INA gene inactivation in isolated strains from frozen leaves and its effects on plant freezing

Freezing is a major environmental stress, which limits plant’s distribution, growth and productivity. Ice nucleation active bacteria can catalyze ice formation at temperatures as high as -2°C. A membrane protein confer the ability of ice nucleation, called ice-nucleating proteins (INPs), which is encoded by a single gene. Mutation in this gene will lead to delaying of ice nucleation. In this study, leaf tissues of several plants with freezing symptoms were collected from different locations and 40 bacterial isolates with yellow circular colonies and regular margins were isolated from samples. Finally, total of 12 isolates belong to Xanthomonas were selected for ice nucleate activity (INA) by Dropletfreezing test and presence of INA gene was surveyed by PCR. According to the obtained results, isolate 28 was targeted to mutagenesis by using Tn5 transposon. After mutagenesis, isolates with ability to grow on kanamycin, which lack of INAx gene in PCR were considered as mutated isolates and their freezing effects were evaluated on bean seedlings. Results showed that isolates with mutated INA gene cannot induce freezing on bean seedlings, while primary identified isolate (isolate 28) could do it. These results show that if we could replace wild type ice nucleation active bacteria with mutated forms (just different in ice nucleation activity), we could, probably, prevent freezing and subsequent economic losses