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

Abstract

Abstract. 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.

Fluorescent reporter analysis revealed the timing and localization of AVR‐Pia expression, an avirulence effector of Magnaporthe oryzae

“…We clearly demonstrated that fungal penetration is not required for AVR‐Pia expression, as it is induced before penetration and also occurs in the appressoria of Δ pls1 . Previously, AVR‐Pia expression has been reported to occur in well‐melanized appressoria of M. oryzae inoculated onto glass slides (Sornkom et al ., ), when the ability of fungal penetration was blocked by impenetrable artificial membranes. Therefore, AVR‐Pia expression is clearly independent of fungal penetration.…”

“…Seeds of Shin‐2 rice, which lacks the Pia resistance gene, were sown in plastic seedling pots containing soil with fertilizer (Honens seedling soil; Honen Agri, Co., Niigata, Japan) and grown in a glasshouse for 60 days, or until the seventh leaf stage, when intact leaf sheaths were inoculated by injecting a conidial suspension, as described previously (Sornkom et al ., ). The inoculated leaf sheaths were prepared for microscopy by excising thin layers of sheath epidermal cells that had been in contact with the conidial suspension, and the excised leaf sheath pieces were placed on slides and visualized using a BX‐50 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a U‐MNIBA3 filter set.…”

“…Onion epidermis was prepared as described previously (Sornkom et al ., ), inoculated with diluted conidial suspensions from each transformant (10 4 −10 5 cells/mL) by dropping about 10 drops (1 μL/drop) of conidial suspension over the membranes and incubated at 27 °C for 24 h. Fluorescence visualization was then performed as in the rice sheath assay.…”

Screening of endophytic fungi as potential antagonistic agents of Pyricularia oryzae and evaluation of their ability in producing hydrolytic enzymes

“…For molecular identification, mycelium of P. oryzae was inoculated into 2YEG medium (consisting of 2 g/L yeast extract and 10 g/L glucose) in an erlenmeyer flask and incubated at 100 rpm for 3 days at 27°C. Mycelia were then collected on filter paper according to Sornkom et al (2015), while endophytic fungi that had the highest inhibitory ability in the antagonist test were grown on potato dextrose broth (PDB) media (containing the same composition as PDA without the agar). Then, the suspension was incubated for 3 days at 27°C.…”

Speculation of Nano-gap Sensor for DNA sequencing technology: A Review on Synthetic Nanopores

“…DNA/RNA sequencing, protein or peptide analysis, nanoparticles detection, and single molecular sieving. Nanopore technology is expected to be the upcoming platform for biotechnology and medical application since it furnishes the ability to analyze the characteristics of biomolecules or even their sub-units at the molecular-scale resolution [3][4][5][6]. Same as other miniaturized systems, it grants following benefits, e.g.…”