Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

Abstract

The antifungal glycoalkaloid α-tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades α-tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (Ͼ98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, α-tomatineinducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by twodimensional gel electrophoresis. A protein with a molecular mass of 64 kDa accumulated in the α-tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading α-tomatine to lycotetraose and tomatidine.

Tomatidine and lycotetraose, hydrolysis products of α‐tomatine by Fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells

“…We next examined the effect of Td and Lt on the infection of tomato with the Japanese radish yellow pathogen, F. oxysporum f. sp. raphani (FOR#21), which does not infect tomato plants and does not have tomatinase [12]. Tomato cuttings (plants with excised root systems) were exposed to conidial suspensions of the fungus in the presence or absence of Td and Lt, and then transferred onto the selective medium for the growth of Fusarium species to detect the fungus colonized in the xylem vessels of tomato cuttings.…”

“…Tomatidine and all other reagents were from Wako Pure Chemical (Osaka, Japan). Lycotetraose was prepared from α‐tomatine by enzymatic hydrolysis with crude tomatinase [12] followed by gel‐filtration with Sephadex LH20 (Pharmacia) and silica gel chromatography. Stock solutions of α‐tomatine, tomatidine, and lycotetraose were made at 10 mM in dimethylsulfoxide (DMSO) (α‐tomatine) and methanol (tomatidine and lycotetraose).…”

Molecular Biology in Plant Pathogenesis and Disease Management

“…radicis lycopersici (FORL) have been reported to produce tomatinases with same mechanisms involving hydrolysis of ␣-tomatine (Lairini et al 1996). But the gene encoding tomatinase seems to be different between these two formae speciales (Ito et al 2004b). The tomatinase gene FoToml capable of degrading ␣-tomatine to less toxic metabolites, is present in FOL and also in some strains of F. oxysporum belonging to formae speciales nonpathogenic to tomato.…”

Tomato as a model plant for plant-pathogen interactions

“…Homologous recombination involving double crossover events between chromosomal DNA and vector DNA frequently occurs, allowing targeted gene disruption. Restriction enzyme mediated integration (REMI; Lu et al, 1994;Kawabe et al 2004) and Agribacterium mediated integration (Takahara et al 2004) are frequently used to generate random insertion of plasmid DNA into fungal genome. Since most fungal nuclei are haploid, primary transformants immediately Rohe et al 1996), and BSD (blasticidin S deaminase; Kimura et al 1994).…”

“…Probably pectinases are encoded by more than one gene, and lack of a particular pectinase is compensated by the pectinases encoded by other genes (Agrios 2005;Kawabe et al 2004). A chemical defense, tomatine, is enzymatically detoxified by Botrytis cinerea, Septoria lycopersici, and FOL (Figure 1; Roldán-Arjona et al 1999;Ito et al 2004;Agrios 2005).…”