Morphological and expression pattern analyses among wild type, <i>hxk1</i> and different filamentation pathway mutants.

<p>A) Morphology of wild type, filamentation pathway mutants under different hypha-inducing conditions. The starved cells of wild type (<i>HXK1</i>/<i>HXK1</i> ) and <i>hxk1</i> mutant<i>(hxk1/hxk1), ras1/ras1, ras1/ras1 hxk1/hxk1, efg1/efg1, efg1/efg1 hxk1/hxk1, cph1/cph1, cph1/cph1 hxk1/hxk1, tpk2/tpk2, tpk2/tpk2 hxk1/hxk1l</i> and their respective complemented strains in which one functional copy of <i>HXK1</i> is reintroduced in the native locus were resuspended in non-inducing and various induction media such as 2.5 mM GlcNAc in salt base, Spider pH 7.2 and Serum (20%). Cells were induced at 37°C for 4 hours in case of GlcNAc, for 2 hours at 37°C in case of Spider and Serum and in case of YPD for 2 hours at 30°C. <i>hxk1</i> mutants except <i>efg1/efg1 hxk1/hxk1</i> were hyperfilamentous in filamentation inducing conditions like Spider and Serum. In response to GlcNAc, all <i>hxk1</i> mutants failed to form germ tubes due to its inability to catabolize the sugar, but the complemented strains were hyper filamentous. B) Expression of hyphal specific genes in different filamentation inducing media in filamentation pathway mutants. Northern blot analysis to show the expression of Hyphal Specific Genes (HSGs) like <i>HWP1, ECE1</i> or <i>RBT4</i> for the cells grown in various filamentation inducing media. Total RNA was isolated from wild type, CAF2–1 (<i>HXK1</i>+/<i>HXK1</i> ) and <i>hxk1</i> mutant (<i>hxk1/hxk1</i>), <i>ras1/ras1, ras1/ras1 hxk1/hxk1, efg1/efg1, efg1/efg1 hxk1/hxk1, cph1/cph1, cph1/cph1 hxk1/hxk1, tpk2/tpk2, tpk2/tpk2 hxk1/hxk1</i> strains induced in YPD, GlcNAc, Spider and 20% Serum. 20 µg of RNA was loaded in each lane and a Northern blotting was performed. As an internal control methylene blue staining of rRNA was used to ensure equal loading of RNA. <i>hxk1</i> mutants except <i>efg1/efg1 hxk1/hxk1</i> showed upregulation of HSGs in filamentation inducing conditions and upregulation was more prominent in Spider medium. C) Quantification of <i>HXK1</i> mediated filamentation : q-RT PCR analysis of HSGs (<i>ECE1</i>, <i>HWP1</i>and <i>RBT4</i>) in <i>hxk1</i> single and double mutants (<i>ras1 hxk1</i>, <i>efg1hxk1</i>, <i>cph1hxk1</i> and <i>tpk2 hxk1</i>) in Spider grown cells compared to their wild type and respective single mutants (<i>ras1</i>, <i>efg1</i>, <i>cph1</i> and <i>tpk2</i>). Comparative expression levels of HSGs for cells grown in YPD have also been shown. <i>ACT1</i> has been selected as the endogenous control. The error bars represent co-efficient of variation.