The Laccaria transcriptome: ESTs and Microarrays

1. The Laccaria transcriptome: ESTs and Microarrays • EST resources • Nylon array • NimbleGen genome expression array • Symbion Chip (JGI)

2. cDNA libraries CAHB, CAHC, CAHF,CAHG, cDNA libraries from mixed tissues (JGI) CAHH, CAPI,CAPN (mainly mycelium) 30826 ESTs LU ? 165 ESTs Lb01-Lb34 cDNA libraries from three week old mycelium (INRA Nancy) 1636 ESTs LbII cDNA library from fruiting bodies stage 1&2 (INRA Nancy) 496 ESTs LbIII cDNA library from cap of fruiting body stage 6 (INRA Nancy) 469 ESTs LG0ADF cDNA library from mycorrhizal roots (Douglas Fir) (INRA Nancy& Génoscope) 747 ESTs LG0ADG cDNA library from mycorrhizal roots (Poplar) (INRA Nancy& Génoscope) 1025 ESTs total 4373 ESTs 14312 TCs 6514 gene models with EST support Only one-third of the predicted gene models have an EST support so far! 10 000 additional ESTs from a fruit body library in 2007

3. NimbleGen Expression Array -22,294 predicted Laccaria gene models used -178,352 60mer (8 oligos per gene models) -for about 30% of the gene models we expect a cross-hybridization

4. Evaluating probes Each probe is evaluated for uniqueness in two ways: Simple frequency count. Each probe is counted for the exact number of times it appears in the genome of interest. "Careful uniqueness" test. Each probe is compared to the target genome and given a pass/fail score for uniqueness. The goal of the "careful uniqueness test" is to screen probes which may be unique, but not in ways that effectively prevent non-specific hybridization. a. Oligos that have long runs (>5) of homopolymers or that are too GC rich or AT rich are avoided.  b. We calculate a self-annealing score by comparing the oligo to its reverse complement. Oligos that are more than ~60% self-complementary are eliminated. In addition, we remove probes that require large numbers of cycles to synthesize. Our target is three times the oligo length, i.e. 72 cycles for a 24mer. Bases are always added in the order A, C, G, and T. If probes exceed our target cycle limits, we can truncate them by removing bases from the 3' end. Bases are synthesized on the array 3'-5', with the 3' end closest to the glass. Selecting probes To select probes, we combine the scores from the evaluation testing and calculate a rank score. When choosing multiple oligos from a given region, we select the first oligo and then recalculate the rank score, adding a positional weight based on proximity to other selected probes. Once a probe has been selected in a given region, its neighbor's scores are decreased so that the tendency is to pick evenly spaced probes. We select longer oligos, those 25mer - 70mer in length, based on the composite scores of the 24mer probes that make up the longer oligo. For example, a 60mer is selected as composite of 37 overlapping 24mer probes.

5. NimbleGen Expression Array • -22,294 predicted Laccaria gene models • -178,352 60mer (8 per gene models) • -for about 30% of the gene models we expect a • cross-hybridization • -Random Priming Labeling • -Biological material • Laccaria S238N mycelium • Laccaria 81306 mycelium • Laccaria S238N mycorrhiza (Douglas fir,Poplar) • Laccaria S238N fruit bodies (early stages, late stages) • Laccaria S238N in contact with helper bacteria

6. Symbion Chip (JGI) • Goal: Study processes involved in symbiosis of L.bicolor (Lb) and P.trichocarpa (Pt) by analysis of transcriptomes of both genomes placed on one Nimblegen chip build at LLNL • Inputs: ~20K Lb genes, ~45K Pt genes, 400,000 spots on chip

7. Chip Design Status • %GC content of each genome 34% for Pt; 47% for Lb • Range of probe lengths 50-70 for Pt; 34-44 for Lb • Labeling reaction, probe placement strategy: random priming; probes span whole transcript (not 3’ biased) • Acceptable range of Tm for probes: 71.8 - 75.3 • Number of probes/transcript: 5-6 (one per exon; no replicates)

8. Target sequence selection • Identify common 12-mer sequences in each genome (usually associated with repeats) and mask them • Pick candidate probes from remaining sequence, adjusting length for optimal Tm • Remove probes with homopolymer runs, palindromes, other undesirable features • Calculate mask cycles needed for NimbleGen synthesis, discard probes outside range for probe length. • BLAST probes against both genomes and find unique probes for each transcript • Select unique probes distributed across exons with high Tm • Calculate free energies of hybridization to target, hairpins, and homodimers • Select desired number of probes for each transcript, with G closest to overall median.

9. Biological material cDNA samples corresponding to RNA from free-living mycelium of Laccaria bicolor S238N, non-mycorrhizal plantlets of Populus trichocarpa and ectomycorrhizal roots of Laccaria/Populus at pre-contact, early and late stages of development will be provided to JGI array facilities by INRA-Nancy and UAH. Owing to the limited amount of symbiotic tissues, complex probes will be prepared by reverse transcription using SuperScript II reverse transcriptase and the SMART-PCR cDNA Synthesis Kit (Clontech). A limited number (13–18) of PCR cycles will be used to ensure that the cDNA synthesis is in the linear phase of amplification using qPCR of selected number of marker genes • Mycorrhizal roots (from greenhouse experiments) • Control roots (from greenhouse experiments) • Control mycelium (from Petri dishes)

10. Existing nylon arrays Laccaria nylon array I: 4608 ESTs spotted (mycelium cDNA library 951 ESTs, each spotted four times) Laccaria nylon array II: 4992 ESTs spotted (mycelium cDNA library 768 ESTs, fruiting body cDNA libraries 4224 clones (965 sequenced))

11. L. bicolor S238N grown on different nitrogen and carbon concentrations Two genotypes of Laccaria bicolor (S238N / 81306) associated with the same Douglas fir seedling (Pseudotsuga menziesii) Experiments M.Peter Forest stand with introduced genotypes of L. bicolor (S238N / 81306) Two genotypes of L. bicolor associated with separate Douglas fir seedlings and fertilized with different nitrogen concentrations

12. Most abundant transcripts in Laccaria S238N Mycorrhiza (Douglas fir)

13. Comparison of most abundant transcripts in different tissues and under different nutritional conditions

14. versus

15. Coming soon… ?

17. Functional Genomics of Ectomycorrhizal Symbioses 10,000 Expressed Sequence Tags (ESTs) in various ectomycorrhizas => ForEST Project, french CNS - Genoscope (Evry) Douglas fir - Laccaria ECM Greenhouse, 9 month-old 2273 ESTs 654 Laccaria 1355 Douglas fir 264 nd Poplar - Laccaria ECM in vitro 6-8 weeks-old 2810 ESTs 910 Laccaria 1665 Poplar 235 nd Poplar - Pisolithus ECM in vitro 1 month-old 6652 ESTs 3823 Pisolithus 2497 Poplar 332 nd 2 % cell-wall 4.5 % MTs 40 % 42 % 36 % 20 % 15 % 22 % Martin et al., unpublished

18. Functional Genomics of Ectomycorrhizal Symbioses Expressed Sequence Tags (ESTs) in ectomycorrhizas Most abundant transcripts are coding for… Poplar - Pisolithus ECM Pisolithus => Cell-wall SR proteins (HydPm2 & 3, SRAP32 = 3.5 % of fungal ESTs) Metallothionein-like proteins (4 % of fungal ESTs) Actin & cytoskeletton-related proteins GTPases, cyclophilin & transduction signaling enzymes Ribosomal proteins (2 % of fungal ESTs) C, N metabolism enzymes & transporters Poplar => Ca2+-related signaling proteins, Ser/thr Kinases & RLKs Auxin, ABA, ethylene response proteins Aquaporines Metallothioneins (3.5 % of plant ESTs) Extensins RedOx regulation enzymes (peroxidases, thioredoxins, GST, SOD) Defense-related genes (PR- proteins, HR-induced proteins, PopTi3) Transcription factors (Myb, WRKY, ERFs, …) Ribosomal proteins (3 % of plant ESTs) C, N metabolism enzymes & transporters

19. Functional Genomics of Ectomycorrhizal Symbioses Expressed Sequence Tags (ESTs) in ectomycorrhizas Most abundant transcripts are coding for… Poplar - Laccaria ECM Laccaria => Symbiosis-related proteins, GTPases (ras-related proteins) Ribosomal proteins (2 % of fungal transcripts) C, N metabolism enzymes & transporters NO Metallothioneins, NO Hydrophobins Poplar => Similar transcripts than in Poplar/Pisolithus ECMs Less Metallothioneins MAPKs & RLKs + R-genes CNL & TNL Douglas fir - Laccaria ECM Laccaria => Ribosomal proteins (2 % of fungal transcripts) ≠ C, N metabolism enzymes than in Poplar/Laccaria ECM (No MTs, Hydrophobines) Douglas fir => Similar transcripts than in Poplar ECMs but… Less defense-related proteins & MTs Phenylpropanoid pathways enzymes Different metabolism-related transcripts => 60 % of plant ESTs = unknown function (vs. 25-30 % in Poplar)

20. Functional Genomics of Ectomycorrhizal Symbioses Expressed Sequence Tags (ESTs) in various ectomycorrhizas Conclusions Poplar associated with 2 ECM fungi Similar EST profiles Different sets of signaling pathways enzymes and putative receptors Important expression of metallothioneins => role in ECM? Several defense response-induced proteins => Defense or stress reaction? ‘interaction-related proteins’ Laccaria & Pisolithus associated with Poplar Different sets of cell-wall proteins, metallothioneins & signaling proteins Laccaria ECMs on 2 different trees Different sets of C & N metabolism & transport genes are expressed Douglas fir ECM vs Poplar ECMs About 60 % of Douglas ESTs are of unknown function Less defense-related genes in 9-month old Douglas fir ECM

21. Microarrays Planned microarrays PICME ARC Seibersdorf research GmbH Custom tailored DNA Chips 1636mycelium cDNA library 965 fruiting body cDNA library 1772 mycorrhizal cDNA libraries (747 Douglas Fir/1025 Poplar) 4373 ESTs ? ~150-200 Euros Additional EST sequencing by the french Génoscope (fruit body, mycorrhiza)

22. Functional classification of TCs using BlastX and KOG 50% unknown F. Duchaussoy EuKaryotic Orthologous Groups (KOG)

23. Most abundant transcripts 31 TCs with more than 50 ESTs 410 TCs with contain between 10 and 50 ESTs 10695 TCs with one or two ESTs

24. Major TC: 585 ESTs (14 ESTs Lb, 2 ESTs LG0ADG ,569 ESTs mixed libraries) ???

25. Most abundant transcripts Contig ESTs gene model putative function 1323708:239 585 eu2.Lbscf0029g00760 Transcription initiation factor TFIID, subunit BDF1 and related bromodomain proteins 1323711:21 348 estExt_fgenesh2_pg.C_60136 Ribosomal protein S12/S23 1323711:20 281 estExt_fgenesh2_pg.C_60136 Ribosomal protein S12/S23 1323708:238 242 fgenesh3_pg.C_scaffold_6000144 - 1323708:237 206 estExt_fgenesh2_pg.C_250083 Nucleolar GTPase/ATPase p130 1323708:236 199 estExt_fgenesh2_pg.C_250083 Nucleolar GTPase/ATPase p130 1316099:2 157 no hit - 1323708:232 136 fgenesh3_pg.C_scaffold_10000205 jgi|Phchr1|7088|fgenesh1_pg.C_ scaffold_12000272 1323708:233 133 fgenesh3_pg.C_scaffold_10000205 jgi|Phchr1|7088|fgenesh1_pg.C_ scaffold_12000272 1323708:235 133 eu2.Lbscf0068g00130 jgi|Phchr1|7088|fgenesh1_pg.C_ scaffold_12000272

26. 20 614 gene models 14312 (11697) TCs 6514 gene models with EST support -Transcripts from the same gene model in different TCs (bad quality,allelic, assembling errors?) -no overlapp between ESTs from 5’ and 3’ end of the gene -Several ESTs from one gene due to altered splicing? Only one-third of the predicted gene models have an EST support so far!