Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum

Abstract

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this fermtin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.Cuticle constitutes the first barrier between a higher plant and its environment. The mode of penetration of this barrier by phytopathogenic fungi has been a controversial subject for a long time (4). Penetration is either by the mere physical force of fungal growth or by enzymic dissolution of the cuticle. The nature of the opening created by the penetrating fungus has been used as a supporting evidence for the enzymic penetration theory (5). However, in the absence of convincing evidence to support either side, the arguments remained unsettled. The structural component of plant cuticle is a biopolyester, cutin, derived from hydroxy and epoxy fatty acids (3) and therefore this polymer constitutes the major physical barrier between the invading fungus and the plant. Recently we have isolated in homogeneous form cutinases from the extracellular fluid of several fungal pathogens grown on cutin as the sole source of carbon (6, 7). We were able to prepare antibodies against these pure enzymes and using ferritin-labeled antibody a specific test was performed to determine whether a fungal pathogen excretes cutinase during the cuticular penetration phase of the infection process. The results presented in this communication provide convincing evidence that Fusarium solani f. pisi excretes cutinase during infection of the host tissue. MATERIALS AND METHODSCutinase-I was isolated in homogeneous form from the extracellular fluid of F. solani f. pisi grown on apple cutin as the sole source of carbon (6). Rabbit antiserum was prepared against the pure cutinase (7) and the immunoglobulin fraction was isolated from this antiserum by repeated ammonium sulfate fractionation (2). Ferritin was conjugated to the immunoglobulin using xylylene metadiisocyanate as the coupling agent (1 Epicotyls from 5-day-old etiolated Pisum sativum seedlings were excised and rinsed in distilled H20 several times. The cuticular surface of these epicotyl segments, 0.5 cm long, was inoculated with small (about 40-,ul) droplets of a thick suspension of micro-and macroconidia of F. solani f. pisi from 15-to 20-day-old cultures grown on 2% potato dextrose agar. The inoculated segments of epicotyl were placed in a Petri dish in humid atmosphere. After varying periods of incubation at room temperature the infected segments were immersed in the ferritin-conjugated anticutinase-I preparation for 30 min. Controls were similarly treated with nonconjugated ferritin or ferritinconjugated immunoglobulin fraction prepared from a rabbit which was not immunized with cutinase-I. The incubated specimens were rinsed in 0.1 M Na-phosphate buffer (pH 7.2) for 30 min and fix...

Evolution of the cutinase gene family: Evidence for lateral gene transfer of a candidate Phytophthora virulence factor

“…The sequence around the catalytic domain is highly conserved and provides a signature pattern for cutinases (Kolattukudy et al, 1985). A critical role for fungal cutinases in the penetration of unwounded host tissues was demonstrated for some fungi by the use of antibodies, inhibitors and by using cutinase deficient fungal mutants (Dickman and Patil, 1986;Dickman et al, 1983;Maiti and Kolattukudy, 1979;Shaykh et al, 1977). In addition, recent gene disruption studies on the cutinase gene of Pyrenopeziza brassicae, an ascomycete, showed molecular evidence that cutinase activity is required for pathogenicity (Li et al, 2003).…”

Infection structures of fungal plant pathogens – a cytological and physiological evaluation

“…Factors present in cuticle or cell wall, such as dihydroxy C^g-fatty acid, 18-hydroxy-9,10-epoxy Cj^-acid or 9,10,18-trihydroxy Cjg-acid, pectin, or cellulose, have been added to culture media in order to induce the formation of cutin-or cell wall-degrading enzymes in byphae (Forster & Rashed, 1985 ;Kolattukudy, 1985 ;Crawford & Kolattukudy, 1987;Koller & Parker, 1989;Urbanek, 1989;Perez Artes & Tena, 1990;Yazdi, Woodward & Radford, 1990;Riou, Freyssinet & P^evre, 1991). Altbough it is unclear whether or not hypbae formed in liquid culture resemble functional germ tubes, differences between cutinase formed in liquid culture and tbat formed on the host have not been detected with immunological techniques (Sbaykh, Soliday & Kolattukudy, 1977).…”

Dancing Together. Social Controls in Parasitic Plant Development

“…The identified xenognosins were unlike any plant hormone known to mediate developmental commitments (Lynn et al, 1981), and surprisingly were not found to be present in host root exudates. Active compounds did appear after host wounding, leading to the proposal that the parasite released oxidative enzymes, similar to cutinase exudation by Fusarium solani during haustorial penetration (Shaykh et al, 1977), to liberate xenognosins from the host cell surface (Chang and Lynn, 1986). This hypothesis was tested directly by screening for enzymatic activity.…”