The Analysis On The Phenotypes Of Magnaporthe Grisea Mutated By T-DNA Insertion And The Location Of The T-DNA Inserting Loci

The most damaging disease of the rice crop is the rice blast which is caused by the heterothallic fungus Magnaporthe gnsea(Herbert) Barr. This disease caused great economic damage on the rice production annually. M grisea has been utilized as a model fungal pathogen for elucidating molecular bases of plant-fungus interactions because the interactions between its races and cultivars of rice follow the gene-for-gene relationship and also due to its genetic and molecular tractability. Knowledge of the molecular mechanism in pathogenicity of the fungus not only theoretically helps the rice blast control, but also provides some cues to understand the pathogenicity of other plant pathogenic fungi.In the study, we mutated M grisea with T-DNA insertion Agrobacterium tumefaczens-mediated transformation(ATMT). We increased the number of the T-DNA inserted mutants to 6,855. The transformation efficiency in average was 300 transformants per 1.0 x106 conidia of the fungus. We selected 1,600 from all the mutants for analysis on the morphological development and assay on virulence to the rice. By morphological comparison with the wild type(Y34), we obtained a total of 173 morphological mutants, including the colony morphological abnormal, change in conidium production and the appressorium formation ability decrease. By inoculation the conidia of the mutants on the susceptible rice variety Nipponbare and resistant variety C101 (as control), we screened out 49 pathogenicity-related mutants, in which 23 completely lost and 26 significantly decreased their virulence to Nipponbare.We randomly selected 28 from the 173 mutants for detailed morphology and development observation, and also for mating type test. We discovered 15 mutants exhibited color abnormal in colony, 7 mutants decreased growth rate, 2 mutants showed abnormal in conidium morphology and sporulation, 2 mutants produced abnormal apperssorium, 3 mutants completely lost their appressorium formation ability. By cross cultivation of the mutants with the mating type testers(P131 and 1528), we found 3 mutants (Y34-0211,Y34-1469 and Y34-0635) completely lost the ability to turn their anamorph into teleomorph phase, while this ability of other mutants tested remained the same as the wild type.We amplified the genome DNA from the 28 mutants by PCR using part of the hph gene sequence as primers. This resulted in fragments the same sequence as the hph gene, suggesting that the hph gene has been inserted into these genomes. Using part of the T-DNA left border sequence as interlaced primers and AD9 as arbitrary primer for...