Functional Validation Of Candidate Pathogenicity-related Genes In Magnaporthe Oryzae

Rice blast is one of the three major diseases of rice caused by Magnapothe oryzae, which severely threatens rice production. Breeding long-lasting disease-resistant cultivars and finding out more efficient disease control strategies are therefore urgently required for rice production. For this purpose, it is necessary to deeply research the molecular mechanisms of the disease resistance of rice and the pathogenicity of M. oryzae. In recent years, many candidate pathogenicity-related genes of M. oryzae have been identified using various approaches such as T-DNA insertional mutagenesis, microarray, suppression subtractive hybridization (SSH) and quantitative trait locus (QTL) mapping, providing important information for deep research on the molecular mechanisms of pathogenicity in M. oryzae. However, the exact functions of these genes still need to be verified individually, and the reliability of the methods has yet to be assessed. For this purpose, in this study, the candidate pathogenicity-related genes obtained by different approaches were compared and the functions of some candidate genes were validated by knocking them out using the method of target gene replacement. The main results are as follows:1. By comparing the results of QTL mapping and microarray expression profiling in M. oryzae,9 candidate pathogenicity-related genes were found previously. In this study,4 genes of these genes were knocked out, but no visible phenotypic changes on vegetative growth, asexual reproduction and infection ability in their knockout mutants, suggesting that these genes are not related to pathogenicity. The results also implies that the the success rate of finding pathogenicity-related genes may be very low based on the positional overlapping between QTLs and differentially expressed genes.2. Two lists of candidate pathogenicity-related genes were previously obtained by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes) in M. oryzae. There were two types of mutations: Type I is that a gene is disrupted by the T-DNA inserted inside it and therefore the gene's function is usually lost; Type II is that a gene remains intact in structure, but its function is possibly affected by the T-DNA inserted beside it. We compared the two gene lists and found that only 13 genes were overlapped between them, and most of these overlapped genes belonged to Type II mutation. For validating gene functions, we successfully knocked out four Type-II overlapped genes, but none of them exhibited visible changes on phenotype and pathogenicity. This suggests that there should be a large proportion of genes that were not related to pathogenicity in the two lists and the two gene lists were not positively correlated as expected. Hence, the candidate pathogenicity-related genes identified by T-DNA insertional mutagenesis and microarray expression profiling should be accepted cautiously before they have been confirmed by strict validation.3. A nuclear envelope protein gene MGG₀6001 was selected from the list of candidate pathogenicity-related genes identified by SSH in M. oryzae previously, and its function was validated by knocking it out. The growth rate in complete medium and conidiation of the mutants were much lower, whereas the ability of the pathogenicity, conidia germination and appressoria formation were normal. The results indicate that the nuclear envelope protein plays important roles in fungal growth and conidiation.