Analysis Of The Roles Of Splicing Factor Branchpoint Binding Protein In Development Of Rice Blast Fungus

Rice is one of the most important food crops of the world and the main staple foodfor almost a half of the world’s population. Asia which has55%of world’s populationproduces and consumes almost90%of the rice mainly in China and India.International Food Policy Research Institute predicts that by the year2030riceproduction will need to increase by38%due to rapid population growth in these mainrice-growing and consuming regions. One of the major berriers to rice production isblast disease caused by the fungus Magnaporthe oryza. Blast disease, which threatensthe average rice need and food security, is a problem in almost everywhere in theworld. To date much is known about this rice pathogen such as conidia morphology,appressoria melanization, spore tip mucilage secretion and the spore penetration.Recent completion of genomic sequence of this pathogen has provided theresearchers with the advantages to deduce the functions of individual or sets of genesand link with the growth, development and pathogenicity. Some of these genes havebeen found to play parts in various molecular processes such as cyclic-AMPmolecular pathways, mitogen-activated protein kinases （MAPK） pathways andpre-mRNA splicing pathways. The interaction of these molecular events in the fungusitself and between the fungus and the host plant finally lead to disease. It is expectedthat by disruption of some of these events at some point will lead the fungus to fail tocolonize on the hosts tissues and open the way to disease control. Because of therelative importance of the pre-mRNA splicing in multi-intronic eukaryotic geneexpression, we have attempted to elucidate the role of branchpoint binding protein（BBP） as one of the most important splicing proteins, in disease development. BBP from M. oryzae （MGG₀6748） is identified as a putative splicing factor with itsorthology-a mammalian splicing factor1（SF1）, which is a member of the STARfamily and participates in the assembly of the spliceosomal E complex. The apparentsimilarities of the BBP/SF1among eukaryotes permits the researchers to propose itscontribution to the pre-mRNA splicing and hence gene expression in M. oryzae.Therefore, the aim of this work was to deduce the role of this putative splicingfactor BBP in disease development process using gene deletion technique and RNAinterference.1. Knockout vector made up of1165bp each left and right flanks spaced byHygromycin single insert marker driven by trpc promoter wasconstructed and used for gene deletion but no knockout mutants obtained.This failure to obtain knockout mutants can be explained by lethality of agene or inaccessible locus due to DNA methylation or presence ofrepetitive sequences and heterochromatin.2. M. oryzae transformants expressing sense and others expressing hairpinRNA constructs of splicing factor BBP gene were created.3. The effect of the individual RNA interference species constructs invegetative growth and sporulation were evaluated. The result with RNAinterference techniques did not give any differences between the wildtype and transformants carrying sense and hairpin RNAi species of theBBP. It is suggested that the RNA interference of the BBP gene was notachieved due to inappropriate part of a gene selected for producinginterference effect.