|
Thomas Smith's Laboratory |
Background:
Several strains of Ustilago maydis (corn smut fungus) and Saccharomyces cerevisiae (yeast) have been classified as 'killer' strains by their ability to kill similar strains of fungi in culture. This killer phenotype is caused by persistent infections of the host cells by double-stranded (ds) RNA virions. These virions are unusual in that they are not expressed externally but instead are transmitted vertically through basidiospores or horizontally through anastomosis. The multisegmented viral genome is encased by an unusual type of capsid composed of 120 copies of the envelope protein gag. In a collaboration with the Steven, Baker, and Wickner groups, we determined the structures of two of these viruses have been determined using cryo-electron microscopy and image reconstruction techniques (see the images below). With two identical copies of gag in each icosahedral asymmetric unit, these capsids were found to contradict Caspar and Klug's rules for icosahedral equivalency without any topological evidence of acceptable pseudo-symmetry such as P=4. It was speculated that these viral capsids do not act as protective carapaces but rather as cytoplasmic compartments for viral genome replication. Unlike most non-fungal viruses, some of these dsRNA fungal viruses have a symbiotic relationship with their host. These viruses are solely dependent upon host survival for replication, and therefore lend the host a selective advantage by encoding small toxins that are secreted by the host cell. The fungal toxins do not affect the host cell strain but do kill similar strains of fungi in that locale. The figure below shows the image reconstructions of the virus from Saccromyces (d and e) and Ustilago (f and g). These capsids are like 'mini nuclei' in that they are essentially holey cages that replicate the dsRNA genome inside the particle and extrude the RNA out through the holes.
|
 |
Structure:
The figure below on the left is a ribbon diagram of the entire KP4 and on the right is a stereo figure of the structure that can be viewed with red/blue stereo glasses. We have determined the atomic structure of KP4 to 1.9Å resolution. KP4 belongs to the alpha/beta-sandwich family, and has a unique topology comprising a five-stranded antiparallel beta-sheet with two antiparallel allpha-helices lying at ~45° to these strands. The structure has two left-handed cross-overs and a basic protuberance extending from the P-sheet. In vivo experiments demonstrated abrogation of toxin killing by Ca2 + and, to a lesser extent, Mg2+. These results led to experiments demonstrating that the toxin specifically inhibits voltage-gated Ca2+ channels in mammalian cells. Similarities, although somewhat limited, between KP4 and scorpion toxins led us to investigate the possibility that the toxic effects of KP4 may be mediated by inhibition of cation channels. Our results suggest that certain properties of fungal Ca2+ channels are homologous to those in mammalian cells. KP4 may, therefore, be a new tool for studying mammalian Ca2+ channels and current mammalian Ca2 + channel inhibitors may be useful lead compounds for new anti-fungal agents.
|
|
 |
KP4 effects on fungus:
From the structural results we thought that KP4 might be acting via calcium channels. To this end we performed a Ustilago killing assay (to the right). The toxin was placed on the white filter blots and incubated with plates of sensitive strains of Ustilago (P2). The dark clearing zone around the white filter blots shows killing. Calcium and to a lesser extent magnesium blocked KP4 action. We followed this up with more accurate growth curve experiments and calcium uptake experiments.
|
 |
Effects of KP4 on L-type calcium channels:
Shown here are whole cell calcium current measurements on animal cells expressing different types of voltage gated calcium channels. Note the remarkable specificity that KP4 has on L-type channels (panel A). Interestingly, similar results were found with the plant defensin Def1 that is half the size of KP4 and belongs to the scorpion toxin family.
|
|
Effects on Plant Roots.
Calcium signaling is known to be important for plant root and root hair growth and development. Interestingly, we found that KP4 and the plant defensins all affect plant root growth. Also in all cases, calcium was found to abrogate this inhibition of root growth. These antifungal agents may affect calcium directly or indirectly.
|
 |
Effects on root hairs:
A finer analysis of the effects of KP4 and the plants defensins was performed by looking at the growth of root hairs. Here we monitored the polarization of a Rab GTPase/GFP fusion in the presence and absence of these antifungal agents. All were very potent at first depolarizing the extending root hair and then stopping its growth. Interestingly, Def2 is a weak antifungal agent and yet is quite effective at inhibiting root hair extension. Could these proteins be doing more in the plant than only protecting against fungal infections?
|
 |
Our Relevant Publications:
-
Allen, A., Snyder, A. K., Preuss, M., Nielsen, E. E., Shah, D. M., Smith, T. J. (2008) Plant and virally encoded fungal toxins inhibit plant root growth. Planta. 227: 331-339.
-
Spelbrink, R., Dilmac, N., Allen, A., Smith, T. J., Shah, D. M., Hockerman, G. (2004) The plant defensin from alfalfa blocks a mammalian calcium channel. Plant Physiol. 135:2055-2067.
-
Gage, M. J., Bruenn, J., Fisher, M., Sanders, D., Smith, T. J. (2001) KP4 fungal toxin inhibits growth in Ustilago maydis by blocking calcium uptake. Mol. Microbiol 41(4):775-786.
-
Gage, M. J., Rane, S. G., Hockerman, G. H., Smith, T. J. (2002) The virally encoded fungal toxin KP4 specifically blocks L-type voltage-gated calcium channels. Mol. Pharmacol. 61:936-944.
-
Gu, F., Khimani, A., Rane, S. G., Flurkey, W. H., Bozarth, R. F., Smith, T. J. (1996) The Structure of a Virally Encoded Fungal Toxin from Ustilago maydis that Inhibits Fungal and Mammalian Ca2+ Channels. In Protein Toxin Structure . Springer, New York (M. Parker, ed.) pp.291-303.
-
Gu, F., Khimani, A., Rane, S. G., Flurkey, W. H., Bozarth, R. F., Smith, T. J. (1995) Structure and function of a virally encoded fungal toxin from Ustilago maydis: a fungal and mammalian Ca2+ channel inhibitor. Structure 3:805-814.
-
Cheng, R.H., Caston, J.R., Wang, G., Gu, F., Smith, T.J., Baker, T.S., Bozarth, R.F., Trus, B.L., Cheng, N., Wickner, R.B., and Steven, A.C. (1994) Fungal virus capsids: cytoplasmic compartments for the replication of double-stranded RNA formed as icosahedral shells of asymmetric Gag dimers. J. Mol. Biol. 244:255-258.
-
Gu, F., Sullivan, T.S., Che, Z., Ganesa, C., Flurkey, W.H., Bozarth, R.F., and Smith, T.J. (1994) The characterization and crystallization of a virally encoded Ustilago Maydis KP4 toxin. J. Mol. Biol. 243:792-795.
|
|
