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

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.