INTRODUCTION OF THE SERINE GREEN FLUORESCENT PROTEIN (SGFP) Pyricularia grisea GENE NTO RA DC4 I CE ISOLATED FROM USING Digitaria ciliaris Agrobacterium tumefaciens-MEDIATED GENETIC TRANSFORMATION STEPHANIE , UTUT WIDYASTUTI and SURYO WIYONO1 2,3* 4 1Program Study of Graduate School, Institut Pertanian Bogor; Bogor 16680, IndonesiaBiotechnology, 2Research Center for Bioresources and Biotechnology, Institut Pertanian Bogor; Bogor 16680, Indonesia 3Department , Faculty of Mathematics and Natural Science , of Biology s Institut Pertanian Bogor; Bogor 16680, Indonesia 4Department of , Faculty of Agriculture, Institut Pertanian Bogor, Bogor 16680, IndonesiaPlant Protection R 4eceived 24 July 2013/Accepted 12 September 201 ABSTRACT Blast disease (caused by ) has long been known as a serious problem for up rice. Now, it also Pyricularia grisea land attacks lowland rice. However, the mechanism facilitating this range expansion is still unknown. One option is to insert a marker into so that it can be used to monitor the spread of infection. (P. grisea P. grisea S sGFP erin Green Fluorescent Protein ) gene of the study of has been used to monitor gene expression specific tagged proteins for fungal cell. genome of dc 4 from In this study, the gene has been integrated into the The sGFP igitaria c . P. grisea D iliaris p using thelasmid was introduced into triparental mating method (TPM). Genetic transformation sGFP A. tumefaciens was performed by co-cultivating spore dc4 with LBA4404–pCAMB-s . dc4 s of The P. grisea A. tumefaciens GFP P grisea. transformant was selected by using selection medium 300 µg/m hygromycin. The integration of a containing L of the the the c thesGFP sGFP sGFP gene into genome was confirmed by PCR using 's spe ific primer pair, -Nos terminator primer pair and -Tubulin primer pair as internal control. Expression of from the an the gene the β sGFP P. grisea dc4 transformant w detected with blue light fluorescent microscopas y. Keywords: Agrobacterium tumefaciens- , Digitaria ciliaris, Pyricularia grisea , erine Green mediated transformation dc4 S Fluorescent Protein INTRODUCTION Pyricularia grisea Magnaporthe grisea (teleomorph ), synonymous with Cav, is a rice Pyricularia oryzae plant pathogen in many countries and is known as the agent causing last disease (Rho 2001). et al.b The disease is estimated to be responsible for 30% of annual yield loss, the equivalent in food volume to meet the needs of 60 million people (Dagdas 2012). In Indonesia, blast infestation et al. reached 36,727 ha of the total 13,153,080 ha area of rice cultivation in 2012 (Ministry of Agriculture 2013). Blast disease has been known as a serious problem for up rice, however, land recent results showed that the last pathogen also b attacks rice planted in low s or irrigated land land (Sobir 2003). To date, the mechanism of blast et al. disease transmission from upland rice to lowland land has not been examined. P griseayricularia is a pathogen on more than 50 species of wild grass in the vicinity of rice field (Couch & Khon 2002 ). has a wide variety P. grisea of hosts besides rice plant; among them are Triticum aestivum Zea mays Eleusine coracana, , (finger millet), (cultivated grass), Setaria italica Brachiaria mutica et al (Couch & Khon 2002; Listiyowati . 2011). Because of this wide host range, cereal grasses and wild grasses around the area of cultivation are also prone to infection and P. grisea therefore, could become alternative hosts as well as alternative inocula sources for spreading disease.* Corresponding author : ututsuharsono2002@yahoo.com BIOTROPIA Vol. 22 No. 1, 2015: 73 - 79 DOI: 10.11598/btb.2015.22.1.329 73 mailto:ututsuharsono2002@yahoo.com BIOTROPIA Vol. 22 No. 1, 2015 74 Research showed that grasses growing around rice field could become temporary hosts for fungi that cause last disease (Listiyowati 2011). et alb . Those fungi were capable of infecting rice plants and showed some genetic structure alterations from the original grass isolate. isolated P. grisea from grass (dc4) is able to infect rice D. ciliaris plants that are moderately resistant and susceptible plants. This isolate of dc4 that switches host undergoes genetic alterations, which are marked by changes in SCAR marker for Cut1, PWL2, Erg2 as well as physiological race based on the ability to infect differential rice varieties in Indonesia (Listiyowati 2011). This et al. is presumed as a genomic adaptation response of the fungi isolate to the new host. However, the mechanism that causes such change is still unknown. Therefore, as an initial step it is necessary to insert a marker into to P. grisea monitor infection.P. grisea According to Lorang (2001), the et al. GFP gene could be used as a tool in studying interactions between fungi and plants. has GFP also been used to monitor the virulence of blast- causing fungi. gene has several advantages as GFP a marker. gene detection does not require any GFP substrate addition to obtain the visualization; it does not require special treatment of the tissue and its presence in the cell is not harmful to the cell itself. These advantages make a great GFP marker in gene transformation and expression (Lorang 2001). Today, the gene has been et al GFP. shown to be expressed in 16 species of 12 genera of fungi including (Lorang 2001). M. grisea et al . The gene has been successfully used as GFP important marker for several fungi that cause diseases and is among them (Balhadere & P. grisea Talbot 2001). sGFP gene transfer into fungi genome requires a biological vector. has Agrobacterium tumefaciens been long used as a biological vector for gene transfer in plants. Besides transferring genes to its host plant, is also able to transfer A. tumefaciens DNA to yeasts and filamentous fungi (Combier 2003). The objective of this research was to introduce gene into dc4 sGFP P. grisea isolated from using D. ciliaris A. tumefaciens-mediated genetic transformation. Subsequently, there would be a gene introduction into the fungi sGFP genome. The success of this gene introduction could be developed to facilitate molecular studies of pathogen-host interactions. MATERIALS AND METHODS Triparental Mating The introduction of binary plasmids that contain gene into was donesGFP A. tumefaciens with the TPM assay described by Hanum (2011). Three bacteria: DH donor ing E. coli 10B contain pCAMB- plasmids, DH1 ing sGFP E. coli contain helper pRK2013 plasmids and A. tumefaciens LBA4404 as the recipient strain were used in TPM assay. The three bacteria were grown in solid LB to prevent conjugation. A. tumefaciens sGFP transformants were then identified using colony PCR with specific primers -F and -R.sGFP sGFP Fungal Strains and Culture Conditions P. grisea D. ciliaris race dc4 isolated from was provide by d Mycology Laboratory, Department of Biology, Faculty of Mathematics and Natural S c i e n c e s , I n s t i t u t P e r t a n i a n B o g o r . Fungal cultures were grown on oatmeal agar medium (oma: 30 g of oatmeal for 1 L). Fungi were grown in OA medium and incubated for 7- 10 days at 28 ºC. For production of conidia, fungal cultures were grown under light of a near- UV lamp for 5 6 days to using sterilized water ( ).Munandhar 1998et al. Transformation One colony of containing A. tumefaciens pCAMB- plasmids was grown on 2 mL of sGFP Minimal Medium (Hooykaas 1979) et al. sup lemented withp 100 µg/mL streptomycin, 60 µg/mL kanamycin, and 50 µg/mL hygromycin at 28 C for 48 hours with 250 rpm rotation ino no light condition. The bacterial culture was diluted with 5 mL Induction Medium (IM) containing 200 µM acetos ringone toy an optical density of 0.15 at A600. The bacterial culture of A. tumefacien -sGFPs was grown for 4-6 an additional hours at 28 C with 250 rpm rotation o to reach an A600 . (Betts 2007) of 0 5 . . Approximately et al 100 µl of dc4 spores (10 spores/mL) was P. grisea 6 mixed with 100 µL of cells A. tumefaciens-sGFP (A600 = 0.5) then added with a etosyringon 200c e µM, and incubated for 30 minutes with no light. The co-cultivated cultures were then into plated IM medium and incubated at 28 C for 48 hours. o The co-cultivation result was transferred into Complete Medium containing 300 µL/mL Introduction of the ( ) gene nto Serine Green Fluorescent Protein sGFP Pyricularia grisea i Stephanie – et al. hygromycin and 200 µg/mL cefotaxime and incubated at 28 C for 5-7 days. pore o Single s selection was done by spreading 100 L µ conidia suspension on OA medium that contained 300 µg/mL h gromycin and incubated for 5-7 days y until dc4 appeared (Rho P. grisea et al single spores . 2001 modified by Betts 2007).et al. Analysis of Transformant Genomic DNA of mycelium from P. grisea transformant and non-transformant were P. grisea isolated for verification using PCR. DNA isolation was done with the method described by Listiyowati (2011) using 2% et al etyl rimethyl . c t a b mmonium romide (CTAB). The isolated DNA sGFP gene as then w amplified through PCR using -F and -R primers as well as sGFP sGFP combined primers of F and Nos-R. The sGFP- PCR mixture used consisted of 1 µL (100 ng) genomic DNA, 0.5 mM forward primers, 0.5 mM reverse primers, 5 µL PCR mix (Fermentas) and added with ddH O up to 10 L of total volume. 2 µ The PCR program to amplify fragments sGFP consisted of: pre-denaturation at 94 ºC for 1 minute, denaturation at 94 C for 1 minute, o annealing at 53 C for 30 seconds, elongation at o 72 C for 1 minute and final elongation at 72 C o o for 5 minutes; this process was run in 35 cycles. The PCR results were through visualized electrophoresis 1% (b/v) agarose gel at 100 on volt for 30 minutes in TAE 1 and continued with gel immersion in 0.5 mg/L EtBr for 20 minutes and were visualized under UV transluminator (Shanti 2008). M yicroscop To visualize , P. grisea transformants they were subcultured in OA medium containing 300 µg/mL hygromycin. The culture was incubated at room temperature for 6 days. The transformants were grown under light of the a near-UV lamp for 5 6 days. The acquired spores were then to observed using an Olympus BH2-RFCH microscope on the fluorescent setting with a 515 bandpass emission fillter (blue light) . RESULTS AND DISCUSSION Triparental Mating (TPM) The plasmid CAMB- was introduced p sGFP into using the TPM method (Fig. 1). A. tumefaciens Plasmids in DH10B were contained E. coli moved into LBA4404 through A. tumefaciens conjugation with the help of pRK2013. E. coli Triparental mating generated several colonies growing in a medium containing 50 µg/mL kanamycin, 50 µg/mL streptomycin and 50 µg/mL hygromycin. The only bacterium able to grow in that selective medium was A. tumefaciens LBA4404 containing pCAMB- plasmids. sGFP Colonies from TPM that were able to obtained grow in the selective medium were then analyzed with PCR. The results of the PCR analysis showed that colonies of with -F and -A. tumefaciens sGFP sGFP R primers produced amplicon with the size of 643 bp (Fig. ). The results of this amplification 1 had the same size as the positive control (Fig. ). 1 This showed that pCAMB- plasmid w sGFP as successfully introduced into A. tumefaciens through the TPM method. This method was previously used by Hanum (2011) to move the binary plasmid pMSH- into MmCuZn-SOD A. tumefaciens et al.. According to Wise (2006) TPM is a quite efficient method for transferring gene in a non-conjugated plasmid into A. tumefaciens. 1000 bp 750 bp 500 bp 250 bp 643bp Figure . PCR identification of the gene introduced into LBA4404 colon using TPM1 ies . M: marker 1 kb; sGFP A. tumefaciens K+: Plasmid pCAMB- (positive control); K-: ddH O (negative control); 1-5: transformant sGFP A. tumefaciens2 sGFP 75 Transformation P. griseaGenetic transformation of dc4 using A. tumefaciens w s achieved using the method a described in Rho (2001) and Betts (2007). et al et al. . One hundred microliter (100 µL) of spores (10 6 per mL) were co-cultivated with 100 µL of bacterial cells in an induction medium that had been added to 200 M acetosyringone for 48 µ hours. An acetosyringon concentration of e 200 µM is important in fungal transformation to induce virA genes of so that T-A. tumefaciens DNA transfer may take place (Knight . 2009). et al An acetosyringon concentration of 200 µM is e commonly used in fungal transformation with A. tumefaciens as a biological vector and it has been shown to have consistent results (Covert . et al 2001; dos Reis . 2004; Knight . 2009; et al et al Xiaoran . 2012)et al . P. grisea dc4 grown in hyg omycin and r cefotaxime selective medium was then cultured to produce spores. The spores were then spread in OA medium containing 300 µg/mL hygromycin and incubated for 5-7 days until some single spores that were transformed were obtained (Fig. 2). Selection of spore was done to single s eliminate false transformant fungi (Betts et al. 2007). Mycelium growth on hygromycin medium indicated the success of transformation (Tucker & Orbach 2007). Some s that single spore were not able to grow on hygromycin medium because the genes having important roles in growth might undergo some damage or their activities might be interrupted by the presence of foreign genes. P. griseaSingle s s pore of dc4 transformant and P. grisea dc4 non-transformant were cultured in a medium containing 300 µg/mL hygromycin then the growth diameter was observed at day 7, 14, and 21. The growth of transformant P. grisea increased rapidly which was 14.8 mm at day 7 became 28.7 mm at day 14 and became 37.3 mm at day 21. non-transformant growth was P. grisea slower which was 6.5 mm at day 7, 12.55 mm at day 14 and 17.3 mm at day 21 (Fig ). . 3 Hygromycin added into the OA medium was able to inhibit the growth of non-P. grisea transformants compared to dc4 P. grisea transformant (Fig 4). This was caused by sGFP . the inhibiting activity of hygromycin on the P. grisea dc4 non-transformant. The use of an appropriate selection marker is essential for the success of transformation. Selection using an antibiotic is performed to eliminate non- transformant cells as well as to ensure the resistance level carried by the transformant (Frandsen 2011). In this research dc4 P. grisea transformants were resistant to hyg o-sGFP r mycin because of the addition of the hygromycin-resistance gene contained in the pCAMB- plasmid. Betts (2007) used sGFP et al. the concentration of 300 µg/mL for the selection of both transformant and non-M. grisea transformant. In contrast, Shanti (2008) used a concentration of 225 µg/mL hygromycin to inhibit 173 (originated from rice plant) P. grisea non-transformant and transformant generated through speroplas. The difference in growth rate between the transformant on hygromycin medium and non-hygromycin medium (Fig. 4) was likely due to the influence of the number of gene copies integrated, however, further evidence for this is required. F 2 Single spore race igure . of dc4 at day 5 grown in OA medium containing 300 µg/mL hygromycinP. grisea BIOTROPIA Vol. 22 No. 1, 2015 76 Figure . Average growth rate of dc4 non-transformant and transformant in OA selective medium 3 P. grisea sGFP containing 300 µg/mL hygromycin Figure 4. Growth of dc4 non-transformant (NT) and transformant (T) in OA medium containing 300 µg/mL P. grisea hygromycin (hgr+) and without hygromycin (hgr-) Analysis of ransformantT Putative transgenic dc4 from grass that P. grisea had been selected in hygromycin medium was then analyzed using PCR with specific primers sGFP- sGFP- sGFP-F and R as well as F and Nos-R primers to detect the presence of gene. sGFP PCR molecular analysis generated amplicons of 643 bp and 899 bp which were in alignment with the positive control amplicon (Fig. ). PCR 5 of non-transformant DNA did not generate sGFP gene amplicons. This result showed that the sGFP gene was inserted into the fungal genome. The gene was used as an internal B-tubulin control to ensure the amplified DNA was in good condition. The PCR using Bt1aF and B-tubulin Bt1aR primers generated amplicons of 550 bp (Fig ). 5 . P. grisea sGFP dc4 with gene insertion was the bright green fluorescence observed using a fluorescence microscope (Fig. 6). This showed that the gene was well integrated in the sGFP fungal genome and constitutively expressed in P. grisea sGFPdc4. The use of requires a promoter which is needed for expression. The sGFP pCAMB- plasmid receiving gene sGFP sGFP insertion from the pCT74 plasmid was able to express the gene under the control of the sGFP ToxA promoter (Lorang 2001).et al. 77 Introduction of the ( ) gene nto Serine Green Fluorescent Protein sGFP Pyricularia grisea i Stephanie – et al. CONCLUSIONS Pathogenic dc4 from had been P. grisea D. ciliaris successfully transformed with the gene sGFP using -A. tumefaciens-mediated genetic trans formation. The gene had been integrated sGFP into the genome. Mycelia had P. grisea fluorescence been observed under a fluorescence microscope. ACKNOWLEDGEMENTS Thanks to Dr. Osbourn from John Innes Institute, England who provided plasmid pCAMB- . Thanks to Margaret Cargill and sGFP Pattric O'Connor for early reading this manuscript. This research was funded by I- MHERE B2C titled “Biological Role of Rice Blast Disease to Develop Rice Plant Tolerance to Rice Blast” under the name of Dr Utut Widyastuti, ag reement letter No : 12- IT3.24.4/SPP-I-MHERE/2012. REFERENCES Balhadere PV, Talbot NJ. 2001. PDE1 encodes a P-type ATPase involved in appressorium-mediated plant BIOTROPIA Vol. 22 No. 1, 2015 78 Figure . PCR results of dc from transformation5 4 . M: Marker 1 kb; K+ dan : plasmid pCAMB-P.grisea sGFP sGFP-Nos sGFP -Tubulin P. grisea P. grisea , K+ : ras 173 (positive control); K-: ddH2O (negative control); 1: DNA dc4 β nontransformant; 2-3: DNA dc4 transformant P. grisea sGFP Figure . dc4 transformant mycelium 7 hours after spore harvest observed under microscope without and with 6 P. grisea sGFP fluorescent infection by the rice blast fungus . Magnaporthe oryzae Science Direc 44:1035-49t . Betts MF, Tucker SL, Galadima N, Meng Y, Patel G, Li L, Donofrio N, Floyd A, Nolin S, Brown D .et al 2007. Development f igh hroughput ransformation o a h t t s f i m iystem or nsertional utagenesis n Magnaporthe oryzae. Science Direct 44:1035 49. - . Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R. 2 0 0 3 . - m e d i a t e d A g r o b a c t e r i u m t um e f a c i e n s transformation as a tool for insertional mutagenesis in the symbiotic ectomycorhizal fungus Hebeloma cylindrosporum. Microbiol Lett 220:141-48. Couch BC, Khon LM. 2002. A multilocus gene genealogy concordant with host preference indicates segregation of a new species, , Magnaporthe oryzae from Mycologia 94(4):683-93.M. grisea. Couch BC, Fudal I, Lebrun MH, Tharreau D, Valent B, Van Kim P, Notteghem JL, Kohn LM. 2005. Origins of host-specific populations of the blast pathogen Magnaporthe oryzea in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice. Genetics 170:613-30. Covert SF, Kapoor P, Lee MH, Goh J, Yoo SY, Park J, Jung K, Kim H, Park SY, Rho HS . 2001. et al Agrobacterium tumefaciens- Fusarium mediated transformation of circinatum. Myco Res 105:703-9. dos Reis MC, Pelegrinelli Fungaro MH, Delgado Duarte RT, Furlaneto L, Furlaneto MC. 2004. Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus . J Beauveria bassiana Microbiol Methods 58:197-202. Dagdas YF, Yoshino K, Dagdas G, Ryder LS, Bielska E, Steinberg G, Talbot NJ. 2012. Septin-mediated plant cell invasion by the rice blast fungus Magnaporthe grisea. Science. 336:1590-5. Departemen Pertanian, Direktorat Jenderal Tanaman Pangan. 2012. Evaluasi rakiraan erangan tama OPT P S U Utama Tanaman Padi, Jagung, Kedelai MT 2011/2012. Jakarta (ID): Deptan. Frandsen RJN. 2011. A guide to binary vectors and transformant for targeted genome modification in fungi using -mediated Agrobacterium tumefaciens transformation. Microbiol Methods 87:247-62.J Hanum S. 2011. Isolasi pengklonan dan analisis ekspresi gen penyandi copper-zinc superoxide dismutase (cuZn- SOD) dari Disertasi. Bogor Melastoma malabathricum. (ID): Institut Pertanian Bogor. Hao X, Ji Y, Liu S, Bi J, Bi Q, Pan J, Zhu X. 2012. Optimized integration of T-DNA in the taxol-producing fungus . African J Biotec Pestaliopsis maliocola h 11(4):771-6. Hooykaas PJJ, Roobol C, Schilperroot RA. 1978. Regulation of the transfer of Ti-plasmid of Agrobacterium tumefaciens . Microbiol 110:99-109. Knight CJ, Bailey AM, Foster GD. 2009. -Agrobacterium mediated transformation of the plant pathogenic fungus Plant Pathol 91(3):745-Verticillium albo-atrum. 50. Listiyowati S, Utut W, Rahayu G, Hartana A, Jusuf M. 2011. Diversity of SCAR markers of raced Pyricularia grisea from following cross infection to Digitaria ciliaris rice. Microbiol Indones 5(1):1-8. Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, Rollins JA, Wolpert TJ, Johnson KB, Rodriguez RJ, Dickman MB . 2001. Green f u rescent protein l oet al is lighting up fungal biology. Appl Environ Microbiol 67:1987-94. Munandhar HK, Jorgensen HJL, Smedegaard-Petersen V, Mathur SB. 1998. Seedborne infection of rice by Pyricularia grisea and it's transmission to seedling. Plant Dis 82:1093-99. Olmedo-Monfil V, Cortes-Penagos C, Herrera-Estrella A. 2004. Three decades of fungal transformation. Balbas P, Lorence A, editor. Recombinant Gene Expression. Reviews and Protocols Volume 267 Methods in : Molecular Biology. ess pTotowa (US): Human Pr . 297- 309. Rho HS, Kang S Lee YH. 2001. -, Agrobacterium tumefaciens mediated ransformation of he lant athogenic t t p p f .ungus, . Mol Cells 12:407-11Magnaporthe grisea Sesma A, Osbourn AE. 2004. The rice leaf blast pathogen undergoes developmental processes typical of root- infecting fungi. Natur 431:582-86.e. Shanti NR. 2008. Introduksi gen - ( ke dalam serin GFP sGFP) genom . kripsi. Bogor : Institut Pyricularia grisea S (ID) Pertanian Bogor. Sobir, Andrianyta H, Amir M. 2003. SCAR (Sequence Characterized Amplified Region) analysis for blast resistant evaluation on 12 genotypes of rice. Bul Agron 31(1):21-5. Tucker SL, Orbach MJ. 2007. -mediated Agrobacterium transformation to create an insertion library in Magnaporthe grisea. Ronald PC, Lorence A, editor . Plant-Pathogens Interaction Methods and Protocols; Methods and Protocols Volume 354 Methods in olecular iology . : M B Totowa (US): Human Pr . 57-67.ess p Wise AA, Liu ZZ, Binns AW. 2006. Three methods for introduction of foreign DNA into . Agrobacterium Methods in Mol iol 43-54B . 79 Introduction of the ( ) gene nto Serine Green Fluorescent Protein sGFP Pyricularia grisea i Stephanie – et al.