| Magnaporthe grisea is a well-known ascomycete that cause rice blast. A better understanding of the molecular basis of this disease is beneficial for rice blast control. As a model fungal pathogen, M. grisea has typical infection processes, including conidiation, germination and appressorium differentiation. Isolation of functional genes of M. grisea is helpful to the understanding of other fungal-plant interactions. The pre-penetration processes of M. grisea, especially appressorium differentiation and formation, have been well studied; the post-penetration processes which is essential for infection cycle of M. girsea, including hypha differentiation and colonization et al, still left much to be understood. Colletotrichum lindemuthianum, another serious plant pathogen that cause anthracnose of common bean, has similar pre-penetration processes to M. grisea. And many homologous genes (e.g. PMK1 and CMK1) control the infection cycles of M. grisea and C. lindemuthianum respectively. CLTAl is a crucial gene involved in post-penetration processes of C.lindemuthianum. The null mutant of CLTAl cannot differentiate secondary hyphae and lost pathogenicity on its host. The clarification of homologues to CLTAl and its function in M. grisea is beneficial for understanding the molecular metabolism in post-penetration of M. grisea and difference between M. grisea's infection cycle and C. lindemuthianum's. In this study, MGTA1 gene from M. grisea, a sequence homologue of CLTAl was cloned. The expression pattern of MGTAl gene was analyzed by eGFP fusion and the role of MGTA1 in pathogenicity was analyzed by targeted gene replacement. The results are showed as follows:1. A sequence homologue (with 51% identity) was found by using the amino acid sequence of CLTA1 to search from M. grisea genome databases, assigned MGTA1. The genomic DNA clones covered the promoter regain and the full length of MGTA1 ORF and cDNA clones were obtained by PCR strategy. Nucleotide sequence analysis to the genomic DNA and cDNA of MGTA1 revealed an open reading frame of 2772 bps, including 5 introns, 6 extrons, and a coding sequence of 2370 bps, coding a 790 amino acid peptide. Three major conserved domains (DNA binding domain, middle homology region and activation domain) of the zinc cluster family were found inMGTA1 protein. Sequence features indicated MGTA1 protein is a potential transcriptional activator belonging to the fungal zinc cluster family.2. One copy was detected by genomic southern blot in the genome of Guyll or each of other 6 strains from different hosts.3. RT-PCR and MGTA1 (p)-eGFP fusion analysis showed that MGTA1 gene was expressed in hyphae tips, conidia and mature appressoria with different levels. Fluorescence of MGTA1-eGFP indicated MGTA1 protein probably locates in karyon.4. One hundred and seventy-seven hygromycin-resistance transformants were obtained by MGTA1 gene replacement vector construction and fungal transformation twice. Eight MGTA1 knock out mutants were identified by PCR and Southern blot, and a double insert transformant T48 included among which. 90% of MGTA1 coding regain was replaced in each △MGTA1 mutants. The probability of knock-out for each transformation was 6.67% and 2.30% respectively.5. The colony modality, growth rate and conidiation of most △MGTA1 (but T44) on different media were unchanged compared to Guy11. The conidiation of T44 reduced greatly on different media (more than one order of magnitude). And the conidiation of T90 also reduced with a lower degree than T44.6. The colony modality and growth rates of △MGTA1 mutants were unchanged compared to Guyl 1 under the condition of N starvation, C starvation or high temperature (33℃). The colony of T44 showed a slight fainter color than Guyl 1 on the media with fibrin as single C source.7. Conidia of △MGTA1 mutants were allowed to germinate and form appressoria on Terylene in water drops. △MGTA1 mutants germinated slower slightly than Guyl 1 but the final germinate percentages were unchanged.△MGTA1 mutants formed appressoria slower, a...
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