İNŞaat mühendiSLİĞİ anabiLİm dali

Quellıng Of Trıchothecene Productıon İn Fusarium Specıes
In this study, tri4 and tri5 genes associated with trichothecene biosynthesis in Fusarium species were quelled with siRNA mediated mechanism. RNAi was triggered by six siRNA molecules designed specific to exonic sites of the genes in F. graminearum 4F and F. culmorum 9F and 20F isolates. For this purpose, effects of different in vitro conditions on tri4 and tri5 expression were investigated in 38 Fusarium isolates. Fusarium cultures grown on pH 5.6 and at 25°C was used as control group. Effects of temperature on trichothecene production was tested by cultivating of mycelia on medium with pH value of 5.6 at 8°C and 15°C. Also, the effects of pH was investigated by cultivating the Fusarium species on medium with pH 3.0 and 7.0 at 25°C. Another factor effecting trichothecene production was addition of H₂O₂ (0.5 mM) to medium. Maximum tri4 and tri5 expression levels were determined in control groups. Maximum expression levels of tri4 and tri5 genes were detected in 4F among screened 14 F. graminearum isolates. While the highest tri4 expression was determined in 20F among 24 F. culmorum isolates, maximum tri5 expression was found in 9F. Consequently, these three isolates were selected for induction of RNAi mechanism. In order to design specific siRNA molecules, full length tri4 and tri5 fragments were amplified from 4F, 9F and 20F isolates, sequenced and subjected to CLUSTALW analysis. Sequencing results revealed that there was high levels of similarity (94-99%) between tri4/tri5 genes belonging to reference genome and gene sequences that of 4F, 9F and 20F isolates. Sequence data belonging to two genes of these three isolates were deposited under database by gaining accession numbers (KJ677970, KJ677971, KJ677972, KJ677973, KJ677974 and KJ677975). siRNA molecules used in study were generated according to these sequence data via bioinformatic tools. Among hypothetically identified 60 exonic siRNA molecules, four siRNA for tri4 (Tri4E1:5'-ACAATACGGGCGTGAGTCATGTCAA-3', Tri4E2:5'-GAACCCTGAGAGAAGTTGGTGATGT-3', Tri4E3:5'-ACATGCTCCTTGTACTTCAAGTTCT-3', Tri4E4:5'-CATCTTGGAGATCTCCCAGAGAT-3') and two siRNA for tri5 gene (Tri5E1:5'-AAGCGACTACAGGCTTCCCTCCAAA-3', Tri5E2:5'-CAGGATCTGATGACTACCCTCAATT-3') were selected for using in quelling. These six double stranded siRNA were co-transfected as a single or multiple together with helper plasmids (pEGFP75 and pAN7-1) into protoplast cultures with totally 30 combinations for tri4 and six for tri5. Transfection efficiency was found ranged from 500.00 to 99.3310.06/5x10⁴ in 36 experimental sets. Transfectants were selected by fluorescence microscopy and spectrofluorimetric analysis. Fluorescence emitting by GFP protein in cells was measured as spectrofluorimetrically. RFU (relative fluorescence unit) values of transfectants were found between 1.37±0.07 and 2.89±0.06. Besides, PCR was used in selection of transfectants with pAN7-1 and pEGFP75. For that purpose, 0.7 kb fragments of hph gene in pAN7-1 plasmid and GFP gene in pEGFP75 plasmid were amplified. Nearly a hundred per cent tri4 suppression in 30 experimental sets was shown by real time PCR analysis. Down regulation of tri5 gene for both of two species was detected as 65-95% in remaining 6 experiment groups. At the same time, it was shown that DON mycotoxin, final product of trichothecene biosynthesis, was not produced in tri4 and tri5 silenced Fusarium samples by thin layer chromatography. As a result, it was demonstrated that both tri4 and tri5 genes could be used in quelling of trichothecene biosynthesis pathway, in this study. Moreover, it was concluded that tri4 gene could be more useful target than tri5 for RNAi. It was thought that significant decreases in the expressions of tri4 and tri5 genes observed as a result of siRNA transfection were carried out in post-transcriptional level in Fusarium species. This study has a great importance in terms of being not only the first report related to siRNA mediated gene silencing in Fusarium species but also an applicable model in struggling with phytopathogenic species.