There’s a Fungus Among Us:

An Investigation of Endocytic Proteins

William Porter, Blake Commer, and Dr. Brian D. Shaw
Bioenvironmental Sciences, Department of Plant Pathology and Microbiology, Texas A&M University

Abstract
Aspergillus nidulans is a model organism for filamentous fungi. Little is known about
how fungi grow. They grow in a polarized fashion with the newest cells being formed
at the tip of finger-like projections known as hyphae. The current model of growth is
known as endocytic recycling where some proteins are involved in the endocytosis of
materials around the fungus and others place the endocytosed cargo at the tip,
creating new growth1. This research focused on the endocytic proteins involved in
this process using A. nidulans.
Using fusion polymerase chain reactions (PCR), we were able to select key proteins
thought to be involved with this process and tag them with Green Fluorescent Protein
(GFP). Using fluorescent microscopy, we could then identify localization patterns of
Figure 4. Localization of AN1909::GFP in
these proteins and assess whether or not they were likely to be associated with
the conidiophore. AN1909 localizes to the
endocytosis. In this research we will be testing the hypothesis that AN1909 is
leading edge of each cell type in the
associated with endocytic recycling.
Figure 3. Comparison of fluorescent conidiophore.
microscopy to bright field image of a
Research Objectives and Outcomes hypha. AN1909::GFP localizes in the
collar.
Objectives Methods Accomplishments
• Familiarize myself with • Design and prepare • Successfully completed Connections to Academic Knowledge
fungi and their roles as primers for fusion PCR many PCR reactions
plant pathogens • Amplify the PCR • Became familiar with • Increased my understanding of genetics
• Gain experience running fragment to insert the fungal development • Increased my understanding of fungi
PCR foreign DNA • Successfully tagged
– Life cycle
• Identify the role of • Transform through AN1909 with GFP
AN1909 in relation to protoplasting • We have supported our – Reproduction
endocytosis in A. • Document the localization hypothesis that AN1909 – Anatomy
nidulans of the protein in vivo is associated with
• Increased confidence with a microscope
• Extract DNA to verify endocytic recycling
GFP insertion at the • Learned Sterile Technique
native locus
Connections to Discipline
This project focused on genetics, biology, and plant pathology. Fungi are some of the most
devastating plant pathogens. By understanding the cell biology of fungi, we will be able to
better counter their roles as pathogens. The polarized tip growth is very specific to fungi. If
we are able to discover how this process works we will then be able to halt it, solving many
of the problems caused by fungi. Due to the use of PCR and genetic modification in this
project, an understanding of genetics is required.

Relationship to Career Goals

This research experience has prepared me for pursuing a graduate degree in plant
Figure 1. Time-lapse images of hyphal tip growth. AN1909::GFP localizes to the endocytic pathology. Working in the lab changed my career goals and I am now much more
collar. As the hypha grows the protein is retained in the collar. This localization supports the interested in pursuing research at higher levels.
hypothesis that AN1909 is associated with endocytosis. (time is in hh:mm:ss)

Figure 2. Hypha with

References
multiple lateral 1. Shaw, Brian D., Da-Woon Chung, Chih-Li Wang, Laura A. Quintanilla, and Srijana
branches. Upadhyay."A Role for Endocytic Recycling in Hyphal Growth." A Role for Endocytic
AN1909::GFP Recycling in Hyphal Growth. Elsevier, 19 Feb. 2011. Web. 24 Mar. 2017.
localizes in the collar
of all of the developing
hyphae. Acknowledgements
The scale bar = 5 μm
on all figures Sponsors for high impact experiences for BESC and the BESC poster
symposium include the Department of Plant Pathology and Microbiology, the
College of Agriculture and Life Sciences, the Office of the Provost and
Executive Vice President for Academic Affairs.