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Our interest in HDACs springs from the discovery that HC-toxin, a critical virulence determinant for Cochliobolus carbonum, is a potent and specific inhibitor of HDACs from maize and other organisms. This has led us to study HDAC function in filamentous fungi and plants, especially in
relation to pathogenesis.We are working on HDACs to address the following four questions. This work is being done with our collaborators Peter Loidl, Gerald Brosch, and Stefan Graessle at the University of Innsbruck, Austria.
1. Why does inhibition of maize HDACs permit the development of disease?
C. carbonum is an exceptionally virulent pathogen on susceptible maize (Figure 1). The cyclic tetrapeptide HC-toxin plays the central role in this disease: Maize that is insensitive to HC-toxin is resistant to C. carbonum, and isolates of C. carbonum that do not make HC-toxin are avirulent. Unlike other host-selective toxins, however, HC-toxin is not toxic to plant (or animal) cells (Wolf and Earle, 1991). Thus, simple killing of its host cannot explain the exceptional virulence of C. carbonum.
HC-toxin is a specific inhibitor of histone deacetylases of the RPD3/HDA1 classes (Brosch et al., 1995). This class of HDAC is found in all eukaryotes including yeast, mammals, and plants. Clearly, HDACs have a a critical role in defense of plants (at least maize) against pathogens. One hypothesis is that HC-toxin is a suppressor of HDAC-regulated defense responses (Brosch et al., 1995), but, if so, the nature of those responses is not known.
Understanding the role of HC-toxin in the disease process depends on our understanding of HDAC function in general. This has proven to be a ‘moving target’. First, plants, like other organisms, have multiple HDACs of the RPD3/HDA1 class (at least 15 in Arabidopsis and maize), and all are apparently sensitive to HC-toxin. Second, HDACs have many substrates. In addition to histones, HDACs can deacetylate other proteins including a number of transcription factors such as NF-kappaB (Chen et al., 2001). Tubulin is also a substrate for a specific human HDAC.
Figure 1. Cochliobolus carbonum infecting maize in the field. The genotype of the (dead) maize in the foreground is hm1/hm1. Photograph taken by Guri Johal at Purdue University in 2003.
2. What virulence functions in C. carbonum are regulated by HDACs?
One of the HDACs of C. carbonum, HDC1, is required for full virulence. An hdc1 mutant shows reduced growth on alternate carbon sources, reduced expression of cell wall degrading enzymes, and reduced leaf penetration efficiency (Baidyaroy et al., 2001) (Figure 2). What are the critical virulence factors controlled by HDC1 - are they cell wall degrading enzymes, either individually or collectively, or perhaps unrelated biochemical processes?
Figure 2. Reduction in virulence of the hdc1 mutant. On the far left in both panels are maize (genotype hm1/hm1) sprayed with wild type (HDC1). The other leaves and plants were sprayed with independent hdc1 mutants. Note that the wild type kills the plants within one week whereas the mutants do not (panel B).
From Baidyaroy et al. 2001.
Figure 3. Conidia of wild type (A and C) and hdc1 mutant conidia (B and D). Note that the hdc1 conidia are smaller and shorter. However, they germinate as well as wild type (D). Although ccsnf1 and hdc1 mutants share many phenotypes (reduced virulence, reduced growth on alternate carbon sources, reduced expression of cell wall degrading enzymes), the hdc1 but not the ccsnf1 mutants show this altered conidial morphology. From Baidyaroy et al., 2001.
Figure 4. Scanning EM micrographs of the behavior of hdc1 conidia on corn leaves. All of the conidia have germinated and formed normal appressoria. Three (A,B, and D) have formed appressoria over the junctions between cells, which is normal.
The phenotypes of the hdc1 mutant of C. carbonum is strikingly similar to those of a ccsnf1 mutant. ccSNF1 encodes a protein kinase that is required for expression of glucose-repressed genes, which includes most of the cell wall degrading enzymes of C. carbonum (Tonukari et al., 2000).There are several regulatory points at which Snf1 and HDACs converge in yeast. For example, Snf1 phosphorylates and thereby inactivates Mig1 (known as CreA in filamentous fungi) under conditions of low glucose. Under high glucose (repressing) conditions, Mig1 recruits the general repressor complex Ssn6/Tup1, which in turn recruits several HDACs (Watson et al., 2000; Wu et al., 2001). Furthermore, under inducing conditions, Snf1 also contributes to gene activation by phosphorylating histone H3, thereby permitting subsequent acetylation by the histone acetyltransferase Gcn5 (Lo et al., 2001). In both of these processes histone acetylation is associated with gene activation and deacetylation with repression, which is not consistent with our observed gene down-regulation in the hdc1 mutant of C. carbonum (Baidyaroy et al., 2001). Therefore, to date the yeast models have not appeared to be applicable to C. carbonum.
3. How does C. carbonum protect itself against its own potent HDAC inhibitor?
Having HDACs, the fungus must have a mechanism to keep from killing itself when making HC-toxin. We have discovered that whereas the HDAC activity of most other organisms, including Aspergillus nidulans and Neurospora crassa, are fully sensitive to HC-toxin and trichostatin, the HDAC activities of some C. carbonum isolates are resistant.
Anion exchange fractionation of the HDAC activity of filamentous fungi reveals two major peaks of activity, one that is resistant and one that is sensitive to HC-toxin (Brosch et al., 2001). There is evidence for both intrinsic and extrinsic resistance mechanisms. For example, extracts from
resistant C. carbonum can protect HDAC activity in extracts from sensitive strains (Baidyaroy et al., 2002).
4. Is HDAC inhibition by plant pathogenic fungi a general virulence strategy?
A number of fungi make HDAC inhibitors. Several of them are, or are closely related to, plant pathogens (Figure 5). The existence of these HDAC inhibitors raises the intriguing possibility that HDAC inhibition is a virulence strategy not restricted to C. carbonum.
Figure 5. Other HDAC inhibitors made by other filamentous fungi. All four are plant pathogens.
References on HDACs and HC-toxin:
Baidyaroy, D., G. Brosch, J.-H. Ahn, S. Grassle, S. Wegener, O. Caballero, P. Loidl, and J.D. Walton (2001) A gene related to yeast HOS2 is necessary for extracellular depolymerase statement and virulence in a plant pathogenic fungus. Plant Cell 13:1609-1624.
Baidyaroy, D., G. Brosch, S. Graessle, P. Trojer, and J.D. Walton (2002) Characterization of inhibitor-resistant histone deacetylase activity in plant-pathogenic fungi. Eukaryotic Cell 1:538-547.
Brosch, G., R. Ransom, T. Lechner, J.D. Walton, and P. Loidl (1995) Inhibition of maize histone deacetylase by HC-toxin, the host-selective toxin of Cochliobolus carbonum. Plant Cell 7:1941-1950.
Brosch, G., M. Dangl, S. Graeesle, A. Loidl, P. Trojer, E.-M. Brandtner, K. Mair, J.D Walton, D. Baidyaroy, and P. Loidl (2001) An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum. Biochemistry 40:12855-12863.Graessle, S., M. Dangl, H. Haas, K. Mair, P. Trojer, E.-M. Brandtner, J.D. Walton, P. Loidl, and G. Brosch (2000) Characterization of two putative histone deacetylase genes from Aspergillus nidulans. Biochim. Biophys. Acta 93405:1-7.
Lechner, T., A. Lusser, A. Pipal, G. Brosch, A. Loidl, M. Goralik-Schramel, R. Sendra, S. Wegener, J. D. Walton, and P. Loidl (2000) RPD3-type histone deacetylases in maize embryos. Biochemistry 39:1683-1692.
Ransom, R.F., and J.D. Walton. (1997) Histone hyperacetylation in maize in response to treatment with HC-toxin or infection by Cochliobolus carbonum. Plant Physiol. 115:1021-1027.
Wolf S.J., and Earle, E.D. (1991). Effects of Helminthosporium carbonum race 1 toxin on host and non-host cereal protoplasts. Plant Sci. 70: 127-137.
Some HDAC papers from other labs:
Yoshida, M., Kijima, M., Akita, M., and Beppu, T. (1990) Potent and specific inhibition of
mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265,
17174-17179.Vidal, M., and Gaber, R.F. (1991) RPD3 encodes a second factor required to achieve
maximum positive and negative transcriptional states in Saccharomyces cerevisiae. Mol. Cell.
Biol. 11, 6317-6327.Kijima, M., Yoshida, M., Suita, K., Horinouchi, S., and Beppu, T. (1993) Trapoxin, an
antitumor cyclic tetrapeptide, is an irreversible inhibitor of mammalian histone deacetylase. J.
Biol. Chem. 268, 22429-22435.Kwon, H.J., Owa, T., Hassig, C.A., Shimada, J., and Schreiber, S.L. (1998) Depudecin
induces morphological reversion of transformed fibroblasts via the inhibition of histone
deacetylase. Proc. Natl. Acad. Sci. U.S.A. 95, 3356-3361Selker, E.U. (1998) Trichostatin A causes selective loss of DNA methylation in Neurospora.
Proc. Natl. Acad. Sci. U.S.A. 95, 9430-9435.Bernstein, B.E., Tong, J.K., and Schreiber, S.L. (2000) Genome wide studies of histone
deacetylase function in yeast. Proc. Natl. Acad. Sci. U.S.A. 97, 13708-13713.Watson AD, DG Edmondson, JR Bone, Y Mukai, Y Yu, W Du, DJ Stillman, SY Roth (2000)
Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Genes Develop
14:2737-2744.Wu, J., Suka, N., Carlson, M., and Grunstein, M. (2001) TUP1 utilizes histone
H3/H2B-specific HDA1 deacetylase to repress gene activity in yeast. Mol. Cell. 7, 117-126.Deckert J, K Struhl (2001) Histone acetylation at promoters is differentially affected by specific
activators and repressors. Mol Cell Biol 21:2726-35.Jenuwein, T., and C.D. Allis (2001) Translating the histone code. Science 293:1074-1080.
Lo, W.-S., L. Duggan, N.C. Tolga Emre, R. Belotserkovskya, W.S. Lane, R. Shiekhattar,
S.L. Berger (2001) Snf1 - a histone kinase that works in concert with the histone
acetyltransferase Gcn5 to regulate transcription. Science 293:1142-1146.Chen, L.-F., W. Fischle, E. Verdin, W.C. Greene (2001) Duration of nuclear NF-kappaB
action regulated by reversible acetylation. Science 293:1653-57.
Revised by JW 9/19/04
