# data[].attributes.body
RESULT

1. Lipid mobilization is impaired in developing appressoria
of the Dpth2 mutant.
-Dpth2 mutants, lipid bodies are not metabolized within appressoria
at the same rate as in a wild-type M. grisea strain.

2. Addition of glucose can partially remediate the Dpth2
mutant phenotype.
-Glucose take-up by the fungus can partly restore the functional
competence of appressoria, and in particular their ability
to form penetration hyphae.

3.Pth2 mutants produce penetration hyphae that are
chitin-deficient.
-chitin synthesis during invasive hypha formation is impaired in the Dpth2 mutant.

# data[].attributes.headline
Farhana Result-PTH2 is required for cuticle penetration and infection ofthe leaf epidermis

# data[].attributes.subject
Farhana Result-PTH2 is required for cuticle penetration and infection ofthe leaf epidermis

# data[].attributes.body
-To investigate the role of PTH2 in rice blast, scientist inoculate seedlings of the blast-susceptible rice cultivar
CO-39 and the barley cultivar Golden Promise, with a pth2 mutant.
-No rice blast disease symptoms were
observed which consistent with previous
analysis of a pth2 insertion mutant.
-moreove, reintroduction of the PTH2 gene restored
fully the ability to cause rice blast disease
- This result can be concluded that PTH2 encodes the major CAT
activity in M. grisea, which is essential for development of
rice blast disease.
- The reasons pth2 mutants fail to
cause rice blast disease is further investigated
-The result shown that conidiogenesis was reduced 10-fold (6 x10^5
conidia ml-1
) in pth2 mutants compared with Guy 11
(3 x 10^6 conidia ml-1
), but spores germinated normally
and developed appressoria.
- The investigation further carried out by allowing the appressoria of the pth2 and Guy 11 to form on intact rice leaf surfaces and those in
which the cuticle was first removed by abrasion
-The disease
symptoms were then recorded 72–102 h after inoculation.
- It seems that no disease lesions were observed on non-
abraded leaves of either rice or barley seedlings inocu-
lated with pth2 mutant
- However,small disease lesions were observed on abraded leaf
sections inoculated with the pth2 mutant but less developed than those
formed by Guy 11.
-This suggests that the initial events of
plant infection are most likely to be affected by loss of
Pth2 because the capacity for growth in rice leaf tissue is
still present in pth2 mutants, but a much-reduced
rate compared with the isogenic wild type.
-To visualize invasive hyphae within rice epidermal
cell layers was performed by epifluorescence microscopy
using aniline blue
-Rice leaves were inoculated with conidia from Guy
11 and pth2 mutant and the fungus allowed to develop
for 4 days.
- The Guy 11 penetration hyphae were
observed ramifying throughout the rice leaf tissue
- In the pth2 mutant, no
penetration hyphae were observed indicating
that rice cuticle penetration and formation of penetration
hyphae are affected by loss of Pth2.
-Epidermal penetration assays were performed with the
pth2 mutant and Guy 11 by inoculating onion epidermal
strips with a concentration of 1 x10^4 conidia ml-1
-In Dpth2 infections,
50 ± 7% appressoria produced penetration hyphae after
24 h compared with 92 ± 3.5% in Guy 11 infections. After
48 h, 61 ± 4% of the Dpth2 mutant had successfully
penetrated.
- These observations indicate that pth2
mutant appressoria retain the capacity for production of
polarized penetration pegs
-This penetration pegs able to breach a
highly yielding surface, such as an onion epidermal strip,
but are reduced in their ability to elaborate penetration of the hyphae to breach the tough outer cuticle of the rice leaf
and enter leaf tissue.

# data[].attributes.headline
Farhana Result- Pth2 is a peroxisomal CAT

# data[].attributes.subject
Farhana Result- Pth2 is a peroxisomal CAT

# data[].attributes.body
-Studies in S. cerevisiae
shown that CAT2 encodes both peroxisomal and mito-
chondrial CAT isoforms.
-this CAT isoforms contain both an N-terminal
amphiphilic a-helix that acts as a mitochondrial targeting
signal and the tripeptide, AKL, a functional variant of the
type I peroxisomal targeting signal
-To determine the subcellular location of Pth2, Pth2–GFP fusion protein in M.
grisea under the control of its native promoter is constructed & expressed.
-This gene
fusion is fully functional and able to complement all
Dpth2 mutant phenotypes
-The expression of GFP was observed with a punctate distribution of
vegetative hyphae, grown in axenic culture and then it is
transferred to medium containing 50 mM sodium
acetate, and within conidia
-To investigate the
cellular distribution, FoxA-
RFP fusion protein is introduced to the same Pth2–GFP transformant
of M. grisea.
-Colocalization of the
foxA-RFP and Pth2–GFP fusion proteins was observed
in an intracellular vesicles
This result is observed when accumulated at the cortex
of cells
- the diffusion back-
ground Pth2–GFP fluorescence in hyphae of the fungus is observed.
- As a conclusion, Pth2 is located in peroxisomes. The small cytosolic
pool of the protein also possibly exists.

# data[].attributes.body
Pth2 is dispensable for turgor generation within the appressorium.
~ loss of CAT lead to inability to form appressorium
~ cytorrhysis assays - to test whether pth2 is require in turgor generation or not.
~ result, pth2 is not needed for appressorium turgor generation
Appresorial melanin biosynthesis is reduced, but not severely compromised by absence of PTH2.
~ peroxisomal acetyl-CoA is required as a substrate for dihydronephthalene melanin biosynthesis in appressorium function.
~ pth2 generate cytoplasmic acetyl-CoA - channeled to pentaketide melanin biosynthesis pathway.
~ electron microscopy perform in pth2 mutant, guy11, alb1 and buf1 mutant
~in guy11 - thin distinct layer of melanin deposite outside plasma membrane.
~ in pth2 - melanin thickness was reduced compare to guy11
~ in buf1 mutant - thick microfubrillar melanin precursor present
~scytalone solution was added to test reduction in melanisation in pth2 - result : no restoration

# data[].attributes.body
1. PTH2 is regulated by the cAMP response pathway
# 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
# PTH2 expression in mycelium is normally repressed in the presence of glucose, which requires the cAMP-dependent PKA signalling pathway.

2. PTH2 encodes a CAT required for lipid metabolism
# PTH2 gene was removed and
replaced with a 1.4 kb hygromycin phosphotransferase
gene cassette
#the gene then was introduced into Guy 11(wild type)
#growth test revealed pth2 mutants unable to utilize acetate ,triglyceride or long chain fatty acids as sole carbon sources

~ determine whether PTH 2 encodes a CAT
++ Guy 11 - CAT activity is higher compared to pth2 mutants.
++reintroduction of PTH2 gene into pth2 mutant restored CAT activity

# data[].attributes.body
- Initial development of the rice blast fungus occurs on the leaf surface in the absence of external nutrients.

- The transfer of acetyl CoA across intracellular membranes is catalysed by carnitine
acetyl transferases (CATs)
**CAT-  convert acetyl CoA units to acetyl-carnitine for transport across the membrane

- PTH2 gene  -  potentially encoding a CAT
a) PTH2 encodes the major CAT in M. grisea and is necessary for appressoria to function correctly.
b)regulated by the cyclic AMP response pathway and is located in peroxisomes
c)contributes to effective lipid reserve mobilization during appressorium maturation
d)necessary for penetration hypha formation and host invasion.

# data[].attributes.body
-CAT is important for directing Acetyl CoA to correct compartment for utilization. In S. cervisiae Cat2 provides peroxisomaaly generated acetyl CoA transfered to cytoplasm, Cat1 into mitochondria.
-PTH2 encodes major CAT activity