Introduction of Serine Green Fluorescent Protein (sGFP) gene into pyricularia grisea Race dc4 isolated from digitaria ciliaris using agrobacterium tumefaciens-mediated genetic transformation

Abstract

Pyricularia grisea is a rice plant pathogen has been known as the agent causing blast disease. Serin Green Fluorescent Protein (sGFP) gene have been used to monitorised gene expression and virulence of blast-causing fungi that has implication for fungal cell study. This research aimed to introduce sGFP gene into genome of P. grisea dc4 from D. ciliaris using A. tumefaciens. Plasmid sGFP was inserted into A. tumefaciens by triparental mating method (TPM). Genetic transformation was performed by co-cultivating spore P. grisea dc4 with A. tumefaciens LBA4404–pCAMB-sGFP. Pyricularia grisea dc4 transformant was selected by using selection medium that contain hygromycin. The growth test of non transformant P. grisea on OA medium 300 μg/ml hygromycin is slower than P. grisea transformant sGFP that increased rapidly from day 7 until day 21. The integration of sGFP gene into genome was confirmed by PCR using sGFP’s spesific primer pair, sGFP-Nos terminator primer pair and β-Tubulin primer pair as internal control. Expression of sGFP from P. grisea dc4 transformant was detected using fluorescent microscope with a 515 nm bandpass emission filter. Microscopic observation show that the growth of P. grisea dc4 transformant spores started from the basal area which will become the germ tube formation. The transformant P. grisea dc4 showed the ability to infect and to colonize within the host, that is showed by the hyphae fluorescence. This result clearly indicated that P. grisea dc4 which was taken from grass have the ability to infect on susceptible rice varities.