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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.

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&ndash, 10) and temperature (15&ndash, 50 &deg, 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.

The 5-enolpyruvylshikimate-3-phosphate synthases (EPSPSs) encoded by aroA genes are traditionally divided into two classes, and only the class II EPSPS is naturally insensitive to herbicide glyphosate. Here, a novel EPSPS from Isoptericola variabilis, designated as I. variabilis EPSPS, was characterized. Its aroA gene contained 1374 bp with 77% of GC contents, encoding a peptide of 458 amino acids. The EPSPS carried a highly conserved region involved in substrate bindings in EPSPSs and displayed a high sequence and structure similarity to class I EPSPS. However, this EPSPS had high naturally glyphosate-tolerant capability and enzymatic kinetics constants similar to that of class II EPSPSs. Escherichia coli AB2829 harboring the aroA gene grew well in medium containing 150 mM glyphosate. The purified EPSPS had the highest enzymatic activity at pH 8.0 and 30 °C, and retained its activity at pH 6–10 and 20–60 °C. This enzyme had a Km value of 28 μM for substrate phosphoenolpyruvate and 111 μM for shikimate-3-phosphate, respectively, with a rather high Ki value for glyphosate. Cations increased substrate affinity but slightly reduced glyphosate tolerance. The I. variabilis EPSPS may be used for generating transgenic glyphosate-tolerant plants and dissecting mechanisms underlying the glyphosate tolerance.

Glyphosate is the most widely used herbicide for its low cost and high efficiency. However, it is rarely applied directly in rice field due to its toxicity to rice. Therefore, glyphosate-tolerant rice can greatly decrease the cost of rice production and provide a more effective weed management strategy. Although, several approaches to develop transgenic rice with glyphosate tolerance have been reported, the agronomic performances of these plants have not been well evaluated, and the feasibility of commercial production has not been confirmed yet. Here, a novel glyphosate-tolerant gene cloned from the bacterium Isoptericola variabilis was identified, codon optimized (designated as I. variabilis-EPSPS (*)), and transferred into Zhonghua11, a widely used japonica rice cultivar. After systematic analysis of the transgene integration via PCR, Southern blot and flanking sequence isolation, three transgenic lines with only one intact I. variabilis-EPSPS (*) expression cassette integrated into intergenic regions were identified. Seed test results showed that the glyphosate tolerance of the transgenic rice was about 240 times that of wild type on plant medium. The glyphosate tolerance of transgenic rice lines was further evaluated based on comprehensive agronomic performances in the field with T3 and T5generations in a 2-year assay, which showed that they were rarely affected by glyphosate even when the dosage was 8400 g ha(-1). To our knowledge, this is the first demonstration of the development of glyphosate-tolerant rice lines based on a comprehensive analysis of agronomic performances in the field. Taken together, the results suggest that the selected glyphosate-tolerant rice lines are highly tolerant to glyphosate and have the possibility of commercial release. I. variabilis-EPSPS (*) also can be a promising candidate gene in other species for developing glyphosate-tolerant crops.

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.

As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops and can facilitate a more economical and simplified weed control system.

N-acyl-homoserine lactones (AHLs) are the main quorum-sensing (QS) signals in gram-negative bacteria. AHLs trigger the expression of genes for particular biological functions when their density reaches a threshold. In this study, we identified and cloned the qsdH gene by screening a genomic library of Pseudoalteromonas byunsanensis strain 1A01261, which has AHL-degrading activity. The qsdH gene encoded a GDSL hydrolase found to be located in the N-terminus of a multidrug efflux transporter protein of the resistance-nodulation-cell division (RND) family. We further confirmed that the GDSL hydrolase, QsdH, exhibited similar AHL-degrading activity to the full-length ORF protein. QsdH was expressed and purified to process the N-terminal signal peptide yielding a 27-kDa mature protein. QsdH was capable of inactivating AHLs with an acyl chain ranging from C(4) to C(14) with or without 3-oxo substitution. High-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) analyses showed that QsdH functioned as an AHL lactonase to hydrolyze the ester bond of the homoserine lactone ring of AHLs. In addition, site-directed mutagenesis demonstrated that QsdH contained oxyanion holes (Ser-Gly-Asn) in conserved blocks (I, II, and III), which had important roles in its AHL-degrading activity. Furthermore, the lactonase activity of QsdH was slightly promoted by several divalent ions. Using in silico prediction, we concluded that QsdH was located at the first periplasmic loop of the multidrug efflux transporter protein, which is essential to substrate selectivity for these efflux pumps. These findings led us to assume that the QsdH lactonase and C-terminal efflux pump might be effective in quenching QS of the P. byunsanensis strain 1A01261. Moreover, it was observed that recombinant Escherichia coli producing QsdH proteins attenuated the plant pathogenicity of Erwinia carotovora, which might have potential to control of gram-negative pathogenic bacteria.

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.