An effective in-gel assay protocol for the assessment of acid phosphatase (ACPase) isoform expression in the fungus Serendipita indica.

Abstract

The induction of acid phosphatase (ACPase) in the mycorrhizal fungi is an adaptive survival mechanism to cope in a low-phosphate environment. A mycorrhizal fungi Serendipita indica can induce the ACPase enzyme and enhance the phosphate (Pi) level to the host plant through Pi-solubilization mechanism, both intracellular and extracellular (media) levels. The spectrophotometer technique has been widely and commonly used to measure the ACPase enzyme activity in all microorganisms and plants using pNPP as a substrate. However, this technique cannot be useful when studying the involvement of ACPase isoforms in Pi-solubilization. In this article, we developed a single method to identify and express the ACPase isoforms of S. indica that contribute to the Pi-nutrition in the plant. This is native-PAGE electrophoresis with the in-gel assay and staining to detect the isoforms of the ACPase enzyme. The dark red-brown color developed after staining indicates the non-denatured (native) ACPase enzyme. This method utilized a modified minimal media for the de-repression of P-responsive genes such as ACPases with minimum salt contamination in the samples. This method will be helpful for the characterization of secretory and intracellular ACPases in fungi.