Differential localization of mRNA using laser microdissection in the polarized hyphal tip of Fusarium oxysporum

The establishment and maintenance of a stable polar axis is critical hyphal growth in filamentous fungi. The hyphal tip of Fusarium oxysporum , like all filamentous fungi, is a polarized growth form that is approximately 150 microns long and 7 microns wide. The actively growing end of the hyphal tip has a considerable accumulation of vesicles, ribosomes and mitochondria and other organelles that are predominantly involved with, sensing their local environment, secretion of cell wall materials and subsequent nutrient uptake. Interestingly, nuclei are located from 75 to 100 microns from the hyphal tip which is a substantial distance for diffusion alone to explain how many relevant proteins are efficiently positioned to support rapid fungal tip growth. Thus, it is hypothesized that certain mRNA transcripts will be differentially localized in this highly polarized cell type. The objective of this study was to investigate differential mRNA localization of Fusarium hyphal tips to better understand the fundamental basis of cell polarity for normal cell growth and disease of this important plant and human pathogen. This study will contribute to our knowledge on what proteins are most critical in the highly specialized hyphal tip region allowing invasion of host organisms as well as degradation of non-living organic material. To better understand the regulation of protein synthesis via mRNA localization to targeted areas of the hyphal tip cell, we quantified relative amounts of message using laser capture microdissection followed by quantitative real time polymerase chain reaction The results reported here, demonstrate that differential localization of actin (tip high) and tubulin (tip low) mRNA within the leading cell of hyphae and supported the hypothesis. In addition, the feasibility of laser capture microdissection to study small and even subcellular domains for fungal expression profiles was demonstrated which will be instrumental future studies on genes involved in pathogenesis and hyphal tip structure/function.