1. Trehalose and carbohydrate metabolism of L. bicolor
A. Deveau
P. Frey-Klett, J.Garbaye, F. Martin
et al.
UMR INRA-UHP 1136 Interactions arbres micro-organismes UFR 110
© INRA

2. Carbohydrate metabolism
Complete pathways:
glycolysis and TCA cycle
pentose phosphate pathway
malate synthesis
trehalose, glycogen metabolism
Trehalose
and
Glycogen
metbolism
Uncomplete pathways:
mannose (mannose-1-phosphate guanylyltransferase …)
mannitol
F-6-P
Mannose-6-P
Mannitol
Mannitol-1-P
Phosphomannose isomerase
Mannose-6-P reductase
Mannitol-1-P-phosphatase
dehydrogenase
KEGG Pathway database

3. The trehalose in ectomycorrhizal fungi
CH2OH
CH2OH*
glucose
fructose
sucrose +
F6P
glycogen
trehalose
mannitol
glycolysis
plant cortical cell
fungal hyphae
apoplast
G6P
photosynthesis
transport
Main roles:
Storage sugar – supplies of endogenous carbon
Stress protectant by stabilization of cellular membranes and proteins (freezing, dessication, growth restriction)
A signal molecule

4. The trehalose in ectomycorrhizal fungi
Involved in interactions between mycorrhization helper bacteria and Laccaria bicolor ?
Strongly accumulated in Laccaria hyphae
L. Bicolor hyphae
Pseudomonas fluorescens
trehalose
Bacterial effect on
presymbiotic growth
and survival
of the fungus
Chemiotactism
Improvement of
bacterial survival

5. The putative metabolism of trehalose in L.bicolor
Glucose
Trehalose
Tre 2
TP ?
extracellular
Trehalose 6-P
Trehalose
UDP-Glucose
Glucose 6-P
Glucose 1-P Pi
glycogen metabolism
glycolyse
Glucose
intracellular
Hxk 1
Hxk 2
Pgm
Tsl 1
Tps 1
Tps 2
vacuolar
storage
Ugp 1
Tre 1 TP
Glucose

6. Specificities of the L. bicolor metabolism
Glucose
Trehalose
Tre 2
TP ?
Trehalose phosphorylase
extracellular
intracellular
C. neoformans
S. cerevisiae
trehalose transporter
Trehalose 6-P
Trehalose
UDP-Glucose
Glucose 6-P
Glucose 1-P Pi
glycogen metabolism
glycolyse
Glucose
Hxk 1
Hxk 2
Pgm
Tsl 1
Tps 1
Tps 2
TPS3
vacuolar
storage
Ugp 1
Tre 1 TP
Glucose

7. Genes structure : Feature Glk1 Glk2 UGP1 PGM TPS1 TPS2 TSL1 Tre1 Tre 2
Gene length (bp)
2076
2276
2040
1968
1836
3749
3214
3063
2747
2851
P. chrysosporum
2406
502 11
132 90
2127
516 12
129 53
1864
566 4
424 55
2685
783 7 56
324
2532
667 8 75
3540
869 9
274
117
1803
526 5
303
2425
678
367 49 -
1849
474
176
220
Coding size (aa)
479
484
523
570
511
957
999
727
756
738
P. chrysosporum
Exons number per gene 13 12 10 5 6 14 9 17 9 13
P. chrysosporum
Mean exon size (bp)
117
123
157
342
256
205
365
128
252
170
P. chrysosporum
Mean intron size (bp) 53 73 52 69 61 68 65 53 60 53
P. chrysosporum
Notes :
The genes are scattered on 9 different scaffolds. TPS1 and Tre2 on the 11th scaffold, separated by bp.
Two categories of gene structure: high similarity with Phanerochaete gene structure vs low similarity
Variable intron size

8. Bases composition of « large introns » (> 70 bp)
High poly T content
No identity with transposons sequences from Genebank
Intron n° 3 of TSL1 (93 bp)
GTAAGTTTTTTTTGCTATCTTTTTTTTGTTTTTTTTTGTTTTTGTTTTTGTTTTTTTGTT
TGTTTGGGAGATGGGTGCTAATGATGTTTTTAG
Intron n° 8 of TSL1 (100 bp)
GTTTAGGACCTCCGCTTTTTTTCTCTGCGGACATTGATCGCTGCTCTTTCTTTTATTCTC
CGTCTCCCGTTTGTTTTCTTGATGGGCTAACACCTTCAAG

9. Sequence evolution Two groups of protein:
high degree of amino acid identity with other fungi(> 70 %)
Acid trehalase (Tre2)
Trehalose synthase 1 (TPS1)
Trehalose phosphorylase (TP)
Uridyltransferase (UGP1)
Hexokinase 2 (Glk 2)
low degree of amino acid identity (< 65 %)
Neutral trehalase (Tre 1)
Trehalose phosphorylase (TPS2)
Trehalose synthase - regulatory sub-unit (TSL1)
Hexokinase 1 (Glk1)
© Broad Institute

10. Sequence evolution
M. griseus
G. zeae
N. crassa
A. nigra
A. fumigatus
A. nidulans
C. neoformens
L. Bicolor
A. muscaria
Concensus
Trehalose synthase 1 (TPS1)
High level of aminoacid sequence conservation (> 70%)

11. Sequence evolution
Lower level of aminoacid sequence conservation (< 65%)
Hexokinase 1 (GLK1)
N. crassa
U. tritici
M. griseus
A. fumigatus
L. bicolor
P. involutus
C. neoformens
T. reesei
consensus
N. crassa
U. tritici
M. griseus
A. fumigatus
L. bicolor
P. involutus
C. neoformens
T. reesei
consensus
N. crassa
U. tritici
M. griseus
A. fumigatus
L. bicolor
P. involutus
C. neoformens
T. reesei
consensus
N. crassa
U. tritci
M. griseus
A. fumigatus
L. bicolor
P. involutus
C. neoformens
T. reesei
consensus

12. Sequence evolution Lower degree of identity (< 65%)
High degree of identity (>70 %)
Lower degree of identity (< 65%)
Trehalose synthase 1 (TPS1)
Hexokinase 1
61 %
58 %
53 %
42 %
41 %
36 %
38 %
82 %
72 %
62 %
63 %
65 %
66 %
74 %
Ascomycetes
Basidiomycetes
Acid trehalase (Tre2)
Trehalose synthase 1 (TPS1)
Trehalose phosphorylase (TP)
Uridyltransferase (UGP1)
Hexokinase 2 (Glk 2)
Neutral trehalase (Tre 1)
Trehalose phosphorylase (TPS2)
Trehalose synthase - regulatory sub-unit (TSL1)
Hexokinase 1 (Glk1)

13. Summary Carbohydrates metabolism:
No main sugar biosynthesis pathway missing
Trehalose
metabolic specifities -> two ways of synthesis (trehalose phosphorylase)
presence of large introns with polyT repeats
variable degree of conservation of aminoacid sequence
CH2OH
CH2OH*
© INRA

14. The bi-phospho-fructo aldolase, a duplicated gene ?
scaffold 25
scaffold 12
nucleotidique sequence: 77 % of identity
amino acid sequence: 90 % of identity
Exactly same exon/intron structure and sizes
No identity between UTR sequences and surounding sequences
EST for one sequence: scaffold 25