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- Different approaches were used to isolate F.oxysporum
cutinase genes, including PCR amplification of genomic DNA or RT-PCR
- The different number of cutinase genes in the two Fusarium species may point to divergent evolutionary
mechanisms during adaptation of the infection process in root and aerial plant pathogens
- The product of the F. oxysporum lip1 gene has 80% identity to F. graminearum Lip1, an extracellular lipase required for virulence on wheat
- The promoters of both
cut1 and lip1 genes contained the element CCGAGG, which
mediates binding of the fatty acid hydrolysis-regulating transcription
factor FarA from A. nidulans
- The key role of Ctf1 in regulation of cut1 and lip1 was substantiated
by RT-PCR and Northern analysis showing that expression of the
two genes was nearly abolished in the Δctf1 mutant
- Targeted disruption of ctfα in F. solani f. sp. pisi resulted in loss of virulence on pea, but the decreased level of secreted cutinase
was not sufficient to fully explain the loss of virulence
- Ctf1, a major regulator
of cutinase and lipase activity during growth in axenic culture, is not required for pathogenicity of F. oxysporum.
- RT-PCR show that
the lip1 gene is highly expressed during infection of roots
- Ctf1 appears to regulate lip1 under the in vitro conditions studied

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

Induction of extracellular esterase activity was both delayed and attenuated when glucose was supplemented as a carbon source in addition to apple cutin.

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

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

Ctf1 is not a major regulator of lip1 during root infection

Expression of ctf1, cut1 and lip1 during infection of tomato plants by F. oxysporum was determined.

Both ctf1 and cut1 were expressed at very low levels.

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

Plants inoculated with the Δctf1 and ctf1C mutant strains showed a very similar symptom development as the wild-type during the entire period of the experiment.

The wild-type and the ctf1 mutant strains efficiently colonized and macerated the fruit tissue, producing aerial mycelium on the surface

Ctf1 is not essential for virulence of F. oxysporum.

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Cloning of the ctf1 gene encoding a cutinase transcription factor of F. oxysporum f. sp. lycopersici
- The ctf1 gene, encoding a putative cutinase transcription factor of F. oxysporum f. sp. lycopersici, was cloned from a λEMBL3 genomic library and a lambda ZAP cDNA library of isolate 4287, using a probe generated by PCR with primers based on the coding sequence of the ctf1α gene from F. solani f. sp. pisi.
- Sequencing of hybridizing genomic and cDNA clones revealed the presence of an open reading frame (ORF) of 2712 nucleotides, interrupted by five introns of 59, 192, 92, 65 and 56 bp
- The 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, respectively, to the cutinase and fatty acid transcription factors Ctf1α of F. solani  and FarA of A. nidulans
- The algorithm ‘PROSITE’ was used to predict 14 potential consensus phosphorylation sequences for casein kinase II, six potential sites for protein kinase C (PKC), three potential sites for tyrosine kinase, one potential site for cyclic AMP-dependent protein kinase A (PKA) and two potential sites for mitogen-activated protein kinase (MAPK)
- Three putative nuclear localization signals with the residue patterns KRKK (position 86), KRHR (position 636) and PKRK (position 659) were detected, with a probability of 0.7 of Ctf1 being localized in the nucleus as predicted by the PSORT program
- Southern analysis of genomic DNA treated with different restriction enzymes indicated that ctf1 is present as a single copy in the F. oxysporum genome
Construction of ctf1 loss-of-function and overexpressing alleles
- Loss-of-function mutants carrying a disrupted copy of the ctf1 gene were generated using one-step gene replacement by replacing  BamHI fragment within the ctf1 coding region with the hygR cassette.
-  Results in disruption of the Cys6Zn2 DNA-binding motif, which is essential for proper function of CTF1α.
- Two transformants produced PCR banding patterns consistent with homologous replacement of the ctf1 gene
- the hybridizing 8.3-kb BamHI  fragment containing the wild-type ctf1 allele was absent in transformant Δctf1, indicative of homologous integrationmediated gene replacement
-  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 (PgpdA)
- one of the transformants named ctf1C revealed the presence of two hybridizing bands, one corresponding to the wild-type ctf1 allele and an additional 6-kb band, consistent with ectopic insertion of the pPgpdA-ctf1 construct into the genome
- absence of a ctf1 transcript in the knockout mutant and increased levels of transcript in the ctf1C strain, independently of the carbon source
- 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
- Microscopic analysis of the fungal strains grown in liquid media containing different carbon sources, including wheat germ oil, did not reveal any apparent differences in growth and development between the wild-type, Δctf1 and ctf1C strains
- Δctf1 mutant not only grew poorly, producing much less aerial mycelium than the wild-type, but also showed a complete absence of fluorescent droplets, indicative of a failure in hydrolysis of fatty acids
- the transformant ctf1C exhibited significantly increased fatty acid hydrolysis compared with the wild-type strain.
Characterization of F. oxysporum genes involved in fatty acid hydrolysis
- role of Ctf1 in regulation of fatty acid hydrolysis of F. oxysporum further, the cut1 and lip1 genes were cloned encoding a putative cutinase and lipase,
- The lip1 gene encoding a putative extracellular lipase of F. oxysporum was isolated by PCR amplification of F. oxysporum genomic DNA with primers lip16 and lip18, derived from the F. verticilloides genome database
- Lip1 protein contains the characteristic catalytic triad, comprising a serine, a histidine and an aspartic acid, which is highly conserved in lipases
- The promoter regions of F. oxysporum cut1 and lip1 each contained one copy of the CCGAGG DNA element (positions –711 and –495, respectively) mediating transcriptional activation by the transcription factors Ctfα and FarA

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Fungal Pathogen attacking the aerial parts of the plants

first contact between pathogen and host occur on the plant surface-> protected by cuticle layer.

Soil-borne pathogens enter their host plant  through the roots that lack of cuticle layer.

To test the role of cutin-degrading enzymes in virulence of the soil-borne vascular wilt pathogen F. oxysporum

cloned and mutated the gene encoding the transcription factor Ctf1, the deduced amino acid sequence of which shows a high degree of identity with F. solani f. sp. pisi  CTFα ---> regulates the inducible cutinase gene cut1

with A.nidulans FarA --> controls expression of genes involved infatty acid metabolism.

The predicted F. oxysporum Ctf1 protein contains three putative nuclear localization signals, and its amino terminus shows the Zn2(II)Cys6 DNA binding domain, characteristic of regulatory proteins with diverse function

This domain follows the canonical pattern CX2CX6CX8CX2CX6C, with both repeats (CX2CX6C) separated by a variable number of residues (eight in CTFα and CTF1).

The group of transcription factors binds to promoter regions at two conserved palindromic triplets GCC(N)xCCG, where N has a variable length

F. solani f. sp. pisi, the DNA motifs recognized by CTFα have the opposite orientation and are separated only by two nucleotides

FarA and FarB, two transcription factors regulating fatty acid metabolism in A. nidulans, bind to the conserved motif CCGAGG in the promoter regions of the target genes

The promoter of the F. oxysporum ctf1 gene contains a copy of the CCGAGG motif at position –374, suggesting that Ctf1 may regulate its own expression

mutants of  F.oxysporum  carrying either loss-of-function or constitutively expressed allele of ctf1  do not show any differences in growth and development in submerged culture even when grown on fatty acid-containing carbon sources such as olive oil or wheat germ oil

Clear differences were detected on solid media with olive oil as carbon sources

Phenotype of ctf mutants condition are:-( poor growth, reduced aerial hyphae and absent of UV-fluorescent droplet)  was consistent that this mutant were impaired in hydrolysis of fatty acid

Ctf1 controls expression of the major lipolytic enzymes produced by F.oxysporum  under these conditions.

CTFα and FarA regulate genes involved in fatty acid metabolism and therefore affect the production of encoded enzyme activities .

Esterase activity was clearly induced by apple cutin in the wild-type strain, and this induction was abolished by deletion of ctf1

Similar results were obtained for lipase activities

Ctf1 is required for efficient induction of extracellular lipolytic enzymes in F. oxysporum