Structure of the cutinase gene and detection of promoter activity in the 5'-flanking region by fungal transformation

Abstract

The cutinase gene from Fusarium solani f. sp. pisi (Nectria hematococa) was cloned and sequenced. Sau3A fragments of genomic DNA from the fungus were cloned in a k Charon 35 vector. When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene. A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron. Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, Which was translationally fused to flanking sequences of the cutinase gene. When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained. Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter. The results of'Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences. Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA. Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.Several lines of evidence strongly suggested that the ability to produce cutinase might determine whether a fungal pathogen can penetrate intact plant organs and infect them (22). In vitro translation of mRNA from cultures induced to produce cutinase (25) revealed that the primary translation product of cutinase was 2,100 daltons larger than the mature enzyme (13). By using the induced mRNA, a cDNA library was constructed, cutinase cDNA was isolated, and the nucleotide sequence was determined, revealing an open reading frame for a 23,951-dalton protein (44). The availability of the cloned cDNA provided a probe for the cutinase gene.Recently, strong evidence was presented that fungal spores sense cotitact with a plant surface by using the small amount of cutinase carried by the spore. Upon contact with the cuticle, the enzyme generates small amounts of cutin monomers and the most unique monomers trigger cutinase synthesis (47). Dot blot analysis with cloned cutinase cDNA as the probe demonstrated that cutinase gene transcripts became measurable within 15 min after the spores contacted cutin. To determine how the cutin monomers trigger cutinase gene expression, a knowledge of the cutinase gene structure is essential. In this paper we report the cloning of the cutinase gene from Fusarium solani f. sp. pisi and its nucleotide sequence, including the sequences of the flanking regions. We also report the transformation of F. solani f. sp. pisi and Colletotrichum capsici using the hygromycin r...

Regulation of Constitutively Expressed and Induced Cutinase Genes by Different Zinc Finger Transcription Factors inFusarium solani f. sp. pisi(Nectria haematococca)

“…Comparison of Promoters of Cutinase Genes-The 5Ј-flanking region that is known to contain the promoter of cut1 (5,17) showed a high degree of identity with the corresponding segment of cut2 and cut3 (Fig. 2).…”

“…1 filter and washed twice with 0.6 M MgCl 2 . Protoplast preparation and transformation were done as described (17). The transformed protoplasts were plated on mineral medium containing 2% glucose, 1.2 M sorbitol, and 2% agar.…”

“…An overlay of 1% agar containing hygromycin B (300 g/ml) (Calbiochem) was added after 24 h of incubation. Stable transformants were selected as before (17) and verified by PCR using primers designed to amplify the hygromycin gene portion or the gus gene:cut promoter junction portion of the construct. The primers used are as follows: hyg forward, 5Ј-GAA GAA TCT CGT GCT TTC AG-3Ј; hyg reverse, 5Ј-TAC TTC TAC ACA GCC ATC GG-3Ј; cut1 forward, 5Ј-GGG GGA TTC TCT TTC ATG TTT GCG G-3Ј; cut2 forward, 5Ј-GGG GGA TCC TCT TTC TTA TTG GGG G-3Ј; cut3 forward, 5Ј-GGG GGA TCC TCT TTC TTA TTT GCG G-3Ј; and gus reverse, 5Ј-GAT TTC ACG GGT TGG GGT TTC T-3Ј.…”

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

“…Protoplast preparation and transformation were performed as before (22). For the double disruptant, the pelA disruptant was transformed with the pelD disruption construct pPELD::zeo.…”

Cutinase is not required for fungal pathogenicity on pea.

“…With fungal pathogens, only the genes for cutinase production (Dickman et al, 1989;Soliday et al, 1989) and pisatin demethylating ability (Weltring et al, 1988;Schafer et al, 1989) of Nectria haematococca MP VI and the b locus of Ustilago maydis (Kronstad and Leong, 1989;Schulz et al, 1990) have been isolated and characterized as putative pathogenesis determinants. The direct involvement of the b locus in the pathogenic development of U. maydis was unequivocally demonstrated by gene replacement (Kronstad and Leong, 1990).…”