TrehaloseTrehaloseand carbohydrate and carbohydrate

metabolism of metabolism of L. bicolorL. bicolor

A. Deveau

P. Frey-Klett, J.Garbaye, F. Martin

et al.

UMR INRA-UHP 1136 Interactions arbres micro-organismes UFR 110

© INRA

Carbohydrate metabolismCarbohydrate metabolism

KEGG Pathway database

• glycolysis and TCA cycle• pentose phosphate pathway• malate synthesis• trehalose, glycogen metabolism

Complete pathways:

Uncomplete pathways:

• mannose (mannose-1-phosphate

guanylyltransferase …)

• mannitol

F-6-P

Mannose-6-P

Mannitol

Mannitol-1-PF-6-P

Phosphomannose isomerase

Mannose-6-P reductase

Mannitol-1-P-phosphataseMannitol-1-P-phosphatase

Mannitol dehydrogenase

http://www.genome.jp/kegg/pathway/map/map01110.html

Trehalose and

Glycogen metbolism

The trehalose in The trehalose in ectomycorrhizal fungi ectomycorrhizal fungi

glucose

fructose

glucose

fructose

sucrosesucrose

fructose

glucose

+

F6P

glycogen

trehalose

mannitol

glycolysis

plant cortical cellplant cortical cellfungal hyphaefungal hyphae

apoplast

G6P

CH2OHCH2OH*

• Main roles:Main roles:

• Storage sugar – supplies of endogenous carbon

• Stress protectant by stabilization of cellular membranes and proteins (freezing, dessication, growth restriction)

• A signal molecule

photosynthesis

transport

The trehalose in The trehalose in ectomycorrhizal fungi ectomycorrhizal fungi

•Involved in interactions between mycorrhization helper bacteria and Laccaria bicolor ?

• Strongly accumulated in Laccaria hyphae

L. Bicolor hyphae

Pseudomonas fluorescens

trehalosetrehalose

Bacterial effect on Bacterial effect on presymbiotic growth presymbiotic growth

and survival and survival of the fungusof the fungus

ChemiotactismChemiotactism

Improvement of Improvement of bacterial survivalbacterial survival

Tsl 1Tsl 1

Trehalose 6-P

TrehaloseUDP-Glucose

Glucose 6-P

Glucose 1-P

Pi

glycogen metabolism

glycolyse

Glucose

PiHxk 1Hxk 1Hxk 2Hxk 2

Ugp 1Ugp 1

PgmPgmTps 1Tps 1 Tps 2Tps 2

Glucose TrehaloseTre 2Tre 2

TP ?TP ?extracellular

intracellular

vacuolarstorage

Glucose

Tre 1Tre 1 TPTP

The putative metabolism of trehalose in The putative metabolism of trehalose in L.bicolorL.bicolor

TPS3TPS3

Tsl 1Tsl 1

Trehalose 6-P

TrehaloseUDP-Glucose

Glucose 6-P

Glucose 1-P

Pi

glycogen metabolism

glycolyse

Glucose

PiHxk 1Hxk 1Hxk 2Hxk 2

Ugp 1Ugp 1

PgmPgmTps 1Tps 1 Tps 2Tps 2

Glucose TrehaloseTre 2Tre 2

TP ?TP ?extracellular

intracellular

vacuolarstorage

Glucose

Tre 1Tre 1 TPTP

Specificities of the Specificities of the L. bicolor L. bicolor metabolismmetabolism

Trehalose phosphorylase

C. neoformansS. cerevisiae

trehalose transporter

Genes structure :Genes structure :

Feature

Gene length (bp)

Coding size (aa)

Exons number per gene

Mean exon size (bp)

Mean intron size (bp)

2076

Glk1

Notes :Notes :

• The genes are scattered on 9 different scaffolds. TPS1 and Tre2 on the 11th scaffold, separated by 200 000 bp.

• Two categories of gene structure: high similarity with Phanerochaete gene structure vs low similarity

• Variable intron size

Glk2 UGP1 PGM TPS1 TPS2 TSL1 Tre1 Tre 2 TP

2276 2040 1968 1836 3749 3214 3063 2747 2851

479 484 523 570 511 957 999 727 756 738

13 12 10 5 6 14 9 17 9 13

117

53 53

128 252

60

205

68

256

61

365

65

170

53

157

5273

123

P. chrysosporum

P. chrysosporum

P. chrysosporum

P. chrysosporum

P. chrysosporum

342

69

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

56

2425

678

9

367

49

-

-

-

-

-

1849

474

9

176

53

220

Bases composition of « large introns » (> 70 bp)Bases composition of « large introns » (> 70 bp)

•High poly THigh poly T content content•No identityNo identity with with transposonstransposons sequences from Genebank sequences from Genebank

Intron n° 3 of TSL1 (93 bp) GTAAGTTTTTTTTGCTATCTTTTTTTTGTTTTTTTTTGTTTTTGTTTTTGTTTTTTTGTT TGTTTGGGAGATGGGTGCTAATGATGTTTTTAG Intron n° 8 of TSL1 (100 bp) GTTTAGGACCTCCGCTTTTTTTCTCTGCGGACATTGATCGCTGCTCTTTCTTTTATTCTC CGTCTCCCGTTTGTTTTCTTGATGGGCTAACACCTTCAAG

Sequence evolution Sequence evolution

© Broad Institute

• 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)

Two groups of protein:

Sequence evolution Sequence evolution

M. griseusG. zeaeN. crassaA. nigraA. fumigatusA. nidulansC. neoformensL. BicolorA. muscaria

Concensus

M. griseusG. zeaeN. crassaA. nigraA. fumigatusA. nidulansC. neoformensL. BicolorA. muscaria

Concensus

M. griseusG. zeaeN. crassaA. nigraA. fumigatusA. nidulansC. neoformensL. BicolorA. muscaria

Concensus

M. griseusG. zeaeN. crassaA. nigraA. fumigatusA. nidulansC. neoformensL. BicolorA. muscaria

Concensus

Trehalose synthase 1 (TPS1)Trehalose synthase 1 (TPS1)

High level of aminoacid sequence conservation (> 70%)High level of aminoacid sequence conservation (> 70%)

Sequence evolution Sequence evolution

Hexokinase 1 (GLK1)Hexokinase 1 (GLK1)

Lower level of aminoacid sequence conservation (< 65%)Lower level of aminoacid sequence conservation (< 65%)

N. crassaU. triticiM. griseusA. fumigatusL. bicolorP. involutusC. neoformensT. reesei

consensus

N. crassaU. tritciM. griseusA. fumigatusL. bicolorP. involutusC. neoformensT. reesei

consensus

N. crassaU. triticiM. griseusA. fumigatusL. bicolorP. involutusC. neoformensT. reesei

consensus

N. crassaU. triticiM. griseusA. fumigatusL. bicolorP. involutusC. neoformensT. reesei

consensus

Sequence evolution Sequence evolution

82 %

72 %

72 %

62 %

63 %65 %

66 %

74 %

Trehalose synthase 1 (TPS1)

• High degree of identity (>70 %)

Acid trehalase (Tre2)

Trehalose synthase 1 (TPS1)

Trehalose phosphorylase (TP)

Uridyltransferase (UGP1)

Hexokinase 2 (Glk 2)

• Lower degree of identity (< 65%)

Hexokinase 1

61 %

58 %

53 %

42 %

42 %42 %

41 %

36 %

38 %

Neutral trehalase (Tre 1)

Trehalose phosphorylase (TPS2)

Trehalose synthase - regulatory sub-unit (TSL1)

Hexokinase 1 (Glk1)

Ascomycetes

Ascomycetes

Basidiomycetes

Basidiomycetes

• Carbohydrates metabolism:

No main sugar biosynthesis pathway

missing

• Trehalose

• metabolic specifitiesmetabolic specifities -> two

ways of synthesis (trehalose trehalose

phosphorylasephosphorylase)

• presence of large intronslarge introns with

polyT repeatspolyT repeats

• variable degree of variable degree of

conservation of aminoacid conservation of aminoacid

sequencesequence

CH2OHCH2OH*

SummarySummary

© INRA

The bi-phospho-fructo aldolase, a duplicated gene ?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