Identification And Functional Analysis Of A Novel Disease-related Gene MgLDB1 In Magnaporthe Grisea

Rice blast is one of important plant diseases, and its causal agent is Magnaporthe grisea, attributed to ascomycete. A better understanding of molecular basis of this disease is not only beneficial for rice blast control, but also can serve as a model for understanding other plant-pathogenic fungus interaction. T-DNA inserting mutation is effective method to discover disease-related genes. In this study, we discovered a novel disease-related gene, MgLDB1 by T-DNA insertion, then to sudy MgLDB1 function by gene knockout and complement mutation, and found this ubiquitous nuclear-located gene played an essential role on colony growth, conidiation, sexual reproduction, pathogenicity, and cell integration. These results are showed as follows:1 Obtainning a non-pathogenicity mutant, A2-12-3. 3600 transformants were gained by ATMT transformation of M .grisea, The result suggested that 4 transformants, A1-3-9, A1-17-9, GPD1-1, and A2-12-3 lose pathogenicity after pathogenicity assay on barley leaves, and that mutant A2-12-3 cannot product conidia on CM, MM, oatmeal, and barley media.2 To identify T-DNA tagged gene of mutant A2-12-3. After Amplifying and sequencing the flanking sequence of T-DNA, it showed that T-DNA inserted fourth exon of hypothetical protein gene, MG01057.5, composed of 7 exons and 6 introns. Alignment of amino acid sequence suggested low identity among these identical genes, and that the high identity regions were located in 400-750 amino acid of MgLDB1, where there are many conserved amino acid residues which maybe relate to LIM-bind domain. In addition, there was a KRRPS....PRMQKR amino acid cascades, which maybe identify the nuclear located sequence.3 The function of MgLDB1 gene. A series of biological characters of T-DNA inserting mutant A2-12-3 and deletion mutant AK58 showed they shared very similar biological phenotypes and were distinctive with wild type Guy11. The following were: (1) Colony morphology, mutants showed dark-green and slow growth rate. (2) Conidium formation and sexual reproduction Conidium formation of MgLDB1 mutants is disrupted on CM, MM, barley, and oatmeal media, so is sexual reproduction on oatmeal medium. (3) Losing or weakenning pathogenicity On leaves of barley and rice leaves with wound and unwounded, respectively, MgLDB1 mutants completely lost pathogencity on barley leaves with/without wounded, and on unwounded rice leaves, whereas there were lightly pathogenicity on wounded rice leaves. (4)Cell integration enhancement of MgLDB1 mutants Mutants can survive on CM media with 0.04%SDS, however, wild type Guy11 cannot survive on 0.02%SDS; Calcofluor white inhibit Guy11 growth rate on CM with 200ug/ml more seriously than Mutants'; Inhibition of cango red to Guylland mutants wasnot significant; With more protoplast product of Guy11 than mutants' after glucanex disgestion. These suggested mutants enhance the cell integrality. (5) At last, similar growth trends under different osmotic pressure between mutants and Guy11 suggested MgLDB1 gene play little role on regulating osmotic pressure. All these showed MgLDB1 gene regulated a series of important function in M.grisea4 Temporal and Spatial expression of MgLDB1 gene. After observation of fusion expression, MgLDB1-GFP, and of nucleus Stained with DAPI in conidia and mycelia by laser scanning confocal microscope, fusion expression protein was located in nucleus, and this result was consistent with LDB1 reported in animals. In the study of temporal expression of MgLDB1 gene, it expressed during mycelia growth, conidiation, conidium germination, and appressorium formation. These results suggested this gene expressed in different development phases and regulated cell determination and development.5 Complement mutant of MgLDB1. After transformed the protoplast of A2-12-3 with pCB1532-MgLDBl-GFP fusion expression vector (known as complement vector), We obtained 10 complement mutant with GPF expression. As a result, complement mutants restored the wild phenotypes of mutants, such as colony morphology, growth rate, conidia, sexual reproduction, and pathogenicity. These showed MgLDB1 gene regulated diverse biological phenotypes of M.grisea.In summary, MgLDB1 gene played an essential role on colony growth, conidiation, sexual reproduction, pathogenicity, and cell integration.