Minna J. Kemppainen, Alejandro G. Pardo Microbial Biotechnology 3: , 2010

Transcription

1 phg/psilbaγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihprna triggering in the mycorrhizal fungus Laccaria bicolor Minna J. Kemppainen, Alejandro G. Pardo Microbial Biotechnology 3: , 2010 Speaker:Chih-Jui Cheng Adviser: Ching-Tsan Huang, PhD Date :

2 2 Plant-fungi Symbiosis Nutrient acquisition Resistance to environmental stress and pathogens Soil aggregation Soil water retention Ectomycorrhiza Endomycorrhiza Mycorrhizal networks help ecosystems Plants with mycorrhizal fungi have higher capability to survive long-term

3 3 Laccaria bicolor Ectomycorrhiza Mutually beneficial Partner tree: conifers Favorite species for research L. bicolor genome is sequenced (Martin et al., 2008.) Martin et al., 2008 Kingdom: Fungi Division: Basidiomycota Class: Agaricomycetes Order: Agaricales Family: Hydnangiaceae Genus: Laccaria Species: L. bicolor

4 4 Homologous recombination knock out Filamentous fungi show low homologous recombination frequency.

5 5 RNA interference (RNAi) Gene down-regulation mediated by small RNAs microrna (mirna) Small interference RNA (sirna) He and Hannon, 2004

6 6 RNAi trigger Silencing vector Transformation Host Transformant with gene knock-down 5' 3' Promoter target intron inverted target terminator Intronic spacer hairpin RNA (ihprna) Incorporation of an intronic sequence as hprna spacer increasing silencing efficiency. (Smith et al., 2000; Wesley et al., 2001; Lee and Carthew, 2003)

7 7 RNAi trigger Transcription Transformant with gene knock-down Gene silencing

8 8 Applications of RNAi Medicine RNA interference Genetic study Biotechnology

9 9 Flowchart Vector construction ihprna of nitrate reductase (NR) Agrobacterium tumefaciens-mediated transformation (ATMT) Transformants analysis phenotypic evaluation semi-quantitative RT-PCR methylation analysis T-DNA copy number analysis integrated site analysis

10 10 Nitrate reductase (NR) Nitrate reductase NO - 3 NO - 2 Nitrite reductase NH 4 +

11 11 Factor of silencing strength Optimized intronic spacer size Chad A. et al., 2004 CpG methylation reduces gene transcription Susan E. Cottrell, Wälti et al., Kemppainen et al., 2009.

12 12 Vector construction M. oryzae CUT intron Pgpd Pgpd Intron of M. oryzae cutinase is 147 bp L. bicolor NR intron Pgpd Pgpd Intron of L. bicolor NR is 52 bp Pgpd L. bicolor NR intron Pgpd SC: silencing cassette HRC: hygromycin resistance cassette Pgpd: glyceraldehide-3-phosphate dehydrogenase promoter of Agaricus bisporus

13 13 Two-step cloning of the hairpin trigger Ti plasmid Silencing vector ATMT can be used in many homobasidiomycetes Suiable for many homobasidiomycetes

14 phenotypic evaluation 14

15 15 Methylation of gpdii promoter 555 bp 249 bp 511 bp 327 bp H, HpaII (methylation sensitive) phg/psilbaγ show minimum Isoschizomer CpG M, MspI (methylation insensitive)

16 16 NR knockdown correlate to growth Trasformants of phg/psilbaγ wt N A S

17 17 Silencing strength variation (SSV) Strains carrying the same silencing vector show variant silencing effects. Possible factors: Transgene copy number Transgene integration site (euchromatin versus heterochromatin)

18 18 T-DNA copy number and silencing strength No correlation was observed between T-DNA copy number and silencing strength

19 19 T-DNA integrated site Strongly affected category (S) integrated in protein-coding region. Growht phenotype category Genomic T-DNA integration site Integration type Protein ID EST support Proposed protein function Silencing casette orientation strain 62 N scaffold 6: intergenic integration strain 32 A scaffold 59: intergenic integration (repetitive sequence ReconFam374.db.fa.best20.malign ) strain 30 S scaffold 3: intron integration yes not known opposite to the gene transcription strain 26 S scaffold 25: intron integration no calsium ion binding opposite strain 26 S scaffold 11: Putative promoter integration (133bp upstream of the start codon) yes Mucin-like protein 1 precursor opposite strain 21 S scaffold 9: integration in the 3`UTR sequence yes Histone 2ª in gene transcription orientation Strain 26 has two T-DNA integrations.

20 20 Flowchart Vector construction ihprna of inositol-1,4,5-triphosphate 5-phosphatase (5-Pase) Agrobacterium tumefaciens-mediated transformation (ATMT) Transformants analysis phenotypic evaluation semi-quantitative RT-PCR

21 21 Inositol-1,4,5-triphosphate 5-phosphatase (5-Pase) Phosphoinositide control several cellular processes including cell growth. Sanjive Qazi, Barry A. Trimmer, 1999.

22 5-Pase knockdown 22

23 23 Summary psγ shows best silencing efficiency 5-Pase gene knockdown NR gene knockdown Lower methylation level in psγ

24 24 Populus trichocarpa Laccaria bicolor

25 25 Thanks for your attention