Construction of quintuple protease gene disruptant for heterologous protein production in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Aspergillus oryzae has received attention as a host for heterologous protein production. However, A. oryzae has 134 protease genes, which is recognized to be one of the major reasons for the proteolytic degradation of heterologously produced proteins. We previously reported that double disruption of the protease genes (tppA and pepE) improved heterologous protein (human lysozyme) production by A. oryzae. In this study, we performed successive round of five protease genes (tppA, pepE, nptB, dppIV, and dppV) disruption in A. oryzae by pyrG marker recycling with highly efficient gene-targeting background (ΔligD). The multiple disruption of protease genes were confirmed by Southern blot analysis. Furthermore, the quintuple protease gene disruptants showed the maximum production level of bovine chymosin (CHY) that was 34% higher than those of the double protease gene disruptant (ΔtppA ΔpepE). Consequently, we successfully constructed a multiple protease gene disruptant bearing enhanced levels of CHY productivity. We presented the first evidence that the quintuple disruption of the protease genes improved the production level of a heterologous protein by A. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Phytosulfokine-α Promotes Root Growth by Repressing Expression of <i>Pectin Methylesterase Inhibitor</i> (<i>PMEI</i>) Genes in <i>Medicago truncatula</i>

Phytosulfokine-α (PSK-α), a sulfated pentapeptide with the sequence YIYTQ, is encoded by a small precursor gene family in Arabidopsis. PSK-α regulates multiple growth and developmental processes as a novel peptide hormone. Despite its importance, functions of PSK-α in M. truncatula growth remains unknown. In this study, we identified five genes to encode PSK-α precursors in M. truncatula. All of these precursors possess conserved PSK-α signature motif. Expression pattern analysis of these MtPSK genes revealed that each gene was expressed in a tissue-specific or ubiquitous pattern and three of them were remarkably expressed in root. Treatment of M. truncatula seedlings with synthetic PSK- α peptide significantly promoted root elongation. In addition, expression analysis of downstream genes by RNA-seq and qRT-PCR assays suggested that PSK-α signaling might regulate cell wall structure via PMEI-PME module to promote root cell growth. Taken together, our results shed light on the mechanism by which PSK-α promotes root growth in M. truncatula, providing a new resource for improvement of root growth in agriculture.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

A sandwiched-culture technique for evaluation of heterologous protein production in a filamentous fungus

Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein.     

Improved gene targeting frequency in marine-derived filamentous fungus Aspergillus glaucus by disrupting ligD

Filamentous fungi from the marine environment have shown great potential as cell factories for the production of pharmacologically active metabolites, but extremely low frequency of homologous recombination brings difficulty to further molecular biology studies. To bypass this problem and develop a highly efficient gene targeting system in marine-derived filamentous fungus Aspergillus glaucus, LigD, a homolog of Neurospora crassa Mus-53 which is considered to play a significant role in nonhomologous end joining (NHEJ), was coloned and deleted, and frequency of targeted gene replacement (TGR) increased dramatically from <2% to 85% in comparison with that in the wild type, when containing 1000 bp of homologous flanking sequence. Such results strongly indicate that AgLigD is indeed involved in the repair of NHEJ in A. glaucus and functions in this pathway. Furthermore, the AgLigD-defective mutant has no discernible differences with wild type regarding sensitivity to mutagens and UV, growth characteristics and transformation frequency. The AgligD-deficient transformant, as the first NHEJ-defective mutant in the field of marine-derived filamentous fungus, will help in expediting studies of molecular biology of marine-derived microorganisms.     

Improved tryprostatin B production by heterologous gene expression in Aspergillus nidulans

Tryprostatin B, a prenylated diketopiperazine with anti-tubulin activity, has been overproduced in fungal culture by expression of genes of the fumitremorgin cluster from Aspergillus fumigatus in the naïve host Aspergillus nidulans using the alcA promoter. The products of the expressed genes catalyse the first two steps of fumitremorgin biosynthesis, namely the formation of brevianamide F and its conversion to tryprostatin B. Yields of tryprostatin B were up to 250 mg/l, a significant improvement in previously reported levels. This approach illustrates how the availability of fungal genome sequences and knowledge of gene function can be used to achieve the efficient production of biologically active secondary metabolites by genetic manipulation.     

Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Objectives

To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins.

Results

To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations.

Conclusions

We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

     

Vegetative Compatibility and Virulence Diversity of <i>Verticillium dahliae</i> from Okra (<i>Abelmoschus esculentus</i>) Plantations in Turkey and Evaluation of Okra Landraces for Resistance to <i>V. dahliae</i>

Forty-four V. dahliae isolates were collected from symptomatic vascular tissues of okra plants each from a different field in eight provinces located in the eastern Mediterranean and western Anatolia regions of Turkey during 2006- 2009. Nitrate-nonutilizing (nit) mutants of V. dahliae from okra were used to determine heterokaryosis and genetic relatedness among isolates. All isolates from okra plants were grouped into two vegetative compatibility groups (VCGs) (1 and 2) and three subgroups as 1A (13.6%, 6/44), 2A (20.5%, 9/44) and 2B (65.9%, 29/44) according to international criteria. Pathogenicity tests were performed on a susceptible local okra (A. esculentus) landrace in greenhouse conditions. All isolates from VCG1A and VCG2B induced defoliation (D) and partial defoliation (PD) symptoms, respectively. Other isolates from VCG2A gave rise to typical leaf chlorosis symptoms without defoliation. The obtained data showed that the virulence level of V. dahliae isolates from okra was related to their VCG belongings. Eighteen okra landraces from diverse geographical origins were screened for resistance to VCG2B and VCG1A of V. dahliae. The results indicated that all landraces were more susceptible to highly virulent VCG1A-D pathotype displaying D or PD symptoms depending on their susceptibility levels with a mean disease severity index of 3.52 than to less virulent VCG2B-PD pathotype of V. dahliae displaying PD and ND symptoms with a mean disease severity index of 2.52. Significant differences were observed among the landraces; however, none of them exhibited a level of resistance. Okra landraces; Çorum, Hatay Has and Şanlıurfa displayed the lowest level of susceptibility or little tolerance to both D and PD pathotypes. VCG2B of PD was prevailing in the surveyed areas and VCG1A of D was the most virulent of the VCGs identified. Introduction of resistant genotypes to Turkish okra germplasm from different sources and breeding new resistant okra cultivars are critical for the sustainability of okra production.     

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.     

Micropropagation of Daylily (<i>Hemerocallis fulva</i>) from Crown-Tip Explants and Assessment of Somaclonal Variation of <i>in Vitro</i>-Propagated Plants Using SCoT Markers

Determination of the somaclonal variation of in vitro-propagated plants is crucial to determine the appropriatemicropropagation protocol and growth regulators for commercial scale multiplication. In this research, nine multiplication media (MM) augmented with different concentrations of 6-benzyl adenine (BA), Kinetin (Kin), andThidiazuron (TDZ), Three rooting media (RM) supplemented with three levels of α-naphthalene acetic acid(NAA) and three types of soil mixtures (v/v); Coco peat/Vermiculite/Sand (CVS), Peat moss/Perlite/Sand(PPS) and Peat moss/Perlite (PP) were used in the micropropagation protocol of daylily plants. MM2 showedthe maximum shoot length and the number of leaves, while MM9 showed the maximum number of shoots.The RM1 showed the maximum root length and the number of roots. During acclimatization, CVS, PPS, andPP soil mixture showed similar performance except the CVS mixture showed lower performance regarding plantheight and diameter. The genetic fidelity of micropropagated plants was evaluated using Start Codon Targeted(SCoT) Markers. Six SCoT primers amplified 51 scorable bands with an approximate range from 146 bp to1598 bp size. Thirty one out of 51 loci were presented in the mother plants. 40 loci were polymorphic, 11 weremonomorphic and 7 were unique. The amplification patterns of the micropropagated plants demonstrated genetic integrity to the mother plant ranging from 84.32 to 47.06 and somaclonal variations ranging from 52.94 with5 mg/l BA pathway to 15.68 with 1mg/l TDZ pathway, thus demonstrating that the homogeneity and the variation of the micropropagated plants affected by the type and the quantity of the plant growth regulator used duringmultiplication subcultures. This research can be successfully used for other ornamental and medicinal plants’ bulkmultiplication, germplasm conservation, and future genetic improvement.     

Factors influencing in the response of <i>Schizolobium parahybum</i> (Vell) Blake to <i>Ceratocystis paradoxa</i> and <i>C. moniliformis</i>

In the Ecuadorian coast one of the most destructive diseases of the pachaco is vascular wilt or stem rot caused by Ceratocystis complex, so the aim of this study was to determine the factors that affect the efficiency of the reaction of bark pachaco to this disease. This research was conducted under laboratory conditions, using trees pachaco S38, S41, S98, AE-1, AE-2 and AE-3, and pathogenic species Ceratocystis paradoxa and C. moniliformis. The method utilized was tissue stem bark,with bark sections with 4.5 cm², and a suspension of 3x10⁴ units infection and remained in a humid chamber for 96 hours at 25 ± 5 °C. Were determined grades of resistance/ susceptibility using a scale from 0 to 4, depending on the amount of mycelia and peritecio in each plant sample. Three factors were used: four colonies obtained by several transfers from each fungal specie, four ages of colonies of each fungal specie and four volumes of inoculum applied (units of infection), using for each experiment separately Completely Randomized Design with 4 replications factorial arrangement. For comparison between treatment means was used Tukey test at 5% probability of error. For future trials using this technique, you could use 30-day colonies for C. paradoxa and 40 days for C. moniliformis, and an application volume of 100 μL/cm², it would improve the level of response for the formation of perithecium and mycelia in samples cortex.     

Inhibitory Effect of <i>N</i>, <i>N</i>-Dimethylhexadecylamine on the Growth of White-Rot Fungus <i>Trametes versicolor</i> (L.) in Wood

Wood is an organic material that is a source of carbon of organisms called Wood-decay fungi, and to preserve the wood, various toxic compounds to man and the environment have been used. To analyze the effect of N,N-Dimethylhexadecylamine (DMHDA) on wood attacked by the rotting fungus Trametes versicolor L. We used an in vitro system to expose the fungus T. versicolor to different concentrations of the DMHDA (50, 150 and 450 μM). We quantified the diameter of mycelial growth and laccase activity, also, under these experimental conditions we studied morphological details of the organisms using different scanning equipment including scanning electron microscopy. The growth of T. versicolor exposed to DMHDA for 60 days, showed a concentration-dependent dose behavior, also, the electron microscopy analysis revealed that the morphology and mycelial density was affected by the DMHDA, showing a formation of atypical morphological and thickener folds. Finally, the pieces of wood treated with DMHDA and exposed to the fungus had a lower mass loss, after a period of 60 days of exposure, the values obtained were 0.7, 1.0 and 0.5 g of mass lost for the control, LoC and LoDMHDA treatments respectively. Wood-rot fungi have represented economic losses worldwide, the strategies used have been supported by toxic compounds for the environment. The DMHDA both in the Petri dish system and as a wood preservative was shown to significantly inhibit the growth of T. versicolor.     

Effects of <i>Piriformospora indica</i> on the Respiration of <i>Taxus chinensis</i> var. <i>mairei</i> under Water Stress

Seedlings of Taxus chinensis var. mairei were used as experimental materials to study the adaptation of Piriformospora indica to this plant under water stress. The materials were divided into two groups, namely, with or without inoculation with P. indica. Each group was subjected to four different levels of water stress. Vitality and physiological and biochemical indexes of the roots of T. chinensis var. mairei were regularly measured. Under water stress, T. chinensis var. mairei had significantly decreased root vitality; root vitality was higher in inoculated roots than in uninoculated roots. Under intense water stress, the inoculated roots had a higher soluble sugar content than the uninoculated roots. Under water stress, T. chinensis var. mairei experienced decreased activity of aerobic respiratory metabolic enzymes. The activity of anaerobic respiratory metabolic enzymes and alcohol dehydrogenase initially increased and then decreased, whereas that of lactate dehydrogenase increased. The inoculated roots had a higher activity of respiratory metabolic enzymes than the uninoculated roots. As water stress was further intensified, the roots had significantly decreased activity of aerobic respiratory metabolic enzymes and significantly increased activity of anaerobic respiratory metabolic enzymes. The activity of respiratory metabolic enzymes decreased faster in the uninoculated roots than in the inoculated roots. This study demonstrated that Piriformospora indica plays a positive role in enhancing the antihypoxic ability of T. chinensis var. mairei, thereby alleviating plant damage due to water stress.     

Wilt and vascular root rot (<i>Pythium tracheiphilum</i>) of lettuce in Bahía Blanca,Argentina

A new disease of lettuce has been observed in the surroundings of Bahía Blanca, Argentina. The symptoms include dwarfing, general chlorosis, wilting, root rot, and leaf blight, sometimes followed by plant death. Pythium tracheiphilum Matta was found to be consistently associated with those symptoms. The morphological and morphometric characteristics of this oomycete are described. Isolates obtained from field-infected lettuce plants were inoculated to lettuce and caused similar symptoms as those found in natural infections. Seedling emergence was also severely affected following experimental inoculation. Koch’s Postulates were fulfilled by recovering the fungus from inoculated plants. The new disease shows a high destructive potential but currently has a limited prevalence and incidence.