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					NACHT/NB-ARC domain proteins:
the most abundant gene family in Laccaria bicolor


A Kohler & F Martin




                                                  IFR 110
         UMR INRA / UHP 1136 « Tree-Microbe Interactions »
1. Identification of major multigene families

Eugène annotation v. 0.02: BlastP protein models vs. protein models

Top list                            copies
II. BlastP of the deduced AA sequences against NCBI database
                 about 70 gene models with N-terminal NB-ARC/NACHT and C-terminal
                 TPR motifs




The NACHT domain is a 300 to 400 residue predicted nucleoside triphosphatase (NTPase) domain, which is found in animal,
fungal and bacterial proteins. The NACHT domain has been named after NAIP, CIITA, HET-E and TP1. The NACHT domain
consists of seven distinct conserved motifs, including the ATP/GTPase specific P-loop, the Mg(2+)-binding site (Walker A and B
motifs, respectively) and five more specific motifs. The unique features of the NACHT domain include the prevalence of 'tiny'
residues (glycine, alanine or serine) directly C-terminal of the Mg(2+)-coordinating aspartate in the Walker B motif, in place of a
second acidic residue prevalent in other NTPases. A second acidic residue is typically found in the NACHT-containing proteins two
positions downstream.
This family is closely related to pfam00931

pfam00931, NB-ARC, NB-ARC domain a signalling motif found in bacteria and eukaryotes, shared by plant resistance gene
products and regulators of cell death in animals


 TPR, Tetratricopeptide repeat domain; typically contains 34 amino acids [WLF]-X(2)-[LIM]-[GAS]-X(2)-[YLF]-X(8)-[ASE]-
X(3)-[FYL]-X(2)-[ASL]-X(4)-[PKE] is the consensus sequence; found in a variety of organisms including bacteria, cyanobacteria,
yeast, fungi, plants, and humans in various subcellular locations; involved in a variety of functions including protein-protein
interactions, but common features in the interaction partners have not been defined; involved in chaperone, cell-cycle, transciption,
and protein transport complexes; the number of TPR motifs varies among proteins (1,3-11,13 15,16,19); 5-6 tandem repeats
generate a right-handed helical structure with an amphipathic channel that is thought to accomodate an alpha-helix of a target
protein; it has been proposed that TPR proteins preferably interact with WD-40 repeat proteins, but in many instances several TPR-
proteins seem to aggregate to multi-protein complexes; examples of TPR-proteins include, Cdc16p, Cdc23p and Cdc27p
components of the cyclosome/APC, the Pex5p/Pas10p receptor for peroxisomal targeting signals, the Tom70p co-receptor for
mitochondrial targeting signals, Ser/Thr phosphatase 5C and the p110 subunit of O-GlcNAc transferasee.
III. BlastP Uniprot database

        Identification of proteins with similar signature in other organisms



                                                Aspergillus



                                                Giberella

                                                Magnaporthe




             Hypotetical proteins, ATP/GTP binding proteins
         IV. TBlastN against sequenced fungal genomes

                       Identification of similar genes in other species

Database: /seq/annotation/blast_databases/fungi/coprinus_cinereus/      Database: /seq/annotation/blast_databases/fungi/
coprinus_cinereus_1.fasta                                               cryptococcus_neoformans/cryptococcus_neoformans_1.fasta
      431 sequences; 36,254,774 total letters         TPR repeat               341 sequences; 19,223,796 total lettere
                                                       only!            ***** No hits found ******
Sequences producing significant alignments: (bits) Value
Coprinus cinereus 1.58 (scaffold 3)          74 5e-13
                                                                        Database: /seq/annotation/blast_databases/fungi/
Coprinus cinereus 1.61 (scaffold 3)          50 1e-05
                                                                        ustilago_maydis/release2/ustilago_maydis_1_r2.fasta
Coprinus cinereus 1.5 (scaffold 1)           47 7e-05
                                                                               274 sequences; 19,683,350 total letters
Coprinus cinereus 1.219 (scaffold 11)        44 8e-04
                                                                                                               Score E
                                                                        Sequences producing significant alignments:    (bits) Value
Database: /seq/annotation/blast_databases/fungi/fusarium_               Ustilago maydis 1.102 (scaffold 6)              61 3e-09
graminearum/fusarium_graminearum_1
     511 sequences; 36,093,143 total letters
Sequences producing significant alignments: (bits) Value                 Database: magnaporthe_2
                                                                              2273 sequences; 38,760,322 total letters
Fusarium graminearum 1.367 (scaffold 6)      438   e-122                                                      Score E
Fusarium graminearum 1.147 (scaffold 2)      245   2e-64                 Sequences producing significant alignments: (bits) Value
Fusarium graminearum 1.190 (scaffold 2)      180   5e-45                 Magnaporthe grisea contig 2.600               324 2e-88
Fusarium graminearum 1.441 (scaffold 7)      173   6e-43                 Magnaporthe grisea contig 2.387                230 6e-60
………                                                                      Magnaporthe grisea contig 2.1690               99 2e-20
16 Hits                                                                  ………..
                                                                         10 Hits
Database: /seq/annotation/blast_databases/fungi/aspergillus_nidulans/
aspergillus_nidulans_1.fasta
      248 sequences; 30,068,514 total letters
Score E
Sequences producing significant alignments: bits) Value
Aspergillus nidulans 1.150 (scaffold 12)       507 e-143
Aspergillus nidulans 1.16 (scaffold 1)         483 e-136                No Hit in Basidiomycete genomes,
Aspergillus nidulans 1.61 (scaffold 4)          444 e-124
Aspergillus nidulans 1.25 (scaffold 2)         322 e-115                Hits in Ascomycetes
Aspergillus nidulans 1.176 (scaffold 19)        408 e-113
……
19Hits
                      Laccaria_sc25_sc235




  Aspergillus




                                            Streptomyces


 Giberella/Fusarium




                                            Nostoc

Magnaporthe
V. BlastN against Laccaria dbEST
        Identification of transcripts




        CONTIG 1319260:1
      2 ESTs : JGI_CAHG3142.fwd und JGI_CAHG3142.rev
      Best gene model : scaffold_81_scaff.28_exon_113952-111761_1

        CONTIG 1320293:1
        2 ESTs : JGI_CAHH438.fwd und JGI_CAHH438.rev
      Best gene model : scaffold_56_scaff.96_exon_327877-330877_1

        CONTIG 1323425:1
      2 ESTs : JGI_CAHC9232.fwd und JGI_CAHC9232.rev
      Best gene model : scaffold_25_scaff.253_exon_688229-685434_1

      CONTIG 1325787:1
      1 EST : JGI_CAHC710.fwd
        Best gene model : scaffold_66_scaff.38_exon_122068-119314_1
                                  Details of EST support

scaffold_25_scaff.253_exon_688229-685434_1



                                  NB-ARC
EST fwd.                                                    TPR    TPR
                                      EST rev


scaffold_81_scaff.28_exon_113952-111761_1



                                  NB-ARC
EST fwd.
                                      EST rev


scaffold_56_scaff.96_exon_327877-330877_1


                                    NB-ARC
EST fwd.                                                          TPR TPR
                                      EST rev                        TPR  TPR


scaffold_66_scaff.38_exon_122068-119314_1



                                    NB-ARC                  TPR TPR
                                                               TPR
                                                 EST fwd.
   Gene model: scaffold_25_scaff.253_exon_688229-685434_1




EST JGI_CAHC9232.fwd translated 175AS NB-ARC domain in red
YTHHRPLRMSRTQVFPNAHGTAVSGGTFYAADTintrons1IHLNNINNGNRTSDGVIPLMPNP
SNRFTGRADVIAKLKGHFSNADNSAQKRKFFLLHGMGGVGKTQICLKFVEEMSDintrons2YFSSVF
WIDASSIGTITQGLKGICNFPAARSTGLDGSPEYALHWIGSLKENYIMVFDNADVFSP
Alignment of the N-terminal part of the deduced AA sequence of four gene models
   scaffold_81_scaff28_exon_11395   ------------------------------------------------------------
   scaffold_25_scaff253_exon_6882   ------------------------------------------------------------
   scaffold_66_scaff38_exon_12206   ------------------------------------------MGTPPSFSLLSFAP---I
   scaffold_56_scaff96_exon_32787   MPKRPSPVQLSVCGGLLSAPQTVRCPQHRKLPLAELGVSCRSALVASFFRPMSFPPSENT

   Prim.cons.                       MPKRPSPVQLSVCGGLLSAPQTVRCPQHRKLPLAELGVSCRS222222F222SF2PSEN2
                                                                Intron 1
                                            70        80        90       100       110       120
                                             |         |         |         |         |         |
   scaffold_81_scaff28_exon_11395   -----MS----FAGASQFVARDNTFIDITANSi1VHIDYGGRTTSDGVIPLMPNPSNRFTGR
   scaffold_25_scaff253_exon_6882   -----MSRTQVFPNAHGTAVSGGTFYAADTi1IHFNNINNGNRTSDGVIPLMPNPSNRFTGR
   scaffold_66_scaff38_exon_12206   CYTSNMSQLNVLQNARNVAITDSNINVADTi1INYYASVGNRTISDAVIPVKPNSSIRFTGR
   scaffold_56_scaff96_exon_32787   LPPPNMSGVNFLSGAQNVVVSG-DVNVAETi1ITYNVHLSRRVTSGAAVPLMPNSSPRFTGR
                                         **    : .*   . . .      :       . . *...:*: **.* *****
   Prim.cons.                       2222NMS33NV242A4NV2VS23TFNVADTI4YN444GGRTTSD2VIPLMPN2SNRFTGR
                                                                                    Intron 2
                                           130       140       150       160       170       180
                                             |         |         |         |         |          |
   scaffold_81_scaff28_exon_11395   KQVITDLKRHFSNTHDSALRR—KFFLLYGMGGIGKTQICLKFIEEMSGi2CFSSVFWIDAS
   scaffold_25_scaff253_exon_6882   ADVIAKLKGHLSNAHNLAQKR—KFFLLHGMGGIGKTQICLKFVEEMSDi2YFSYVFWMDAS
   scaffold_66_scaff38_exon_12206   TDVLATLKEHFTAESNNKLRRQ-KFFLLYGMGGIGKTQICLRFIEDMSDi2YFSHVFWIDAS
   scaffold_56_scaff96_exon_32787   TAILAKLQDHFMRGSDKQQLRSRKYFLLYGMGGIGKSQICLRFIEDMSDKi2FSHVFWIDAF
                                      ::: *: *:    :     * *:***:*******:****:*:*:**. ** ***:**
   Prim.cons.                       TDV2AKLK4HFSN4224A2RR2RKFFLLYGMGGIGKTQICL2FIE2MSDYFSHVFWIDAS

                                           190       200       210       220       230       240
                                             |         |         |         |         |         |
   scaffold_81_scaff28_exon_11395   SVGTITQGLKGICNLPAAQSSGLDGSPESGLHWIGFLKENYVMVFDNADVLSPAELEAYF
   scaffold_25_scaff253_exon_6882   SLGTITQGLKSICAFPAARSSGLDESPEYALLWIGSLKENYIMVFDNADVLSPAELEAYF
   scaffold_66_scaff38_exon_12206   SVGTITQALKGICNLPEAQSSALDGSPESALWWISSLRGNYAMVFDNADNLTPEELEQYF
   scaffold_56_scaff96_exon_32787   SAGSIIQGLKGICNLSVAQTQLLDGSPESALSWIGSLRDNYVIVFDNADTLRPEELEGYF
                                    * *:* *.**.** :. *::. ** *** .* **. *: ** :****** * * *** **
   Prim.cons.                       SVGTITQGLKGICNLPAAQSSGLDGSPESAL4WIGSL2ENYVMVFDNADVLSP2ELEAYF

                                           250       260       270       280       290       300
                                             |         |         |         |         |         |
   scaffold_81_scaff28_exon_11395   PPGRGGNILITSRNYTMRILTLPENSLEVIEMEEKDAIGLLLKASCLDLCSMEFLTEASK
   scaffold_25_scaff253_exon_6882   PPGRGGNILITSRNSTMRHLTSPENSLEVTELEENDAIELLLKASCLDLSSLMFQAEASK
   scaffold_66_scaff38_exon_12206   PSGLGGNILITSRNSGLKHLTSHENSLEVKEMEENDAISLLLKAACLSESQENLQAEASK
                     sc66 sca38     EST support TPR repeat
VI. Phylogenetics
                    sc27 sca42
                              sc14 sca52
                                               sc7sca 53
                                                                                sc30 sca17 9
                                                                           sc11 sca7
                                                                         sc30 sca17 0
                                                                         sc11 sca17
                                                                                              sc16 sca14 7
                                                                                               sc16 sca15 0
                                                                                             sc16 sca19 8
                                                                                             sc35 7sca2
                                                                                            sc16 sca20 4
                                                                                         sc16 sca11 1
                                                                                         sc16 sca11 3
                                                                                        sc16 sca64                        12x scaffold16
                                                                                          sc16 sca50
                                                                                          sc16 sca52
                                                                                                 sc16 sca45
                                                                                                sc85 sca27
                                                                                              sc16 sca88
                                                                                                    sc6sca 178
                                                                                                     sc33 sca17 2
                                                                                                        sc18 1sca3
                                                                                                      sc24 sca95
                                                                                               sc16 sca12 9
                                                                                                   sc81 sca44
                                                                                                     sc33 sca15 4
                                                                                                     sc81 sca17
                                                                                                      sc14 sca10 9
                                                                                                   sc33 sca14 2
                                                                                                   sc81 sca28
                                                                                                  sc81 sca40
                                                                                                                    EST support NB-ARC
                                                                                                 sc32 sca43
                                                                                                  sc32 sca18
                                                                                              sc11 sca26 3
                                                                                           sc36 sca10 9
                                                                                         sc20 sca17 5
                                                                                               sc6sca 213
                                                                                         sc6sca 179
                                                                                          sc6sca 223
                                                                                              sc1sca 937
                                                                                                sc33 sca65
                                                                                           sc20 sca16 4
                                                                                           sc20 sca13 7
                                                                                            sc13 sca25 1
                                                                                  sc41 sca17 7
                                                                                          sc24 sca57
                                                                                    sc24 sca21 7
                                                                                      sc24 sca10 1
                                                                                       sc1sca 105 3
                                                                                  sc25 sca24 4
                                                                                   sc25 sca25 3
                                                                                 sc25 sca24 2
                                                                                                     EST support NB-ARC
                                                                               sc61 sca95
                                                                          sc3sca 267
                                                                   sc20 sca14 9
                                                          sc61 sca75
                                                      sc56 sca96
                                                                                                sc51 sca48
                                               EST support                                       sc51 sca14
                                                                                                    sc51 sca36
                                               NB-ARC                                               sc51 sca53
                                                                                                    sc50 sca19
                                                                                                                    6x scaffold51
                                                                                              sc51 sca76
                                                                                              sc51 sca80
                                                                                         sc48 sca10 7
                                                                                             sc31 sca16 9
                                                                                             sc87 sca25
                                                                                           sc60 sca90
                                                                                            sc87 sca9
                                                                                          sc24 2sca4
                                                                                           sc87 sca18
                                                                                            sc3sca 244
                                                                                            sc31 sca15 9
                                                                                             sc63 sca65
                                                                                         sc31 sca14 2
                                                                                         sc31 sca12 2
                                                                                                               4x scaffold31
                                                                                           sc31 sca15 2
                                                                                     sc2sca 97
                                                                                  sc5sca 720
                                                                             sc21 sca24 2
                                                                                                                                       Aspergillus
                                                                                                                   Aspergi l lu s AN9 426 .2
                                                                                                                   Q5A TQ8Aspe rg il l us
                                                                                                            Aspergi l lu s AN1 071 .2
                                                                  sc14 sca30
                                                                          sc66 sca21
                                                                                                    Mag nap orth e MG 029 76.4
                                                                                                                                    Magnaporthe
                                                                          sc66 sca22
                                                                         sc34 sca62
                            sc6sca 97
                                                                           Gi bbe re ll a FG0 895 6.1  Giberella
                             sc34 sca15 0
                                sc16 sca29 3
                                 sc4sca 19
                             sc4sca 38
                           sc16 sca28 5
                           sc4sca 68
                    50 changes
                                 Motif discovery tool MEME
Example:



                                       NACHT/NB-ARC




                                TPR-repeats, number of repeats variable




           Motif consensus sequences




                                                              Alignment of motif 3
Prediction of subcellular localization and signal sequences




                                               Any other localization than
                                               Chloroplast, Mitochondrion
                                               or secretory pathway




                                       No nuclear localization signal

                     No predicted
                     transmembrane
                     helices
                                            Summary




- A multigene family with about 70 members was identified (about 230 members with a e-value
  cut-off of e-20)

- Predicted genes contain a N-terminal NACHT/NB-ARC domain and a C-terminal TPR repeat

- Homologous genes are present in the Aspergillus, Fusarium and Magnaporthe genome, but no
  homologs in other basisiomycete genomes were detected

- Transcripts are present in a cDNA library from mixed Laccaria tissues,
  but with a very low frequency

- Software to predict the subcellular localization of the NACHT domain proteins was used.
 No transmembrane helices were predicted. No nuclear localization signal could be detected.
 Any other localization than chloroplast, mitochondrion and secretory pathway is the most
 probable

- Different subgroups exist and the genes are often clustered (i.e. on scaffold 16).

				
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posted:3/3/2012
language:Latin
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