Homology Base Tracking of Tomatinase in the Genome of Fusarium oxysporum f. sp. melonis

Document Type : Research Article


1 دانشگاه صنعتی اصفهان

2 Plant science research institute, Ferdowsi University o f Mashhad

3 Faculty of Agriculture, Isfahan University of Technology

4 Shiraz University


Introduction: Phytopathogenic fungi exposes various proteins to overcome plant defense systems. Production of saponins likes α-Tomatine is one of the tomato preformed defenses barriers which should be detoxicated by the pathogens. It has been revealed before which most of Fusarium species and forma specials could produce tomatinase, a glycosyl hydrolases protein, to de-glycosylate α-Tomatine. Fusarium oxysporium f. sp. melonis (FOM) wildly attached melon cultivars and at the time of this investigation, there was only one report underlining the existence of the gene sequence of tomatinase in the genome of FOM using southern blotting experiment. This study was carried out to track the whole tomatinase gene sequence in the FOM genomic sequence and investigate the probability genetic variation of the gene in the nucleotide and protein sequences.
Materials and Methods: Fusarium oxysporium f. sp. melonis (Fom) race1 have been previously reported in Khorasan, Iran. It was cultured in liquid medium and the mycelia were used for the genomic DNA isolation. Primers were designed based on conserved sequence in upstream and downstream of FoTom1 sequence (AJ012668). PCR was carried out and amplified segments were bi-directional sequenced. The results were then analyzed by Vector NTi software. The sequencing result was aligned with FoTom1 sequence as Refseq and the single nucleotide variations were detected by CLC work bench software. The effects of the mutations on the protein structure were predicted by CLC work bench software.
Results and Discussion: Electrophoresis pattern of PCR products showed a single band of the expected size in the strain FomR1 that was at the same size of the band amplified from FoL genome. The designed primers based on the FoTom1 sequence amplified a specific segment in the Fom genome. Alignment the sequencing results with the Fo-Tom1 from Fusarium oxysporium f. sp. lycopercisi (Fol) in nucleotide level revealed 14 mutations which seven of them were appeared in the protein sequence. Three mutations occurred in the functional domain of the protein. At the position 145, an acidic amino acid with a negative charge was substituted by a polar amino acid. Replacements at positions 218 and 236 were the same in terms of polarity and at position 218; both amino acids have a positive charge.
There were no introns in the coding sequence of FomTom R1 region as same as FoTom1. Pairwise alignment results showed some an-synonyms mutations between two sequences that made some changes in the secondary structure of the translated protein from FomTomR1. The first an-synonym mutation, SNP15 (E→G), inside the signal peptide, converts the alpha helix to a new beta sheet. SNP34 (K→N) and SNP35 (S→N) mutations shortened the alpha helix. The other mutations happened out of the alpha helixes and beta sheets. To predict the effects of the mutations on the FomTomR1function, in-silico analyses were carried out. The results revealed that three mutations occurred in the functional domains of tomatinase in Fom. The mutations in the hydrolysis domain may affect the structure of FomTomR1and can be effective in the protein. The presence of different active saponins components in Melon may be an evolutionary reason for some variation in sequence and structure of the FomTomR1 protein in Fom. To prove the differences in the tomatinase function, the interaction of proteins with various types of melon saponins components should be investigated at the future studies.       
Conclusion: The results showed the tracked sequence could be homolog of the Tomatinse gene in the Fom genome. We named it Fom-TomR1 and the sequenced was submitted in the Genbank with accession number MF178403. For the future study, the gene influences should be investigated in the pathogenesis of FOM on melon cultivars and it could be considered as a general screening index using heterologous expression of the gene.


1- Agrios G.N. 2005. Plant Pathology, 5th ed. Academic Press. 922 pp.
2- Arneson P., and Durbin RD. 1968. Studies on the Mode of Action of Tomatine as a Fungitoxic Agent. Plant Physiology 43: 683-686.
3- Carter J.P., Spink J.P., Cannon M., Daniels J., and Osbourn A.E. 1999. Isolation, characterization and avenacin sensitivity of a diverse collection of cereal-root-colonizing fungi. Appl. Environ. Microbiology 65: 3364–3372.
4- Crombie W.M.L., Crombie L., Green J.B., and Lucas J.A. 1986. Pathogenicity of the take-all fungus to oats: its relationship to the concentration and detoxification of the four avenacins. Phytochemistry 25: 2075–2083.
5- Davies G., and Hernissat B. 1999. Structures and mechanisms of glycosyl hydrolases. Curr Biology 3: 853-859.
6- Ford J.E., McCance D.J., and Drysdale R.B. 1977. The detoxification of a-tomatine by Fusarium oxysporum f. sp. lycopersici. Phytochemistry 16: 545-546.
7- Gonzalez-Mendoza D., Argumedo-Delira R., Morales-Trejo A., Pulido-Herrera A., Cervantes-Diaz L., Grimaldo-Juarez O., and Alarcon A. 2010. A rapid method for isolation of total DNA from pathogenic filamentous plant fungi. Genetics and Molecular Research 9(1): 162-166.
8- Ito S., Kawaguchi T., and Nagata A. 2004. 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. Journal Gen Plant Pathology 70: 195–201.
9- Ito S., Nagata A., Kai T., Takahara H., and Tanaka S. 2005. Symptomless infection of tomato plants by tomatinase producing Fusarium oxysporum formae speciales nonpathogenic on tomato plants. Physiol. Mol. Plant Pathology 66: 183–191.
10- Keukens E.A.J., Vrije T., Van den Boom C., Waard H., Plasmna H., Thiel F., Chupin V., Jongen W.M.F., and Kruijff B.de. 1995. Molecular basis of glycoalkaloid induced membrane disruption. Biochim. Biophys. Acta, 1240: 216–228.
11- Lairini K., Perez-Espinosa A., Pineda M., and Ruiz-Rubio M. 1996. Purification and characterization of tomatinase from F. oxysporum f. sp. lycopersici. Appl. Environ. Microbiology 62: 1604-1609.
12- Lairini K., Perez-Espinosa A., and Ruiz-Rubio M. 1997. Tomatinase induction in formae specials of Fusarium oxysporum non-pathogenic of tomato plants. Physio Mol Plant Pathology 50: 37-52.
13- Lairini K., and Ruiz-Rubio M. 1998. Detoxification of a-tomatine by Fusarium solani. Mycol. Research 11: 1375-1380.
14- Morrissey J.P., and Osbourn A.E. 1999. Fungal Resistance to Plant Antibiotics as a Mechanism of Pathogenesis. Microbiol mol biol R, 63: 708–724.
15- Osbourn A.E. 1996. Saponins and plant defence—a soap story. Trends Plant Science 1:4–9.
16- Pegg G.F., and Woodward S. 1986. Synthesis and metabolism of- tomatine in tomato isolines in relation to resistance to Verticillium albo-atrum. Physiol. Mol. Plant Pathology 28: 333-338.
17- Roldan-Arjona T., Perez-Espinosa A., and Ruiz-Rubio M. 1999. Tomatinase from Fusarium oxysporum f. sp. lycopersici defines a New Class of Saponinases. MPMI 12: 852–861.
18- Safe L.M., Safe S.H., Subden R.E., and Morris D.C. 1977. Sterol content and polyene antibiotic resistance in isolates of Candida krusei, Candida parakrusei and Candida tropicalis. Can. J. Microbiology 23: 398-401.
19- Sandrock R.W., DellaPenna D., and VanEtten H.D. 1995. Purification and characterization of b2-tomatinase, an enzyme involved in the degradation of a-tomatine and isolation of the gene encoding b 2-tomatinase from Septoria lycopersici. Mol. Plant-Microbe Interact 8: 960-970.
20- Steel C.C., and Drysdale R.B. 1988. Electrolyte leakage from plant and fungal tissues and disruption of liposome membranes by- tomatine. Phytochemistry 27:1025-1030.
21- VanEtten H.D., Mansfield J.W., Bailey J.A., and Farmer EE. 1994. Two Classes of Plant Antibiotics: Phytoalexins versus “Phytoanticipins”. The Plant Cell 6: 1191-1192.