Regulation of mycotoxin production and kinome analysis in Fusarium graminearum
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1 Regulation of mycotoxin production and kinome analysis in Fusarium graminearum Chenfang Wang Northwest Agricultural & Forestry University
2 Regulation of mycotoxin production and kinome analysis in Fusarium graminearum Chenfang Wang Jin-Rong Xu, Purdue University Purdue NWAFU Joint Research Center
3 Nationwide Outbreak of Wheat Scab in China
4 DON 陕西杨凌 : ppm 陕西华县 : ppm (FDA: 1ppm human consumption, 5 ppm animal feeds) Organizing a wheat scab initiative/working group
5 Unique features of Fusarium graminearum F. graminearum F. asiaticum - Ascospores primary inoculum - Sexual reproduction critical in its infection cycle - No microconidia - Tissue-specific for flowering heads wheat, barley, Brachypodium, and others
6 Unique genomic features of F. graminearum - Very few repetitive sequences (0.03%, 15x less than Sc) - No active transposable elements - Rare recent duplications - Localized polymorphism and pathogen specialization Cuomo et al., Science. 317:
7 Many TRI genes were up-regulated during plant infection TRI Gene Functions TRI3 C-3 Acetylation TRI9 Unknown TRI4 Trichodiene Oxygenation TRI10 Transcription factor TRI5 Trichodiene Synthesis TRI11 C-15 Hydroxylation TRI6 Transcription factor TRI12 Transporter TRI7 C-4 Acetylation TRI13 C-4 Hydroxylation TRI8 C-3 Deacetylation TRI14 TRI101 Trichothecene 3-0-acetyltransferase TRI6
8 Tri6: Cys2His2 zinc-finger domain Tri10: Fungal specific Zn(2)-Cys(6) binuclear cluster domain TRI5 Expression (Seong et al., 2010) tri6 tri10 double mutants -Both Tri6 and Tri10 are important for TRI5 expression -Tri6 appears to be more important than Tri10
9 0 WT tri6 tri Tri5 * * PH1 dtri6 dtri Tri4 * * 0 PH1 dtri6 dtri Tri101 * * TRI genes down-regulated in the tri6 and tri10 mutants PH1 dtri6 dtri PH1 dtri6 dtri10 Tri11 * * PH1 dtri6 dtri10 Tri3 * * PH1 dtri6 dtri10 Tri1 * * Tri8 * * 0 PH1 dtri6 dtri10
10 Microarray analysis (Seong et al., 2010) 1/37 genes positively regulated by Tri10 87/208 genes positively regulated by Tri6 Tri10: quite specific for trichothecene biosynthesis Tri6: other genes related to plant infection
11 Evolution: co-regulation of isoprenoid and trichothecene biosynthesis genes in F. graminearum Why is Tri6 global? Tri6-Tri10 relationship? (Seong et al., 2010)
12 Tri6-Tri10 interaction may be conditional - Tri6/10 TRI6/10 Tri6/10 TRI genes DON 合成
13 Environmental or host factors affect DON production may converge on Tri6/Tri10 ph: acidic ph induced TRI genes Nitrogen sources ROS + other stresses NH 4 inhibits DON synthesis Polyamines stimulates (Gardiner et al., 2010) Plant factors (flowering heads) (DON not produced in rice scab)
14 ph N ROS/Stresses Plant factors Tri6/Tri10 model PacC A A B Tri6/10 B A. TRI6/10 expression Binding to TRI6/10 promoter B. Tri6/10 activities Interacting with Tri6/10 TRI6/10 Tri6/10 TRI genes
15 A type factors: binding to TRI6 promoter A Tri6/10 TRI6/10 Conserved PacC-binding sites: GCCAAG (-512) TRI6 promoter - yeast one-hybrid assays - searching for conserved promoter elements
16 Tri6/10 B TRI6/10 B type factors: Tri6 interacting proteins - Screening 2 yeast two-hybrid libraries - Affinity purification -MS analysis TRI6-GFP TRI6-3xFLAG TRI6-S-tag (may need to be over-expressed)
17 WT Mutants Three MAPK pathways (Gpmk1, Mgv1, FgHog1) The camp-pka pathway Putative PKA & MAPK phosphorylation sites in Tri6 MAPK docking site Why Tri6 is global regulator?
18 Project 1. Functional analyses of predicted protein kinase genes (Kinome) Project 2. Genes unique to F. graminearum ( Uniquitome ) Targeted gene deletion highly efficient (80%) in F. graminearum
19 Comparative Analysis -Protein Kinase Genes F. oxysporum & F. verticillioides- Ma et al., Nature 464:
20 20 genes - essential Mutants identified for 96 PK gene At least two mutants for each gene >30 of them- complementation assays (subcellular localization) Specifically localized to the septal pore
21 17 phenotypes scored - mutants identified -growth rate -colony morphology (pigmentation) -hyphal growth and branching -conidiation -conidium morphology & germination -sexual reproduction -infection assays with flowering wheat heads -DON production -stress responses: NaCl, H 2 O 2, SDS, CR. Searchable database Wang et al., PLoS Pathogens. 7(12): e
22 42 PK genes are important for plant infection 8.25% no disease disease indexes 1 disease indexes between 1-5 disease indexes > % 57.73% 20.62%
23 Networks of PK-PK interactions predicted with the interlog approach Three MAP kinase cascade
24 PK genes essential in S. cerevisiae or S. pombe but not in F. graminearum F. graminearum genes S. cerevisiae orthologs S. pombe orthologs Number of mutants FGSG_02399 a IPL1 ark1 14 FGSG_00433 RAD53 Cdk cds1 11 FGSG_04947 CAK1 kinase - c csk1 6 FGSG_13318 MEC1 SPBC FGSG_10381 CDC15 cdc7 3 FGSG_08468 b CDC28 cdc2 12 FGSG_03132 b Cdk CDC28 cdc2 8 FGSG_01188 CBK1 orb6 d 7 FGSG_05734 KIC1 nak1 d 5 FGSG_ c sid1 15 FGSG_ c SPCC70.05C 19 FGSG_ c prp4 7 Key regulator of cell cycle
25 Two Cdc28/Cdc2 genes FGSG_03132 mutant : Reduced 30% in conidiation FGSG mutant: similar to Fg04947 (CDK kinase) Defective in pathogenesis (DI <1.5) Blocked in ascosporogenesis WT Mutant Double mutants not viable
26 Cell cycle and fungal pathogenesis Some CDKs: infection-specific functions Hyphal growth: in vitro vs. in planta
27 Project 2. Genes unique to F. graminearum -Unique genes with signal peptide (secreted) -Unique genes with conserved domains Six of them have distinct functions
28 SSP1 mutant: reduced growth rate + colony morphology defects A F. graminearum unique gene important for hyphal growth
29 SSP2 is required for normal sexual reproduction PH-1 aborted Mutant abnormal Empty perithecia (60%)
30 Two F. graminearum specific genes important for plant infection PH-1 ssp3 sdp1 In planta expression
31 Acknowledgements Northwest Agricultural & Forestry University Chengfang Wang Huiquan Liu Qiaojun Jin Shijie Zhang Qian Zheng Rui Hou Qijun Xu + undergraduate students Collaborators Corby Kistler (USDA) Li-Jun Ma (UMass) Purdue University Jin-Rong Xu Xiaoying Zhou
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