Molecular Tagging And Cloning Of Genes Involved In Pathogenicity In Magnaporthe Grisea

To perform insertional mutation to tag pathogenicity related gene in Magnaporthe grisea, the pathogen strain Ml31 was used to establish a high-efficient Restriction Enzyme Mediated Integration(REMI) transformation system with the plasmid pUCATPH. The plasmid pUCATPH was linearized by restriction endonucleases Hind III, Sac I, and Kpn I, respectively. Resulted fragments were introduced individually into Ml31 in the presence of corresponding enzymes. Transformation ration was found to be increased by the incorporation of restriction enzymes, while levels of transformation enhancement varied with enzymes.Based on pathogenicity and pigment production of 200 transfomants, two transformants with altered phenotypes and six transformants with modified pathogenicity were obtained. DNA dot hybridization analysis revealed that the plasmid DNA was integrated into the fungal genomes in these transformants. Moreover, genes involved in pathogenicity or pigment production experimentally were mutated in these mutants. Rep-PCR fingerprint patterns of the trasformants and M131 revealed that mutants were different from M131, and from one another, suggesting that they have mutated at molecular levels. To determine patterns of the plasmid integration, DNA from four transformants were digested with Hind III and EcoR V ,respectively, and tested by dot hybridization using pUCATPH as the probe. It is found that M131 was not homologous to pUCATPH, and the four transformants all showed many bands of varied sizes. These patterns could result from REMI events, tandem integrations, or a combination of both.The transformant 01-36 was nonpathogenic to the rice varity K1 with resistance gene Pita, which was different from M131. It could be deduced that avirulence gene avr-Pita might had mutated in the transformant 01-36. The genome DNA of the strains 01-36 and M131 was PCR amplified using a couple of primers PI and P2 designed according to the sequence of avirulence gene avr-Pita, and a 1057bp fragment was obtained. The amplifiedband from M131 was sequenced and then aligned with the sequence of avr-Pila gene. The result was that the sequence was 98% homologous to one fragment of avr-Pita sequence, but the integration site was not within this fragment. The other amplified fragments of the strains those are incompatible to Kl and from different regions, was subjected to dot hybridization analysis with the probe of tagged avr-Pita. Positive responses showed that they all could included homologous fragments to avr-Pita gene. The interaction fashions between avirulence genes and resistant genes were pilot studied.