Onion Epidermis Research Articles

AaSlt2 Is Required for Vegetative Growth, Stress Adaption, Infection Structure Formation, and Virulence in Alternaria alternata

Slt2 is an important component of the Slt2-MAPK pathway and plays critical regulatory roles in growth, cell wall integrity, melanin biosynthesis, and pathogenicity of plant fungi. AaSlt2, an ortholog of the Saccharomyces cerevisiae Slt2 gene, was identified from A. alternata in this study, and its function was clarified by knockout of the gene. The ΔAaSlt2 strain of A. alternata was found to be defective in spore morphology, vegetative growth, and sporulation. Analysis of gene expression showed that expression of the AaSlt2 gene was significantly up-regulated during infection structure formation of A. alternata on hydrophobic and pear wax extract-coated surfaces. Further tests on onion epidermis confirmed that spore germination was reduced in the ΔAaSlt2 strain, together with decreased formation of appressorium and infection hyphae. Moreover, the ΔAaSlt2 strain was sensitive to cell wall inhibitors, and showed significantly reduced virulence on pear fruit. Furthermore, cell wall degradation enzyme (CWDE) activities, melanin accumulation, and toxin biosynthesis were significantly lower in the ΔAaSlt2 strain. Overall, the findings demonstrate the critical involvement of AaSlt2 in growth regulation, stress adaptation, infection structure formation, and virulence in A. alternata.

The Stylo Cysteine-Rich Peptide SgSnakin1 Is Involved in Aluminum Tolerance through Enhancing Reactive Oxygen Species Scavenging.

Stylo (Stylosanthes spp.) is an important pasture legume with strong aluminum (Al) resistance. However, the molecular mechanisms underlying its Al tolerance remain fragmentary. Due to the incomplete genome sequence information of stylo, we first conducted full-length transcriptome sequencing for stylo root tips treated with and without Al and identified three Snakin/GASA genes, namely, SgSnakin1, SgSnakin2, and SgSnakin3. Through quantitative RT-PCR, we found that only SgSnakin1 was significantly upregulated by Al treatments in stylo root tips. Histochemical localization assays further verified the Al-enhanced expression of SgSnakin1 in stylo root tips. Subcellular localization in both tobacco and onion epidermis cells showed that SgSnakin1 localized to the cell wall. Overexpression of SgSnakin1 conferred Al tolerance in transgenic Arabidopsis, as reflected by higher relative root growth and cell vitality, as well as lower Al concentration in the roots of transgenic plants. Additionally, overexpression of SgSnakin1 increased the activities of SOD and POD and decreased the levels of O2·- and H2O2 in transgenic Arabidopsis in response to Al stress. These findings indicate that SgSnakin1 may function in Al resistance by enhancing the scavenging of reactive oxygen species through the regulation of antioxidant enzyme activities.

The enzyme encoded by Myrmecia incisa, a green microalga, phospholipase A2 gene preferentially hydrolyzes arachidonic acid at the sn-2 position of phosphatidylcholine

The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands’ cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.

Characterisation and Expression Analysis of LdSERK1, a Somatic Embryogenesis Gene in Lilium davidii var. unicolor.

The Lanzhou lily (Lilium davidii var. unicolor) is a variant of the Sichuan lily of the lily family and is a unique Chinese 'medicinal and food' sweet lily. Somatic cell embryogenesis of Lilium has played an important role in providing technical support for germplasm conservation, bulb propagation and improvement of genetic traits. Somatic embryogenesis receptor-like kinases (SERKs) are widely distributed in plants and have been shown to play multiple roles in plant life, including growth and development, somatic embryogenesis and hormone induction. Integrating the results of KEGG enrichment, GO annotation and gene expression analysis, a lily LdSERK1 gene was cloned. The full-length open reading frame of LdSERK1 was 1875 bp, encoding 624 amino acids. The results of the phylogenetic tree analysis showed that LdSERK1 was highly similar to rice, maize and other plant SERKs. The results of the subcellular localisation in the onion epidermis suggested that the LdSERK1 protein was localised at the cell membrane. Secondly, we established the virus-induced gene-silencing (VIGS) system in lily scales, and the results of LdSERK1 silencing by Tobacco rattle virus (TRV) showed that, with the down-regulation of LdSERK1 expression, the occurrence of somatic embryogenesis and callus tissue induction in scales was significantly reduced. Finally, molecular assays from overexpression of the LdSERK1 gene in Arabidopsis showed that LdSERK1 expression was significantly enhanced in the three transgenic lines compared to the wild type, and that the probability of inducing callus tissue in seed was significantly higher than that of the wild type at a concentration of 2 mg/L 2,4-D, which was manifested by an increase in the granularity of the callus tissue.

The low-affinity calcium channel protein AaFig1 is essential for the growth and development, infection structure differentiation, and pathogenicity of Alternaria alternata

Fig1, as a low-affinity Ca2+ channel, is needed for Ca2+ influx and normal mating found in a few species, but its detailed molecular regulating mechanism in fungi, especially in Alternaria alternata, the causal agent of pear black spot, is still unclear. In this study, Fig1 in A. alternata was cloned, identified and named as AaFig1, and its function was characterized by targeted gene knockout strategy. The results showed that the ΔAaFig1 was severely decreased in vegetative growth, biomass accumulation and sporulation. Fluo-4, AM staining indicated that the Ca2+ content in ΔAaFig1 was decreased. Deletion of AaFig1 reduced the response of A. alternata to exogenous Ca2+ but improved the tolerance to osmotic stresses. Interestingly, the appressorium formation of the ΔAaFig1 strain on hydrophobic and pear wax extract coated surface, and infection hyphae formation on the onion epidermis were significantly reduced, meanwhile, the ΔAaFig1 strain could not penetrate the cellophane. In addition, virulence on wound-inoculated pear fruit and intact pear leaves, melanin production and the activities of cell wall degrading enzymes in AaFig1 strain were reduced. In summary, these results indicate that AaFig1-mediated Ca2+ homeostasis positively regulates the growth and development, infection structure formation and pathogenicity of A. alternata. The findings will provide a basis for developing new strategies to control this pathogen in pear fruit.

Onion cryptochrome 1 (AcCRY1) regulates photomorphogenesis and photoperiod flowering in Arabidopsis and exploration of its functional mechanisms under blue light

Cryptochromes (CRYs), as blue-light photoreceptors, play a crucial role in regulating flowering time and hypocotyl and cotyledon development. Their physiological functions have been extensively studied in various plant species. However, research on onions remains limited. In this study, we identified AcCRY1 and conducted preliminary investigations into its function. Our results demonstrate that AcCRY1 possesses a conserved domain typical of cryptochromes with high homology to those found in monocots. Furthermore, we examined the expression level of AcCRY1 in onion. The green tissues is significantly higher compared to non-green tissues, and it exhibits a significant response to blue-light induction. AcCRY1 demonstrates cytoplasmic localization under blue-light conditions, while it localizes in the nucleus during darkness, indicating a strong dependence on blue-light for its subcellular distribution. In comparison to cry1, overexpression of AcCRY1 leads to a significant shorten in seedling hypocotyl length, notable expansion of cotyledons, and acceleration of flowering time. The yeast two-hybrid experiment demonstrated the in vitro interaction between AcCRY1, AcCOP1, and AcSPA1. Additionally, BIFC analysis confirmed their interaction in Onion epidermis. Notably, under blue-light conditions, a significantly enhanced binding activity was observed compared to dark conditions. These findings establish a functional foundation for the regulatory role of AcCRY1 in important physiological processes of onion and provide initial insights into the underlying molecular mechanisms.

The influence of shape and matrix size on the mechanical properties of the 2D epoxy thin film by Monte Carlo simulation method

In this paper, we studied the effect of the 2D epoxy thin films’ shape with equilateral triangle and square structures, and matrix size Lx × Ly of (10 × 9), (20 × 19), (30 × 29), and (40 × 39) with equilateral triangle structure and (10 × 10), (20 × 20), (30 × 30), and (40 × 40) with the square structure on their mechanical properties [such as strain (ɛ), stress (σ), Young stress (E), and shear strain (G)] by using the Monte Carlo simulation method. The results show that when the shape of the 2D epoxy thin film is changed from an equilateral triangle structure to a square structure, the values of σ, E, and G decreased sharply. In addition, when the matrix size is increased from (10 × 9) to (20 × 19), (30 × 29), and (40 × 39) with an equilateral triangle structure and from (10 × 10) to (20 × 20), (30 × 30), and (40 × 40) with a square structure, σ slightly increased, but E and G decreased slightly. These results prove that the influence of structure shape on the mechanical properties of the 2D epoxy thin film is very large. The strain stress on the epoxy 2D thin film with an equilateral triangle structure and with a matrix size of (30 × 29) has a value of σ = 63.3 MPa. This result is consistent with the experimental result that σ of bulk epoxy has the maximum value of σmax = 64.76 MPa. The results are the basis for experimental research in future studies on practical applications of epoxy-thin films. In these cases, when thin films with equilateral triangle structures are used in biomedical fields, high stresses are required (such as replacement material for adaxial onion epidermis and fibrin and collagen with low stress).

Gene expression, molecular docking, and molecular dynamics studies to identify potential antifungal compounds targeting virulence proteins/genes VelB and THR as possible drug targets against Curvularia lunata.

Curvuluria lunata is a melanized fungus pathogenic to both plants and animals including humans, causing from mild, febrile to life-threatening illness if not well treated. In humans, it is an etiological agent of keratomycosis, sinusitis, and onychomycosis in immunocompromised and immunocompetent patients. The development of multiple-drug-resistant strains poses a critical treatment issue as well as public health problem. Natural products are attractive prototypes for drug discovery due to their broad-spectrum efficacy and lower side effects. The present study explores possible targets of natural antifungal compounds (α-pinene, eugenol, berberine, and curcumin) against C. lunata via gene expression analysis, molecular docking interaction, and molecular dynamics (MD) studies. Curcumin, berberine, eugenol, and α-pinene exhibited in vitro antifungal activity at 78μg/ml, 156μg/ml, 156μg/ml, and 1250μg/ml, respectively. In addition, treatment by these compounds led to the complete inhibition of conidial germination and hindered the adherence when observed on onion epidermis. Several pathogenic factors of fungi are crucial for their survival inside the host including those involved in melanin biosynthesis, hyphal growth, sporulation, and mitogen-activated protein kinase (MAPK) signalling. Relative gene expression of velB, brn1, clm1, and pks18 responsible for conidiation, melanin, and cell wall integrity was down-regulated significantly. Results of molecular docking possessed good binding affinity of compounds and have confirmed their potential targets as THR and VelB proteins. The docked structures, having good binding affinity among all, were further refined, and rescored from their docked poses through 100-ns long MD simulations. The MDS study revealed that curcumin formed a stable and energetically stabilized complex with the target protein. Therefore, the study concludes that the antifungal compounds possess significant efficacy to inhibit C. lunata growth targeting virulence proteins/genes involved in spore formation and melanin biosynthesis.

Cloning and functional analysis of expansin TaEXPA9 orthologs in winter wheat in frigid regions

Long-term low temperatures restrict the regrowth of winter wheat (Triticum aestivum L.), thus decreasing agricultural output. Non-enzymatic expansins, which are related to plant growth, have been reported to respond to drought, salinity, and low-temperature stress. We obtained an expansin 3 gene, TaEXPA9. It is located in winter wheat cv. Dongnong with high cold hardiness. We analyzed the expression patterns of TaEXPA9-A/B/D in this cultivar and conducted a subcellular localization analysis of TaEXPA9-A/B/D in the onion epidermis. Transgenic Arabidopsis thaliana line with EXPA9-A/B/D overexpression was obtained to examine the effects of the orthologous genes of these expansins on plant growth and low-temperature stress resistance. The results showed that EXPA9-A/B/D expression significantly increased at 4 °C, it was higher in the roots than in shoots, and EXPA9-A/B/D was localized in the cell wall. The roots were well-developed in the transgenic A. thaliana, and the growth-related markers and setting rate were better than in the wild-type. Recovery was stronger in the transgenic plants after freezing stress. At low-temperature stress, the antioxidant enzyme activities and content of osmoregulatory substances in the TaEXPA9-A/B/D-overexpressing A. thaliana plants were significantly higher than in the wild-type plants, and the degree of membrane lipid peroxidation was lower. In summary, TaEXPA9 orthologous genes participate in the low-temperature stress response, and they might be of great importance in molecular breeding.

Identifying Factors that Determine Effectiveness of Delivery Agents in Biolistic Delivery Using a Library of Amine-Containing Molecules.

Biolistic transfection is a popular and versatile tool for plant transformation. A key step in the biolistic process is the binding of DNA to the heavy microprojectile using a delivery agent, usually a positively charged molecule containing amine groups. Currently, the choice of the commercial delivery agent is mostly limited to spermidine. In addition, the detailed delivery mechanism has not been reported. To help broaden the selection of the delivery agent and reveal the fundamental mechanisms that lead to high delivery performance, a library of amine-containing molecules was investigated. A double-barrel biolistic delivery device was utilized for testing hundreds of samples with much-improved consistency. The performance was evaluated on onion epidermis. The binding and release of DNA were measured via direct high-performance liquid chromatography analysis. This study shows that the overwhelming majority of the amine library performed at the same level as spermidine. To further interpret these results, correlations were performed with thousands of molecular descriptors generated by chemical modeling. It was discovered that the overall charge is most likely the key factor to a successful binding and delivery. Furthermore, even after increasing the amount of the DNA concentration 50-fold to stress the binding capacity of the molecules, the amines in the library continued to deliver at a near identical level while binding all the DNA. The increased DNA was also demonstrated with a Cas9 editing test that required a large amount of DNA to be delivered, and the result was consistent with the previously determined amine performance. This study greatly expands the delivery agent selection for biolistic delivery, allowing alternatives to a commercial reagent that are more shelf-stable and cheaper. The library also offers an approach to investigate more challenging delivery of protein and CRISPR-Cas via the biolistic process in the future.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

In Situ Cell Detection Using Terahertz Near-Field Microscopy

The photoconductive antenna microprobe-based terahertz (THz) near-field scanning microscope has proven to overcome the diffraction limit and demonstrated the potential in distinguishing cellular types and states. Here, using the onion epidermal cells as a sample model, detection of single epithelial cell in the onion skin has been realized at amplitude signal around 1 THz. The boundaries of the target cells in a monolayer onion epidermis are clearly depicted with the spatial resolution of 9 <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">μ</i> m (λ/33). Differentiated THz dielectric response shows clearly the distinctive intracellular and intercellular regions, mainly related to the biowater distribution and cellular heterogeneity, which can be used for <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in situ</i> subcellular identification <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">.</i> This finding may have significant implications for the advancement of THz near-field high-resolution bioimaging technology.

BsTup1 is required for growth, conidiogenesis, stress response and pathogenicity of Bipolaris sorokiniana

Tup1, a conserved transcriptional repressor, plays a critical role in the growth and development of fungi. Here, we identified a BsTup1 gene from the plant pathogenic fungus Bipolaris sorokiniana. The expression of BsTup1 showed a more than three-fold increase during the conidial stage compared with mycelium stage. Deletion of BsTup1 led to decrease hyphal growth and defect in conidia formation. A significant difference was detected in osmotic, oxidative, or cell wall stress responses between the WT and ΔBsTup1 strains. Pathogenicity assays showed that virulence of the ΔBsTup1 mutant was dramatically decreased on wheat and barely leaves. Moreover, it was observed that hyphal tips of the mutants could not form appressorium-like structures on the inner epidermis of onion and barley coleoptile. Yeast two-hybrid assays indicated that BsTup1 could interact with the BsSsn6. RNAseq revealed significant transcriptional changes in the ΔBsTup1 mutant with 2369 genes down-regulated and 2962 genes up-regulated. In these genes, we found that a subset of genes involved in fungal growth, sporulation, cell wall integrity, osmotic stress, oxidation stress, and pathogenicity, which were misregulated in the ΔBsTup1 mutant. These data revealed that BsTup1 has multiple functions in fungal growth, development, stress response and pathogenesis in B. sorokiniana.

Histological characterization of the early‐stage infection events of Setosphaeria turcica in maize

AbstractNorthern corn leaf blight (NCLB) caused by Setosphaeria turcica is a major foliar disease of maize. The early‐stage infection events of this pathogen on maize leaves are unclear. We investigated the optimum temperature for conidial germination and appressorium formation, and characterized penetration and growth of S. turcica in maize leaf sheath and onion epidermis cells, including use of histological staining to assess plant cell viability. The results showed that the optimum temperature for conidial germination and appressorium formation was 20°C. On the maize leaf sheath, the appressoria were formed by germinated conidia, and penetration on the epidermal cells occurred at 8 h postinoculation (hpi). Round vesicles developed beneath the appressoria. Between 16 and 24 hpi, the branched invasive hyphae invaded three to five adjacent cells at most infection sites. The invasive hyphae tended to move along the cell wall and crossed from one cell to another. In the onion epidermis cells, the appressoria formed at 8 hpi, and in most cases the epidermal cells were penetrated through the juncture of the cell walls. At 16–24 hpi, the primary hyphal terminus swelled to a vesicle. The maize leaf sheath cells died at 8 hpi, whereas the onion cells did not. Our findings documented in detail the penetration and invasive hyphal growth in maize leaf sheath and onion epidermis, as well as viability of plant cells, at the early stages of infection, and provide a foundation for elucidating the underlying mechanism of S. turcica–maize interactions.

MicroLED chromatic confocal microscope.

In this Letter, a microLED-based chromatic confocal microscope with a virtual confocal slit is proposed and demonstrated for three-dimensional (3D) profiling without any mechanical scanning or external light source. In the proposed method, a micro-scale light-emitting diode (microLED) panel works as a point source array to achieve lateral scanning. Axial scanning is realized through the chromatic aberration of an aspherical objective. A virtual pinhole technique is utilized to improve the contrast and precision of depth reconstruction. The system performance has been demonstrated with a diamond-turned copper sample and onion epidermis. The experimental results show that the microLED panel could be a potential solution for portable 3D confocal microscopy. Several considerations and prospects are proposed for future microLED requirements in confocal imaging.

Ionic liquid impregnated Allium cepa peel for supercapacitor application

This paper report a new biodegradable epidermis of onion (Allium cepa) doped with low viscosity ionic liquid as novel electrolyte for EDLC application. The porous membrane used in present study was epidermis of onion (onion peel membrane) and low viscosity ionic liquid (IL) i.e. 1-ethyl 3-methyl imidazolium thiocynate. The electrical, structural, optical characterization was carried out in detail. The optical micrographs show addition in amorphosity with adding IL. Fourier transform infrared spectroscopy (FTIR) revealed the interaction between ionic liquid and membrane as well as composite nature of membrane electrolyte. Impedance spectroscopy revealed the twofold enhancement in conductivity value by doping IL. Electrical double layer supercapacitor (EDLC) has been fabricated using carbon nanotube (CNT). Cyclic voltammetry and low frequency impedance spectroscopy data further affirm the reasonable value of specific capacitance (Csp = 9.2F/g).

Development of a hybrid piezoelectric and pneumatic miniature optical scanner for endoscopic applications

Gastrointestinal (GI) cancers are among some of most significant public health issues worldwide. Early cancer diagnosis will increase GI cancer patients’ survival rate, however, developing endoscopes for early cancer detection is a challenge as it is difficult to design a high-resolution optical scanner. In this paper, we report a new 3D optical scanner using piezoelectric (for in-plane, X-Y axes) and pneumatic (for out-of-plane, Z-axis) mechanisms. Feedback control is implemented to reduce disturbances and provide a stable position for the pneumatic actuator. The scanner shows a Z and X-Y resolution of 1.5 μm and 1.4 μm, respectively. The field-of-view (FOV) is around 100 × 100 (μm)2, with a frame rate of 1.25 fps (frame per second). Paper fiber, and onion epidermis images were obtained by the new optical scanner.

Isolation and functional characterization of four microbial type terpene synthases from ferns

Typical plant terpene synthases (TPSs) are responsible for the production of terpenes, a major class of plant secondary metabolites. However, various nonseed plants also harbor genes encoding microbial terpene synthase-like (MTPSL) enzymes. Here, a scan of 31 ferns transcriptomes revealed 40 sequences putatively encoding MTPSLs. Two groups of sequences were recognized based on the key conserved motifs. Four representative genes were isolated from each of the four species Adiantum capillus-veneris, Cyclosorus parasiticus, Drynaria bonii and Microlepia platyphylla. Following their heterologous expression in E. coli, the recombinant proteins were tested for monoterpene synthase and sesquiterpene synthase activity. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by classical plant TPSs when provided with GPP and FPP as substrates. Subcellular localization experiments in the leaf epidermis of Nicotiana benthamiana and onion (Allium cepa) inner epidermal cells indicated that AcMTPSL1 and DbMTPSL were deposited in both the cytoplasm and nucleus, whereas CpMTPSL1 and MpMTPSL were localized in the cytoplasm, chloroplasts and nucleus. AcMTPSL1 was up-regulated in plants exposed to methyl jasmonate treatment, suggesting a role for this gene in host defense. This study provides more information about the catalytic function of MTPSLs in nonseed plants and for the first time, the subcellular localization of MTPSLs was experimentally characterized.

MoNFR, encoding a putative NADPH-ferrihemoprotein reductase, is required for the pathogenicity of Magnaporthe oryzae

A wild type Magnaporthe oryzae CY2 with normal growth and sporulation but weak pathogenicity is not virulent to more than over 1000 rice cultivars. Based on the transformant library of M. oryzae CY2 by Agrobacterium tumefaciens-mediated transformation (ATMT), we screened a transformant T23 with normal infectious hyphae and pathogenicity to rice line IRBL-19. Co-segregation and southern blotting suggested that T-DNA is inserted with single copy, and pathogenicity may require the mutated gene MoNFR. Bioinformatics analysis predicted that the gene MoNFR encoded a putative NADPH-ferrihemoprotein reductase with an indole amine 2,3-dioxgyenase (IDO) domain, a flavin adenine dinucleotide binding domain (FAD_binding domain), a nicotinamide adenine dinucleotide binding domain (NAD_binding domain) and a cytochrome-b5 domain. Vectors were constructed to knock-out the target gene. Two mutants with MoNFR disruptant were obtained from the wild type strain CY2 by targeted gene replacement with hygromycin B phosphotransferase (HPH) gene. Compared with strain T23 and CY2, the MoNFR mutants showed no significant difference in colony morphology, growth rate and mycelium color but conidia produced in culture were 4.5–5 times more and higher percentage of appressorial and infectious hyphae formation was observed on onion epidermis. They retain the pathogenicity to IRBL-19, and are similar in virulence to strain T23. These results indicate that MoNFR plays a key role in the pathogenicity of M. oryzae.

Cell phone digital microscopy using an oil droplet.

We introduce an accessible cell phone imaging method using small droplets of microscope immersion oil and consumer-grade oils. Oil droplets were more resistant to evaporation than water droplets, and they resolved cellular structures that were visible using a 20x/0.75 objective. We optically characterized the droplets using a cell phone screen and resolution target. We further obtained cellular resolution images of an onion epidermis and a zea stem cross-section sample. Our droplet-based method enables stable optical imaging for diagnostic and educational purposes without custom setups, specialized components, or manufacturing processes.