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For functional characterization of predicted genes encoding hypothetical proteins in fungal genomes, it is complementary to genetic studies to determine their expression and subcellular localization patterns in different developmental or infection stages. It is also important to identify and characterize other proteins that are physically associated with or functionally related to these genes in vivo by co-immunoprecipitation or affinity purification analyses. In this chapter, we described a set of yeast shuttle vectors and protocols to generate fusion constructs by the yeast gap repair approach. Because of the simplicity and efficiency of yeast gap repair, these vectors and the general methods described in this chapter are suitable for functional genomics studies in filamentous fungi.

Pathogenic fungi cause diseases on various organisms. Despite their differences in life cycles, fungal pathogens use well-conserved proteins and pathways to regulate developmental and infection processes. In this review, we focus on Rack1, a multifaceted scaffolding protein involved in various biological processes. Rack1 is well conserved in eukaryotes and plays important roles in fungi, though limited studies have been conducted. To accelerate the study of Rack1 proteins in fungi, we review the functions of Rack1 proteins in model and pathogenic fungi and summarize recent progress on how Rack1 proteins are involved in fungal pathogenesis. [ABSTRACT FROM AUTHOR]

Various surface signals are recognized by M agnaporthe oryzae to activate the Pmk1 MAP kinase that is essential for appressorium formation and invasive growth. One of upstream sensors of the Pmk1 pathway is the MoMsb2 signalling mucin. However, the activation of MoMsb2 and its relationship with other sensors is not clear. In this study, we showed that the cleavage and transmembrane domains are essential for MoMsb2 functions. Cleavage of MoMsb2 was further confirmed by western blot analysis, and five putative cleavage sites were functionally characterized. Expression of the extracellular region alone partially rescued the defects of M omsb2 in appressorium formation and virulence. The cytoplasmic region of MoMsb2, although dispensable for appressorium formation, was more important for penetration and invasive growth. Interestingly, the M omsb2 cbp1 double mutant deleted of both mucin genes was blocked in Pmk1 activation. It failed to form appressoria on artificial surfaces and was non-pathogenic. In addition, we showed that MoMsb2 interacts with Ras2 but not with MoCdc42 in co-immunoprecipitation assays. Overall, results from this study indicated that the extracellular and cytoplasmic regions of MoMsb2 have distinct functions in appressorium formation, penetration and invasive growth, and MoMsb2 has overlapping functions with Cbp1 in recognizing environmental signals for Pmk1 activation. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, MADS-box genes are known to play major regulatory roles in various biological processes by combinatorial interactions with other transcription factors. In this study, we functionally characterized the FgMCM1 MADS-box gene in F usarium graminearum, the causal agent of wheat and barley head blight. Deletion of FgMCM1 resulted in the loss of perithecium production and phialide formation. The F gmcm1 mutant was significantly reduced in virulence, deoxynivalenol biosynthesis and conidiation. In yeast two-hybrid assays, FgMcm1 interacted with Mat1-1-1 and Fst12, two transcription factors important for sexual reproduction. Whereas F gmcm1 mutants were unstable and produced stunted subcultures, F gmcm1 mat1-1-1 but not F gmcm1 fst12 double mutants were stable. Furthermore, spontaneous suppressor mutations occurred frequently in stunted subcultures to recover growth rate. Ribonucleic acid sequencing analysis indicated that a number of sexual reproduction-related genes were upregulated in stunted subcultures compared with the F gmcm1 mutant, which was downregulated in the expression of genes involved in pathogenesis, secondary metabolism and conidiation. We also showed that culture instability was not observed in the F vmcm1 mutants of the heterothallic F usarium verticillioides. Overall, our data indicate that FgMcm1 plays a critical role in the regulation of cell identity, sexual and asexual reproduction, secondary metabolism and pathogenesis in F . graminearum. [ABSTRACT FROM AUTHOR]