Onion Epidermis Research Articles

Assembly of a simple scalable device for micromechanical testing of plant tissues.

Tensile testing is widely used to evaluate the mechanical properties of biological materials including soft primary plant tissues. Commercially available platforms for tensile testing are often expensive and limited in customizability. In this chapter, we provide a guide for the assembly and use of a simple and low-cost micromechanical testing apparatus suitable for research and educational purposes. The build of the setup is presented with scalability and universality in mind and is based on a do-it-yourself mind frame towards mechanical tests on plant organs and tissues. We discuss hardware and software requirements with practical details on required components, device calibration and a script to run the device. Further, we provide an example in which the device was used for the uniaxial tensile test of onion epidermis.

Modeling the nonlinear elastic behavior of plant epidermis

Cell growth and organ development in plants are often correlated with the tensile behavior of the primary cell wall. To understand the mechanical behavior of plant material, various mechanical testing techniques have been employed, such as tensile testing of excised tissue samples. The onion (Allium cepa L.) epidermis has emerged as a model system for plant tissue mechanics. In this study, we performed tensile tests on strips of adaxial onion epidermis. While the tissue appeared stiffer in the direction along the major growth axis compared with the transverse direction, the tensile strength of tissue was not significantly different between the two orientations, indicating a nontrivial link between the cell wall and tissue mechanical anisotropy. Importantly, we observed the stress–strain behavior of the onion epidermis under tension to be highly nonlinear. Several hyperelastic models were fitted to the test data to evaluate their capacity to describe the nonlinear deformation of onion epidermis. The Yeoh hyperelastic model could successfully simulate the uniaxial tensile test data. This study suggests that accounting for nonlinearity in the deformation of the primary tissue may be essential for the accurate interpretation of mechanical test data, and a better understanding of the mechanics of the primary plant cell wall.

Chromatic confocal microscopy using liquid crystal display panels.

A chromatic confocal microscopy system in combination with two liquid crystal display (LCD) panels is proposed and demonstrated for surface profiling. The major advantage of this system is no mechanical translation is needed for three-dimensional (3D) imaging. Axial scanning is realized thanks to the chromatic aberration in the objective, whereas lateral scanning is achieved by turning on different pixels on LCDs. Chromatic aberration of the objective lens is used to provide wavelength-to-depth coding, and decoding is realized by using a dispersion prism. System performance is validated with a 50μm step standard and the capability of 3D imaging is demonstrated with an onion epidermis.

Isolation and functional characterization of CsLsi2, a cucumber silicon efflux transporter gene.

Silicon has been proven to exert beneficial effects on plant growth and stress tolerance, and silicon accumulation varies among different plant species. Cucumber (Cucumis sativus) is a widely used dicot model for silicon accumulation, but little is known about the molecular mechanism of its silicon uptake. Previously, we isolated and characterized CsLsi1, a silicon influx transporter gene from cucumber. In this study, we cloned a putative silicon efflux transporter gene, CsLsi2, from cucumber and investigated its role in silicon uptake. The expression pattern, transport activity, and subcellular and cellular localizations of CsLsi2 were investigated. The transport activity of CsLsi2 was determined in Xenopus laevis oocytes. The subcelluar and cellular localizations were conducted by transient expression of fused 35S::CsLsi2-eGFP in onion epidermal cells and expression of ProCsLsi2::CsLsi2-mGFP in cucumber, respectively. CsLsi2 was mainly expressed in the roots. Expression of CsLsi2-eGFP fusion sequence in onion epidermis cells showed that CsLsi2 was localized at the plasma membrane. Transient expression in Xenopus laevis oocytes showed that CsLsi2 demonstrated efflux but no influx transport activity for silicon, and the transport was energy-dependent. Expression of CsLsi2-mGFP under its own promoter revealed that CsLsi2 was mainly expressed on endodermal cells, showing no polar distribution. In combination with our previous work on CsLsi1, a model for silicon uptake in cucumber roots is proposed. The results suggest that CsLsi2 is a silicon efflux transporter gene in cucumber. The coordination of CsLsi1 and CsLsi2 mediates silicon uptake in cucumber roots. This study may help us understand the molecular mechanism for silicon uptake in cucumber, one of the few dicots with a relatively high capacity for silicon accumulation.

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

High yield accelerated reactions in nonvolatile microthin films: chemical derivatization for analysis of single-cell intracellular fluid.

The identification of trace components from biological media can require derivatization under mild conditions for successful analysis by mass spectrometry (MS). When aqueous droplets (ca. 500 nL) containing a sugar and an amine as reagents are allowed to evaporate they may form long-lasting microthin films in which derivatization reactions can occur fast relative to reaction rates in bulk solution. Evidence is presented that these reactions are heterogeneous in nature and comparisons are made with reactions in pastes and in neat reagent mixtures. Moreover, these thin film reactors can be made stable for the long periods of time that may be necessary to give high product yields. The situation is typified by imine formation from reducing sugars which have reaction times of much more than 1 hour provided that small concentrations (e.g. 20 ppm) of nonvolatile solvents are included. After evaporation of almost all the water, the reaction occurs at an approximately constant rate for the first hour. The rate is two orders of magnitude faster than the reaction in the corresponding homogeneous saturated bulk solution. Conversion of the reagent to the Schiff base product is 67% to 96% efficient in these long-lasting thin films, in sharp contrast to the corresponding derivatization efficiencies in the bulk of less than 1%. This method was used to chemically derivatize and thus to identify, using tandem mass spectrometry, 29 reducing sugars in ca. 1 nL of intracellular fluid from a single onion epidermis cell. A formal description of the kinetics of reversible and irreversible second order reactions in thin films is provided. The effects of thermodynamic and kinetic factors are separated and the measured apparent acceleration factor is shown to represent the ratio of intrinsic rate constants for the microthin film reactor relative to the bulk reaction.

Arabidopsis phosphatidylinositol 4-phosphate 5-kinase 2 contains a functional nuclear localization sequence and interacts with alpha-importins.

The Arabidopsis phosphoinositide kinase PIP5K2 has been implicated in the control of membrane trafficking and is important for development and growth. In addition to cytosolic functions of phosphoinositides, a nuclear phosphoinositide system has been proposed, but evidence for nuclear phosphoinositides in plants is limited. Fluorescence-tagged variants of PIP5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP5K2 with alpha-importins and characterize its nuclear localization sequences (NLSs). The PIP5K2 sequence contains four putative NLSs (NLSa-NLSd) and only a PIP5K2 fragment containing NLSs is imported into nuclei of onion epidermis cells upon transient expression. PIP5K2 interacts physically with alpha-importin isoforms in cytosolic split-ubiquitin-based yeast two-hybrid tests, in dot-blot experiments and in immuno-pull-downs. A 27-amino-acid fragment of PIP5K2 containing NLSc is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mCherry markers fused to RubisCO large subunit. Substitution of basic residues in NLSc results in reduced import of PIP5K2 or other cargoes into plant nuclei. The data suggest that PIP5K2 is subject to active, alpha-importin-mediated nuclear import, consistent with a nuclear role for PIP5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides.

Family of Ricinus communis Monosaccharide Transporters and RcSTP1 in Promoting the Uptake of a Glucose-Fipronil Conjugate.

Enhancing the systemic distribution of a bioactive compound by exploiting the vascular transport system of a plant presents a means of reducing both the volume and frequency of pesticide/fungicide application. The foliar uptake of the glucose-fipronil conjugate N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(β-d-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF) achieved in castor bean (Ricinus communis) and its transport via the phloem are known to be mediated by monosaccharide transporter(s) [MST(s)], although neither the identity of the key MST(s) involved nor the mechanistic basis of its movement have yet to be described. On the basis of homology with Arabidopsis thaliana sugar transporters, the castor bean genome was concluded to harbor 53 genes encoding a sugar transporter, falling into the eight previously defined subfamilies INT, PMT, VGT, STP, ERD6, pGlucT, TMT, and SUT. Transcriptional profiling identified the product of RcSTP1 as a candidate for mediating GTF uptake, because this gene was induced by exposure of the plant to GTF. When RcSTP1 was transiently expressed in onion epidermis cells, the site of RcSTP1 deposition was shown to be the plasma membrane. A functional analysis based on RcSTP1 expression in Xenopus laevis oocytes demonstrated that its product has a high affinity for GTF. The long-distance root-to-shoot transport of GTF was enhanced in a transgenic soybean chimera constitutively expressing RcSTP1.

SmMYB36, a Novel R2R3-MYB Transcription Factor, Enhances Tanshinone Accumulation and Decreases Phenolic Acid Content in Salvia miltiorrhiza Hairy Roots

Phenolic acids and tanshinones are two major bioactive components in Salvia miltiorrhiza Bunge. A novel endogenous R2R3-MYB transcription factor, SmMYB36, was identified in this research. This transcript factor can simultaneously influence the content of two types of components in SmMYB36 overexpression hairy roots. SmMYB36 was mainly localized in the nucleus of onion epidermis and it has transactivation activity. The overexpression of SmMYB36 promoted tanshinone accumulation but inhibited phenolic acid and flavonoid biosynthesis in Salvia miltiorrhiza hairy roots. The altered metabolite content was due to changed metabolic flow which was regulated by transcript expression of metabolic pathway genes. The gene transcription levels of the phenylpropanoid general pathway, tyrosine derived pathway, methylerythritol phosphate pathway and downstream tanshinone biosynthetic pathway changed significantly due to the overexpression of SmMYB36. The wide distribution of MYB binding elements (MBS, MRE, MBSI and MBSII) and electrophoretic mobility shift assay results indicated that SmMYB36 may be an effective tool to regulate metabolic flux shifts.

低温下における玉葱表皮細胞の生存率(平成16年度 第50回低温生物工学会研究報告)

低温下における玉葱表皮細胞の生存率(平成16年度 第50回低温生物工学会研究報告)

Effects of mechanical stretching on average orientation of cellulose and pectin in onion epidermis cell wall: A polarized FT-IR study

The organization of polysaccharides in plant cell walls is important for the mechanics of plant cells. Spectral analysis of cell walls by polarized IR can reveal polysaccharide organization, but may be complicated by dipoles not aligned with the backbone. For instance, analysis of uniaxially-aligned cellulose Iβ film revealed that the dipole transition vector of the 1160 cm−1 band involving stretch vibrations of glycosidic C1–O–C4 linkages is approximately at 30° with respect to the backbone of the cellulose chain, because of coupling with C5–O–C1 bonds in the six-membered rings. In the case of homogalacturonan, the dipole transition vector of the ester carbonyl group vibration (νC=O, 1745 cm−1) is expected to be nearly normal to the homogalacturonan backbone. Using this information and the dichroism equation, the change in net orientation of cell wall polymers upon mechanical stretch was determined by polarized IR analysis. Never-dried abaxial outer epidermal cell walls of the second scale of onion bulb were mechanically stretched along longitudinal or transverse directions with respect to the long axis of the cells and then dried while under mechanical stretch. The average orientations of both 1160 and 1745 cm−1 vibration transition dipoles were rotated by ~5° and ~4°, respectively, along the stretch direction from their initial random distributions upon longitudinal strain by 14%; and by ~4° and ~3°, respectively, upon transverse strain by 12%. These results imply that both cellulose microfibrils and pectins in the cell wall are passively realigned along the stretch direction by external mechanical force. The analytical methodology developed here will be useful to study how cell wall polymers might reorganize during cell wall growth and development.

The conidial mucilage, natural film coatings, is involved in environmental adaptability and pathogenicity of Hirsutella satumaensis Aoki

The Hirsutella genus is very special asexually-reproducing pathogens of insects by reduced sporulation, host specificity and spores covered by a thick mucilage layer. However, the ecological function of conidial mucilage remains elusive. In this study, the possible ecological role of conidial mucilage from the entomopathogenic fungus Hirsutella satumaensis was functionally investigated through tolerance, adherence and insect bioassays involving aerial conidia (AC) and mucilage-free conidia (MFC). Measurements of hydrophobicity using microbial adhesion to hydrocarbons (MATH) indicated that mucilage is main contributor to the surface hydrophobicity of AC. When subjected in tolerance assays to extreme temperatures, high chemical pressure, extended exposure to ultraviolet radiation and cold stress, AC produced more colonies, exhibited higher conidiation and germination percentages than those of MFC. In adhesion assays, MFC displayed an approximately 40% reduction in adherence to locust, dragonfly cuticle and onion epidermis when washed with 0.05% Tween 20. Similarly, Galleria mellonella and Plutella xylostella larvae infected with mucilage-producing AC experienced a relatively higher mortality rate. Our findings suggest that mucilage is critical to the ecological adaptability of H. satumaensis, where it plays positive roles on maintenance of spore surface hydrophobicity, enhancement of spore resistance to extreme environments and strengthening of spore adhesion and host pathogenicity.

Induction of anthocyanin in the inner epidermis of red onion leaves by environmental stimuli and transient expression of transcription factors.

Novel imaging approaches have allowed measurements of the anthocyanin induction in onion epidermal cells that can be induced through water stress or transient expression of exogenous transcription factors. Environmental and genetic mechanisms that allow the normally colourless inner epidermal cells of red onion (Allium cepa) bulbs to accumulate anthocyanin were quantified by both absorbance ratios and fluorescence. We observed that water-stressing excised leaf segments induced anthocyanin formation, and fluorescence indicated that this anthocyanin was spectrally similar to the anthocyanin in the outer epidermal cells. This environmental induction may require a signal emanating from the leaf mesophyll, as induction did not occur in detached epidermal peels. Exogenous transcription factors that successfully drive anthocyanin biosynthesis in other species were also tested through transient gene expression using particle bombardment. Although the petunia R2R3-MYB factor AN2 induced anthocyanin in both excised leaves and epidermal peels, several transcription factors including maize C1 and Lc inhibited normal anthocyanin development in excised leaves. This inhibition may be due to moderate levels of conservation between the exogenous transcription factors and endogenous Allium transcription factors. The over-expressed exogenous transcription factors cannot drive anthocyanin biosynthesis themselves, but bind to the endogenous transcription factors and prevent them from driving anthocyanin biosynthesis.

The autophagy-related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea.

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that the expression of BcATG1 was up-regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re-introduction of an intact copy of the wild-type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.

BcIEB1, a Botrytis cinerea secreted protein, elicits a defense response in plants

BcIEB1 is a very abundant protein in the secretome of Botrytis cinerea but it has no known function and no similarity to any characterized protein family. Previous results suggested that this protein is an elicitor of the plant defense system. In this work we have generated loss-of-function B. cinerea mutants lacking BcIEB1 and we have expressed the protein in yeast to assay its activity on plants. Analysis of the Δbcieb1 mutants did not result in any observable phenotype, including no difference in the virulence on a variety of hosts. However, when BcIEB1 was applied to plant tissues it produced necrosis as well as a whole range of symptoms: inhibition of seedling growth in Arabidopsis and tobacco, ion leakage from tobacco leaf disks, a ROS burst, cell death and autofluorescence in onion epidermis, as well as the expression of defense genes in tobacco. Moreover, tobacco plants treated with BcIEB1 showed an increased systemic resistance to B. cinerea. A small 35-amino acids peptide derived from a conserved region of BcIEB1 is almost as active on plants as the whole protein. These results clearly indicate that BcIEB1 elicits plant defenses, probably as a consequence of its recognition as a pathogen associated molecular pattern.

Transcriptomics-based identification of WRKY genes and characterization of a salt and hormone-responsive PgWRKY1 gene in Panax ginseng.

WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses.

Antisense expression of Gossypium barbadense UGD6 in Arabidopsis thaliana significantly alters cell wall composition.

Uridine diphosphate-glucose dehydrogenase (UGD, EC1.1.1.22 oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), a critical precursor of cell wall polysaccharides. GbUGD6 from Gossypium barbadense is more highly expressed late in the elongation of cotton fibers (15 d post-anthesis (DPA)) and during the stage of secondary cell wall thickening (30 DPA). Subcellular localization analysis in onion epidermis revealed that fluorescently labeled GbUGD6 protein was distributed throughout the cell membrane, as well as the nucleus and vacuoles. Examination of UGD function in Arabidopsis revealed that the antisense GbUGD6 lines had shorter roots, deferred blossoming, compared to wild-type plants. Activities of associated enzymes were also affected by UGD reduction, and biochemical analysis of cell wall samples showed an increase in cellulose levels and a decrease in UGP-GlcA contents. The results of the present study as well as previous studies on UGD support the conclusion that UGD plays a major role in synthesizing polysaccharides synthesis in the cell wall.

Multimodal Broadband Vibrational Sum Frequency Generation (MM-BB-V-SFG) Spectrometer and Microscope.

A broadband sum frequency generation (BB-SFG) spectrometer with multimodal (MM) capabilities was constructed, which could be routinely reconfigured for tabletop experiments in reflection, transmission, and total internal reflection (TIR) geometries, as well as microscopic imaging. The system was constructed using a Ti:sapphire amplifier (800 nm, pulse width = 85 fs, repetition rate = 2 kHz), an optical parameter amplification (OPA) system for production of broadband IR pulses tunable between 1000 and 4000 cm(-1), and two Fabry-Pérot etalons arranged in series for production of narrowband 800 nm pulses. The key feature allowing the MM operation was the nearly collinear alignment of the visible (fixed, 800 nm) and infrared (tunable, 1000-4000 cm(-1)) pulses which were spatially separated. Physical insights discussed in this paper include the comparison of spectral bandwidth produced with 40 and 85 fs pump beams, the improvement of spectral resolution using etalons, the SFG probe volume in bulk analysis, the normalization of SFG signals, the stitching of multiple spectral segments, and the operation in different modes for air/liquid and adsorbate/solid interfaces, bulk samples, as well as spectral imaging combined with principle component analysis (PCA). The SFG spectral features obtained with the MM-BB-SFG system were compared with those obtained with picosecond-scanning-SFG system and high-resolution BB-SFG system (HR-BB-SFG) for dimethyl sulfoxide, α-pinene, and various samples containing cellulose (purified commercial products, Cladophora cell wall, cotton and flax fibers, and onion epidermis cell wall).

Cloning and analysis of β-amyrin synthase gene in Bupleurum chinense

Bupleurum chinense DC. is one of the source plants of a well-known crude drug, Chai hu (Radix Bupleuri), producing triterpenoid saponins (saikosaponins) with a wide-spectrum of pharmacological applications. The biosynthesis of triterpenoid saponins involved with the cyclizing of the precursor 2,3-oxidosqualene to produce the first committed triterpene β-amyrin catalyzed by β-amyrin synthase (β-AS), whereafter diverse of triterpenoid saponins was biosynthesized. In addition, 2,3-oxidosqualene could be catalyzed by cycloartenol synthase directing to the synthesis of phytosterol. β-AS was thus defined as an important branch point between primary and secondary metabolisms, and may play a regulating role in the control of triterpenoid saponins biosynthesis. In this study, the promoter and protein-encoding regions of a β-AS gene (designated bcAS1) were isolated by genome walking and PCR from B. chinense. Several important cis-acting elements for gene regulation were identified within the promoter region including light-responsive, hormone-responsive and various other stress-related elements. Approximate 0.8 kb fragment on upstream of ATG start codon of bcAS1 was sub-cloned into pAN580 vector to replace the 35S promoter driving the expression of green fluorescent protein (GFP) gene. The promoter activity was detected by transient expression in onion epidermis cells by the expression of GFP. Approximately 6 kb length of bcAS1 gene was cloned, containing 18 exons and 17 introns. Although a dozen of β-AS cDNA was isolated, seldom the promoter and gene of it was reported. This work was a valuable foundation for further studies on the regulatory role of β-AS in biosynthesis of saikosaponins.

Use of Native Promoter-eGFP as a Gene Reporter on Onion Epidermis to Analyze Gene Expression of AVR-Pia, an Avirulence Effector of Rice Blast Pathogen

Rice blast disease, caused by Magnaporthe oryzae , is a major rice disease over the world. Recent studies have identified avirulence effectors from the blast fungus that trigger rice immune against the pathogen invasion after specific interaction with resistance (R) proteins in rice. AVR-Pia is one of avirulence effectors that correspond to Pia-resistant protein, inducing hypersensitive response (HR). Enhanced Green fluorescent protein (eGFP) was used as a reporter of AVR-Pia expression in this study. We synthesized expression vector containing native promoter of AVR-Pia ( PRR ) fused to eGFP gene. Rice sheath assay was done to observe the fluorescence and the signal was found in appressoria and invasive hypha of M. oryzae , suggesting that AVR-Pia is expressed in appressorium and invasive hyphae after penetration. Although, rice sheath assay is a reliable way to study rice-pathogen interaction, it is a consuming-time method. Onion epidermis was tested to check the availability to use as model system instead of rice sheath. After inoculation M. oryzae containing PPR::eGFP on onion epidermis, fluorescence was observed in appressoria and invasive hypha of transformants similar to observation on rice sheath. Therefore, onion epidermis can be used as plant cell model to study M. oryzae effectors expression by fluorescence observation.