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Peroxisomal carnitine acetyl transferase is required for elaboration of…
Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphae during plant infection by Magnaporthe grisea
Introduction
- Pathogenic m/o need to tranverse plant cuticle to gain entry to underlying tissue
- To do so, pathogenic fungi evolved the ability to breach plant cuticles using cell wall-degrading enzymes and also through app. formation
- M. grisea use app. to penetrate into the plant cuticle
- Appressorium
~ Dome-shaped
~ Differentiate at the tip of apex of germ tube
~ Bounded by melanin-rich cell wall
~ Accumulate glycerol at high concentration, hence allowing hydrostatic turgor generation, followed by penetration peg into plant cuticle
~ Invasive hyphae proliferates throughout rice leaf, kill plant cells, and produce necrotic lessions on plant leaf
- Initial development of rice blast fungus occurance
~ Absence of external nutrients
~ Lipid bodies was mobilize to appressorium under control of Pmk1 MAP kinase pathway
~ Rapid lipolysis occurs within mature infection cell at the onset of turgor generation
- Triacylglycerol lipase activity regulated by cAMP response pathway
~ mutant lacking cAMP-dependent protein kinase A - non-pathogenic, they form misshapen app that do not degrade lipids. non-f(x)al
- Isocitrate lyase activity
~ rqd. for efficient app f(x)
~ Glyoxylate cycle plays important fole in plant infection by *M. grisea
- Important consequence of lipolysis and fatty acid B-oxidation in app
~ Generation of acetyl CoA - transported to cellular compartments for glyoxylate cycle
- Transfer of acetyl CoA
~ Catalysed by carnitine acetyl transferases (CATs)
~ Convert acetyl CoA units to acetyl-carnitine - transport across membrane
- Carnitine acetyl transferases (CATs)
~ From carnitine acyltransferase family - comprise the carnitine palmitoyltransferases and carnitine octanoyltransferases, highly conserved
- PHT2
~ Forward genetic screen for pathogenicity mutants of M. grisea gene
~ Potential encoding CAT
- Obj:
~ Investigate role of PHT2 in fungal pathogenesis to determine how this enzyme contributes to app dev. and why this is so critical to successful plant infection.
- Overview
~ Demonstrate that PTH2 encodes the major CAT in M. grisea
~ PHT2 necessary for appressoria to function correctly
~ Pth2 is regulated by the cyclic AMP response pathway, located in peroxisomes (abundant in app)
~ Pth2 contributes to effective lipid reserve mobilization during appressorium maturation
~ Necessary for penetration hypha formation and host invasion
Results
Elevated expression of a 2.1 kb PTH2 transcript was observed when the fungus was grown on ethanol acetate, olive oil, oleic acid, triolein, or glycerol as sole carbon sources compared with growth on glucose
Elevated expression of PTH2 was associated with substrate induction as opposed to glucose repression because the presence of glucose in the medium did not affect PTH2 transcript levels when the ungus was exposed to acetate or glycerol
elevated transcript levels of PTH2 observed as soon as 1 h after transfer to acetatecontaining growth medium, with peak transcript 8 h after transfer to acetate
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PTH2 mutants were unable to utilize acetate, triglycerides (triolein) or long chain fatty acids (olive oil) as
sole carbon sources
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Colocalization of the oxA-RFP and Pth2–GFP fusion proteins was observed In intracellular vesicles, which accumulated at the cortex
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Discussion
PTH2 encodes the major CAT activity in M. grisea
- based on enzymatic assays and the inability of pth2 mutants to utilize acetate or lipids as a sole carbon source
The function of PTH2 in rice blast disease:
- Pth2 is required for turgor generation necessary for appressorium-mediated penetration
- PTH2 is involved in the supply of acetate units for melanin biosynthesis via the dihydroxynaphthalene pathway
: appressorium melanization is vital for plant infection by M. grisea
- Pth2 acts to make acetyl CoA available to the glyoxylate cycle which feeds through reactions of the gluconeogenesis pathway to support glucan and chitin biosynthesis
: required for cell wall generation and growth of
penetration hyphae
pth2 mutant:
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- light and electron microscopy confirmed that appressorial melanin biosynthesis does occur in pth2 mutants
- appressorium turgor generation was normal in pth2 mutants
- addition of a melanin biosynthesis intermediate, scytalone, failed to restore lesion formation in pth2 mutants
- pth2 mutants have reduced chitin within infectious hyphae when compared with Guy 11
- completely non-pathogenic in contrast with M. grisea icl1 mutants, which lack isocitrate lyase, but only show a delay in disease symptom expression
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- Cat2 provides a means for peroxisomally generated acetyl CoA to be transferred to the cytoplasm.
- Cat1 channels acetyl CoA into mitochondria for oxidation via the tricarboxylic acid cycle
Procedures
- Carnitine acetyltransferase enzyme assay
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- Assays for infection-related morphogenesis
- Observe M. grisea app dev on plastic coverslips
- Assay app-mediated penetration of sterile onion epidermis (Allium cepa) and detached rice leaves
- Rice and barley infections
- Perform plant infection assays
Spray seedlings of rice (Oryza sativa) cultivar CO-39 and barley (Hordeum vulgare) cultivar Golden Promise
- with suspension of M. grisea conidia using artist's airbrush
- Pth2 subcellular localization
- Identification and targeted gene disruption of PTH2
- Identify 2 independent, non-pathogenic mutants - the PHT2 gene is mutated by RE-mediated integration
- Select HindIII fragment spanning PHT2 gene locus
- Remove and replace 80 bp EagI-NcoI with 1.4 kb hydromycin phosphotransferase gene cassette with resistance to hydromycin B
- Use result construct, pCB965 to transform protoplast Guy 11
- To complete pht2 mutants, clone ApaI–NotI fragment into pCB1532, which carries a sulphonylurea resistance selectable marker gene
- Transmission electron microscopy
- Perform ultrastructural investigation - freeze-substitution fixation
- Inoculate Conidia of Guy 11 pht2 and buf1 into onion epidermis and let to allow app dev
- in 24 h, freeze small square of epidermis in Freon-23, then cool to -180C with liquid nitrogen
- Fungal strains, culture conditions and DNA analysis
- Wild-type and mutant strains of M. grisea stored in lab of N. J. Talbot, Uni of Exeter
- Follow standard procedures for M. grisea growth, miantenance, nucleic acid extraction and transformation
- Perform Gel electrophoresis, RE digestion and DNA gel blot hybridization using standard procedures
Results
:star: Appressorial melanin biosynthesis is reduced, but not
severely compromised by absence of PTH2
pth2 mutant might be affected in appressorium function might be due to a requirement for peroxisomal acetyl-coA as a substrate for dihydroxynaphthalene melanin biosynthesis.
Pth2 could generate cytoplasmic acetyl CoA, which is channeled into the pentaketide melanin biosynthesis pathway.
electron microscopy were performed in a Dpth2 mutant, Guy 11 and the melanin-deficient, non-pathogenic mutants alb1 and buf1, which lack polyketide synthase and trihydroxynaphthalene reductase respectively
electron microscopy were performed in a Dpth2 mutant, Guy 11 and the melanin-deficient, non-pathogenic mutants alb1 and buf1, which lack polyketide synthase and trihydroxynaphthalene reductase respectively
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