The Role Of Isocitrate Lyase Gene(ICL1)in The Pathogenicity Of Magnaporthe Grisea

Magnaporthe grisea is best known as the causal agent of theeconomically important blast disease of rice worldwide. A betterunderstanding of the molecular basis of this disease is not onlybeneficial for rice blast control, but also can serve as a model forunderstanding other fungal-plant interactions. Obviously, theidentification of genes required for pathogenicity is a key step towardachieving this goal. The fungus elaborates a specialized infection cellcalled an appressorium to penetrate the rice cuticle mechanically. Togenerate mechanical force, appressoria produce enormous hydrostaticturgor by accumulating molar concentration of glycerol. How glycerol issynthesized in such large amounts in M. grisea appressorium is anintriguing question. Previous studies on cytobiology and enzymaticactivities showed glycerol generation from lipids in a conidium istheoretically possible. Glyoxylate cycle is involved in the pathway ofglycerol synthesis from fatty acid. Isocitrate lyase(ICL) is a key enzymein glyoxylate cycle, catalyzing the conversion of isocitrate toglyoxylate and succinate, which generate glycerol by gluconeogenesispathway. In this study, M. grisea ICL1 gene encoding isocitrate lyase wascloned and the role of ICL1 gene in pathogenicity was analyzed by targetedgene replacement. The results are showed as follows:1. Two primers designed by conserved oligonucleoitides of isocitratelyase genes from other organisms were used for PCR amplification withM. grisea Guyll genomic DNA as template. The expected 340bp product wasobtained. By using the PCR product as a probe, two positive clones wereisolated from M. grisea Guyll genomic DNA library and conidial cDNAlibrary respectively. Both the ICL1 genomic DNA included promoter regionand open reading frame and cDNA clones were sequenced. The results showedM. grisea ICL1 gene contain 5 exons and 4 introns, and encode 547 predicted amino acids. The ICL1 protein sequence was very high homologous to the proteins of Neurospora crassa(acu3) and Aspergillus nidulans(acufl), 85.5% and 76.3% respectively. The molecular weight of ICL1 protein was about 60KD and similar to that of acu3 and acuD. But acu3 or acuD only had 2 introns witch were similar positions with the first 2 introns of ICL1 gene. Moreover, single copy of ICL1 gene in M. grisea was showed by Southern blot.2. Fifty-five transformants were obtained by fungal transformation with ICL1 gene replacement vector. Three ICL1 gene knock out mutants were identified by both PCR and Southern blot. These A ICL1 mutants could not grow and conidate on the medium with olive oil or sodium acetate as the sole carbon source. This result showed the acetyl-CoA from fatty acid JJ-oxidation could not feed glyoxylate cycle for further metabolism by these A ICL1 mutants, i.e. the fat metabolism had been blocked.3. The colonies of A ICL1 mutants on CM plates showed grey-white and different to the grey-dark colour of Guy 11, and the conidiation of these AICL1 mutants were also reduced. But the growth rate between AICL1 mutants and Guy 11 was no different. Moreover, the results of germination test on plastic coverslips showed the conidial germination and appressorium formation of A ICL1 mutants were delayed compared to Guyll, and the germ tubes of A ICL1 mutants were usually much longer than those of Guy11. Meanwhile the appressorium formation rate of AICL1 mutants was also reduced.4. Conidia of A ICL1 mutants were allowed to germinate on plastic coverslips in water drops, and the water was removed at different intervals during germination and stained with Nile Red for the presence of lipid droplets. The results showed the lipid mobilization of A ICL1 mutants seemed slower than that of Guyll. Furthermore, theappressorium turgor at intervals of 24h and 48h period was measured by a cytorrhysis assay. We found the turgor pressure of A ICL1 mutants was reduced, but this reduction was not significantly high. These preliminary data showed l...