Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence

Abstract

Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate-or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in V... Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.

Cutinase structure, function and biocatalytic applications

“…Cutinases are serine hydrolases specific for primary alcohol esters, the dominant linkage in cutin (Murphy et al, 1996). To date, the majority of the work has been done with a fungal pathogen of peas, Fusarium solani f. pisi (Purdy and Kolattukudy, 1975;Lin and Kolattukudy, 1980;Dantzig et al, 1986;Murphy et al, 1996;Soliday and Kolattukudy, 1983). The production of cutinase seems to be highly regulated by the growth conditions.…”

“…It was shown that inhibitors of cutinase could prevent fungal penetration of plants and thus prevent infection (Soliday and Kolattukudy, 1983). Since this enzyme appears to play an important role in virulence, several studies have been performed to elucidate its physiological and biochemical properties (Purdy and Kolattukudy, 1975;Lin and Kolattukudy, 1980;Dantzig et al, 1986;Murphy et al, 1996;Sebastian et al, 1987;Soliday and Kolattukudy, 1983). Cutinases are serine hydrolases specific for primary alcohol esters, the dominant linkage in cutin (Murphy et al, 1996).…”

“…Cutinases have been purified and characterized from several different sources, mainly fungi (Purdy and Kolattukudy, 1975 ;Lin and Kolattukudy, 1980;Dantzig et al, 1986;Murphy et al, 1996) and pollen Sebastian et al, 1987). Evidence of a bacterial cutinase produced by a phyllospheric fluorescent Pseudomonas putida strain, cohabiting with a nitrogen-fixing bacterium Corynebacterium sp., has also been demonstrated (Sebastian et al, 1987).…”

Induction of Extracellular Lytic Enzymes by <i>Fusarium solani</i>

“…Stability at 28˚C was different for xylanase activity (81.2%), and for polygalacturonase was 61% (Figures 3 and 4). For the induction of extracellular enzymes in Fusarium solani, there are few reports of this species in the literature, and there are referred to F. oxysporum or other species [6,7,18], but reports of F. solani were found as a pathogen associated celery in South America [19], infecting asparagus in five municipalities of Guanajuato [20], and vegetable and flower species in Jujuy, Argentina [21], and some different enzymes [10][11][12][13]. But not found any reports of F. solani in tomato, as the strain that was isolated from the culture of Villa de Arista, S.L.P.…”

“…It has been reported that F. oxisporum produces a variety of lytic enzymes, which depolymerize all components of plant cell walls, such as cellulose, xylan, pectin, polygalacturonic acids and proteins (extensins); and they have been purified and biochemically characterized as several enzymes, one endopolygalacturonase majority (PG1), two exopolygalacturonase (PG2 and PG3), one endoxylanase (XYL1), one endopectate lyase (PL1) [6,7], seven polygalacturonases of Fusarium species of Pinus pinea [8], and the isolation of differentially expressed genes during interactions between tomato cells and a strain of F. oxysporum [9]. With respect to F. solani, studies have reported the isolation of a novel pectate lyase gene from the phytopathogenic fungus F. solani [10], a F. solani mutant recurring in cutinase activity and virulence [11], extracellular lipase by the phytopathogenic fungus F. solani FS1 [12], and a cellulose of F. solani [13]. Therefore, it is important to try to determine cellulolytic enzymes involved in the pathogenesis of F. solani.…”