# data[].attributes.body
cut1 n lip1 gene encoding a putative cutinase and lipase
cut1 isolate from hEMBL3 genomic library of f. oxysporum
_cut1 protein contain the characteristic conserved catalytic triad ser
_lip1 gene encode putative extracellular lipase pf f. oxysporum
_nucloetide sequence of lip1 locus show ORF of 1035 bp interupt by 2 intron
_the promoter region of f. oxysporum cut1 n lip1 contained one copy of CCGAGG DNA element

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- FarA and FarB, two transcription factors regulating fatty acid metabolism in A. nidulans
- Ctf1 may regulate its own expression.
- the zinc finger transcription factors XlnR controlling expression of xylanase genes in F. oxysporum  and Zfr1 regulating fumonisin production in F. verticillioides.
- the role of this transcription factor in activation of genes involved in fatty acid hydrolysis appears to be broadly conserved in ascomycetes
- a similar role of lip1 in virulence of F. oxysporum as reported for its orthologue fgl1 in F. graminearum cannot yet be ruled out.

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Ctf1 is not required for virulence of F. oxysporum on tomato

1. 3 week old tomato plants is immersed with   microconidial suspensions of the different strains.

2.  Severity of wilt symptoms in plants inoculated with the wild-type strain increased steadily throughout the experiment.

3. It begins with with leaf tips turns yellow and curved  then the stalk weakened and decayed, and most of the plants were dead 20 days after inoculation.

4. Same goes to  plants inoculated with the Δctf1 and ctf1c mutant strains showed a very similar symptom development as the wild-type.

5. To allow  to grow invasively on tomato fruit tissue, microconidia strains was injected directly into tomato fruits.

6. The wild-type and ctf1 mutant efficiently colonized the fruit tissue producing aerial mycelium on the surface.

7. As a conclusion,  Ctf1 is not essential for virulence of F. oxysporum.

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Ctf1 is not a major regulator of lip1 during root infection

1.  Both ctf1 and cut1 were expressed at very low levels   during infection of tomato plants by F. oxysporum.

2.  7 and 10 days after inoculation, ctf1 is low in signal in roots after  inoculated with the ctf1c  strain.

3.  10 days after inoculation with the three different strains,  faint cut1 transcript signals were observed  and in stems infected with the wild-type or the ctf1c strain.

4. High  expression of lip1 was detected in plants inoculated with all the strains, particularly in infected roots.

5.  No expression signal was detected in non-infected control roots.

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Cloning of the ctf1 gene encoding a cutinase transcription factor of
F. oxysporum f. sp. lycopersici

1. Cloned based on the coding sequence of the ctf1 α  gene from  F. solani f. sp. pisi

2. F. oxysporum ctf1 gene encodes a predicted protein of 904 amino acids with a molecular weight of 100.6 kDa,
which shows 88.9 and 57.5% identity.

3. F. oxysporum Ctf1 protein contains:-  # a Cys 6 Zn 2 binuclear cluster DNA-binding motif located close to the N-terminus (amino acid residues 52–85)
# a middle homology region (MHR)
# three ‘coiled coil’ regions

Construction of ctf1  loss-of-function and overexpressing alleles

1. Loss-of-function mutants carrying a disrupted copy of the ctf1  gene were generated.

2. knockout construct was introduced into protoplasts of the wild-type strain

3. F. oxysporum strains carrying a constitutively overexpressed  ctf1c allele were produced by fusing the ctf1 coding and terminator regions to the the A. nidulans gpdA promoter

4. Δctf1 mutant lacks a functional
copy of the ctf1 gene, whereas transformant ctf1c constitutively
produces high amounts of ctf1 transcript.

Mutations in ctf1 affect fatty acid hydrolysis

1. Differences betwen wild type and mutants were observed on SM containing 1% olive oil with or without 0.1% sucrose.

2. growing colonies of the wild-type strain observed under UV light contained many fluorescent droplets surrounding the colony, indicating the presence of degradation products from fatty acid hydrolysis

3. ctf1 mutant
- grow poorly
- produce less aerial mycelium
- absence of fluorescent droplets( fail in f.acid hydrolysis)

4. transformant ctf1c
- exhibited significantly increased fatty acid hydrolysis compared
with the wild-type strain.

## conclude that Ctf1 is required for expression of F. oxysporum genes involved in fatty acid hydrolysis.

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Cultinolytic enzymes are secreted by pathogen to facilitate penetration of the outermost cuticular barrier of host.
Fungal pathogen secretes cutinases and lipases that catalyse the hydrolysis of ester bond from fatty acid polymers abd facilitating fungal penetration through cuticle.
3 cutinases genes cut1,cut2,cut3 share high degree identity. cut2 and cut3 are expressed at basal levels and expression of cut1 is induced by cutin monomers and activated by CTF1a
in A. nidulans, CTF1a homolog FarA and FarB regulate the exprression of genes of fatty acids. This suggest that transcriptional regulation of fatty acid metabolism may be widely conserved in fungi.
Knockout of the cutinases gene cut1 resulted in decreased virulence on pea. insertion of cut1 into a pathogen that normally requires wounds in the plants to cause infection allowed penetration.
immunolocalization with antibodies against Cut1 suggested that the cutinaase was targeted to the growing tip of germinating spores of F.solari.
Ctf1a participates in the regulation of other genes essential for pathogenicity.
Ctf1a also regulates virulence factors in a pathogen which attacks the host plant through roots.

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Ctf1 regulates extracellular esterase and lipase activities and expression of cut1 and lip1 during growth in submerged culture

1. To test if Ctf1  regulates extracellular esterase and lipase activities in F. oxysporum,  wild type and mutant is grown in different time period in liquid SM and different carbon sources.

2. Esterase activity when grown in SM, remain at basal level without carbon source (carbon starvation).

3. Esterase activity in wild-type and the ctf1c strain grown on 0.2% apple cutin  increased rapidly.

4.  No induction of esterase activity on apple cutin was detected in supernatants of the Δctf1 strains.

5. When glucose is added  as a carbon source in addition to apple cutin,   induction of extracellular esterase activity was both delayed and attenuated  due to preferential utilization of glucose by the fungus .

6. Induction of lipolytic activity on 2% wheat germ oil was slightly increased in the ctf1c strain as compared with the wild-type, and was not detected in the Δctf1 mutant.

7. Conclusion,  Ctf1 mediates activation of extracellular esterase and lipase activities in F. oxysporum under inducing conditions, but not under carbon starvation or glucose-repressed conditions.

Test for expression of cut1 gene depends on Ctf1

1.  RT-PCR with gene-specific primers cut19 and cut14 with flank intron is used to determine the level of cut1 transcripts in mycelia grown under inducing conditions.

2. Higher levels of RT-PCR product corresponding to the cut1 transcript were detected in mycelia of the wild-type grown on apple cutin than in those grown on glucose, suggesting that cut1 transcription is triggered by inducing carbon sources.

3.   A weak activation of cut1 expression was observed in the Δctf1 mutant.

4.  Mycelia grown for 24 or 48 h on apple cutin confirmed the low level of cut1 induction in the Δctf1 mutant compared with the wild-type and ctf1c strain.

5. Scientist  study the effect of mutations in ctf1 on expression of the lip1 gene. Transcript levels of the lip1 gene were slightly higher in the ctf1c strain than in the wild-type, whereas an extremely low hybridization signal was detected in the Δctf1 mutant.

6. Conclusion,  efficient transcriptional activation of cut1 and lip1 on fatty acid substrates, but not on glucose, is mediated by the Ctf1 transcription factor.