.

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.

Generation of a highly reactive chicken-derived single-chain variable fragment against Fusarium verticillioides by phage display.

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10(-2) µg/mL and contaminating mycelium at a quantity of 10(-2) mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

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.

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.

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes of Fusarium graminearum induced by Tri5 gene deletion.

Fusarium graminearum (FG) is a serious plant pathogen causing huge losses in global production of wheat and other cereals. Tri5-gene encoded trichodiene synthase is the first key enzyme for biosynthesis of trichothecene mycotoxins in FG. To further our understandings of FG metabolism which is essential for developing novel strategies for controlling FG, we conducted a comprehensive investigation on the metabolic changes caused by Tri5-deletion by comparing metabolic differences between the wild-type FG5035 and an FG strain, Tri5(-), with Tri5 deleted. NMR methods identified more than 50 assigned fungal metabolites. Combined metabonomic and quantitative RT-PCR (qRT-PCR) analyses revealed that Tri5 deletion caused significant and comprehensive metabolic changes for FG apart from mycotoxin biosynthesis. These changes involved both carbon and nitrogen metabolisms including alterations in GABA shunt, TCA cycle, shikimate pathway, and metabolisms of lipids, amino acids, inositol, choline, pyrimidine, and purine. The hexose transporter has also been affected. These findings have shown that Tri5 gene deletion induces widespread changes in FG primary metabolism and demonstrated the combination of NMR-based metabonomics and qRT-PCR analyses as a useful way to understand the systems metabolic changes resulting from a single specific gene knockout in an eukaryotic genome and thus Tri5 gene functions.

Difference in TRI13 gene sequences between the 3-acetyldeoxynivalenol producing Fusarium graminearum chemotypes from Canada and China.

Positive-negative PCR assays based on the genes involved in the trichothecene biosynthesis pathway are useful in assessing the risk of trichothecene contamination in grain and are important in epidemiological studies. A single PCR detection method based on the structural gene sequence of TRI13 gene has been developed to predict the 3-ADON, 15-ADON and NIV chemotypes in China. The chemotypic differences are based on the deletions within the TRI13 gene. The objective of this study was to assess the reliability of using this single primer based on the TRI13 gene to differentiate the F. graminearum chemotypes in Canada. In this study, we found that, this single PCR detection method based on the deletions in the TRI13 gene cannot be used to differentiate the 3-ADON and 15-ADON chemotypes in the Canadian F. graminearum isolates; further sequence analysis of the PCR products confirmed that both Canadian 3-ADON and 15-ADON chemotypes have the 61 bp deletion in the TRI13 gene. This 61 bp deletion was absent in the Chinese 3-ADON isolates. Therefore these findings revealed that there are genetic differences between the examined 3-ADON F. graminearum isolates from Canada and China. The observed genetic differences between the 3-ADON chemotype populations in Canada and China may be resulted from a random mutation (insertion/deletion) that took place in one of the populations and accumulated due to genetic drift and/or selection.

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.

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.

Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered structure of cell walls and reduced virulence.

Natural resistance of wheat against Fusarium head blight (FHB) is inadequate and new strategies for controlling the disease are required. Chitin synthases that catalyze chitin biosynthesis would be an ideal target for antifungal agents. In this study, a class I chitin synthase gene (CHS1) from Fusarium asiaticum, the predominant species of FHB pathogens on wheat in China, was functionally disrupted via Agrobacterium tumefaciens-mediated transformation. Specific disruption of the CHS1 gene resulted in a 58% reduction of chitin synthase activity, accompanied by decreases of 35% in chitin content, 22% in conidiation, and 16% in macroconidium length. The Deltachs1 mutant strain had a growth rate comparable to that of the wild-type on PDA medium but had a 35% increase in the number of nuclear cellulae and exhibited a remarkably increased sensitivity to osmosis stresses. Electron microscopy revealed substantial changes occurring in cell wall structures of the macroconidium, ascospore, and mycelium, with the most profound changes in the mycelium. Furthermore, the Deltachs1 mutant displayed significantly reduced pathogenicity on wheat spikes and seedlings. Re-introduction of a functional CHS1 gene into the Deltachs1 mutant strain restored the wild-type phenotype. These results reveal an important in vivo role played by a CHS1 gene in a FHB pathogen whose mycelial chitin could serve as a target for controlling the disease.

Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons.

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO(3) supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l alpha-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.

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.