Genetic Dissection of T-DNA Insertional Mutants Reveals Uncoupling of Dikaryotic Filamentation and Virulence in Sugarcane Smut Fungus

The biotrophic basidiomycetous fungus Sporisorium scitamineum causing smut disease in sugarcane is characterized by a life-cycle composed of a yeast-like nonpathogenic haploid basidiosporial stage outside the plant and filamentous pathogenic dikaryotic hyphae within the plant. Under field conditions, dikaryotic hyphae are formed after mating of two opposite mating-type strains. However, the mechanisms underlying genetic regulation of filamentation and its association with pathogenicity and development of teliospores are currently unclear. This study has focused on the characterization and genetic dissection of haploid filamentous mutants derived from T-DNA insertional mutagenesis. Our results support the existence of at least three genotypes among the six haploid filamentous mutants that differentially contribute to virulence and development of the whip and teliospore, providing a novel foundation for further investigation of the regulatory networks associated with pathogenicity and teliospore development in S. scitamineum.

Agrobacterium Tumefaciens-mediated T-DNA Insertional Mutagenesis of Fusarium Oxysporum

BackgroundFusarium species are important pathogenic organisms, which can cause many diseases in plants and humans. Characterizing the mechanism underlying their pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated genetic transformation has been widely used for the molecular analysis of many species. ResultsIn this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and efficient procedure for the transformation of resistant Fusarium oxysporum mediated by A. tumefaciens. The transformation efficiency was as high as 250 mutants per 104 conidia. A total of 1,450 stably transformed mutants were generated, resulting in a small-scale library of F. oxysporum mutants containing T-DNA tags. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. Additionally, the sequences flanking the inserted T-DNA were obtained by touchdown-TAIL PCR, the insertion sites and genes associated with the phenotypic changes could be determined.ConclusionsThe developed method may enable to analyze gene functions and study biological characteristics, which will lay the foundation for future analyses of the mechanism underlying F. oxysporum pathogenicity and resistance. Furthermore, it may be applicable to investigations of other important pathogenic fungi.

Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly

The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.

Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling inMagnaporthe oryzae

High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity inMagnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity inM. oryzaeidentified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected.