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Filamentous fungi have external stomachs. That is, their major mode of obtaining nutrients is to secrete hydrolytic enzymes and then absorb the products through the plasma membrane.The ability of fungi to grow on almost anything is notorious. This is because they can degrade virtually every known polymer - nucleic acids, proteins, lipids, polysaccharides, etc., even 'inert' subtrates like plastic and rubber.
To do this, fungi make and secrete a huge variety of extracellular depolymerases.
Plant pathogenic fungi make a variety of enzymes that can degrade the polymers of the plant cell wall.
We are studying the role, if any, of these cell wall degrading enzymes (CWDEs) in the disease process.We have identified more than 15 CWDEs and their corresponding genes in the filamentous fungus
Cochliobolus carbonum. Most of the genes were cloned based on amino acid sequences derived from
the purified and enzymatically characterized proteins. Most have been verified by gene disruption.CWDE genes from the Walton lab: All are from C. carbonum
GENE GENE PRODUCT REFERENCE
(see below)Glycosyl
Hydrolyase
FamilyaGENBANK
accession no.PGN1 endo-alpha1,4-polygalacturonase 1, 2, 13 28 L48982 PGX1 exo-alpha1,4-polygalacturonase 13 28 M55979 PME1 pectin methylesterase unpublished - AF159252 XYL1 endo-beta1,4-xylanase 1 5, 10 11 L13596 XYL2 endo-beta1,4-xylanase 2 5, 10 11 U58915 XYL3 endo-beta1,4-xylanase 3 5, 10 11 U58916 XYL4 endo-beta1,4-xylanase 4 5, 10; unpub 10 - XYP1 beta-xylosidase 11, 16 43 AF095243 ALP1 protease 1, trypsin-like 9 - U39500 ALP2 protease 2, subtilisin-like 9; unpub - - MLG1 mixed-linked glucanase 1 12, 18 16 U81606 MLG2 mixed-linked glucanase 2 12, 18 12 AF229447 EXG1 exo-beta1,3-glucanase 1 6, 15, 18 55 L48994 EXG2 exo-beta1,3-glucanase 2 6, 18 5 AF229446 CEL1 beta1,4-glucanase 1 8 7 U25129 CEL2 beta1,4-glucanase 2 19 7 AF336799 ARF1 alpha-arabinosidase 11; unpub 62 AF306763 ARF2 alpha-arabinosidase unpub. 54 AF306764 ccSNF1 protein kinase 17 - AF159253 ccCREA catabolite repressor 20 - AF306571 HDC1 histone deacetylase (HOS2) 20 - AF349677 a See: http://afmb.cnrs-mrs.fr/~pedro/CAZY/ghf.html
Signal Peptides of CWDEs
Almost all secreted proteins have a well-defined signal peptide that targets the nascent protein to the ER, from where it is transported to the plasma membrane. As the protein is imported into the ER, the signal peptide is cleaved. We have found that the SignalP program is quite accurate at predicting signal peptides for the C. carbonum CWDE enzymes. Based on their known N-terminal amino acid sequences, many of them are predicted to undergo a second processing event that removes additional amino acids.
Beta-xylosidase, the product of XYP1, is the only secreted C. carbonum CWDE that lacks a signal peptide that can be predicted by SignalP (see reference 16).
Post-translation Modifications Revealed by Gene Disruption
Targeted mutations of all of the CWDE genes shown above have been constructed. In every case, proof that the correct gene had been cloned and disrupted was the disappearance of the appropriate chromatographic peak of activity. In several cases, notably PGX1 and MLG1, more than one peak of enzymatic activity disappeared in the mutants. This is because many extracellular enzymes are post-translationally modified, particularly by glycosylation, so the different peaks represent different glycosylation isoforms.
None of the CWDE mutants we have made to date show any decrease in virulence.
Possible explanations are:
(1) CWDEs have no role in the disease process. However, much indirect evidence indicates that C. carbonum penetrates maize epidermal cells by enzymatic not mechanical means.
(2) we haven't looked at the right enzymes. Maize cell wall polysaccharides contain many types of glycosidic linkages. Perhaps the enzymes made in culture are irrelevant; maybe there are specialized enzymes just for pathogenesis; maybe the important enzymes are those that break rare but critical linkages in the plant cell wall; etc.
(3) redundancy. Every class of enzyme activity in C. carbonum is due to multiple gene products. That is, there are multiple pectinases, xylanases, beta-glucanases, etc. Multiple knockout strains (e.g., pgn1/pgx1/pme1, xyl1/xyl2/xyl3/xyl4; mlg1/mlg2/exg1/exg2) are still fully virulent and have only slightly reduced growth on the appropriate substrate. Because all mutants retain at least some residual activity, we cannot make a definitive statement about the involvement of any particular activity in pathogenesis.
Light at the end of the tunnel:
Genes that regulate expression of CWDEs are required for full virulenceAnother approach to the question of the role of CWDEs in pathogenicity has been to identify regulatory genes that control multiple CWDE activities. To this end we identified and mutated a homolog of the yeast protein kinase gene SNF1 (Tonukari et al., 2000). In yeast, SNF1 is essential for expression of glucose-repressed genes under inducing conditions. The ccSNF1 mutant has reduced growth on alternate carbon sources, down-regulation of CWDE genes, and reduced virulence due to reduced penetration efficiency. Thus, these studies support a role for CWDE in virulence, without, however, telling us which CWDE genes are important.
From Tonukari et al. (2000).
Furthermore, and unexpectedly, disruption of one of the histone deacetylase genes of C. carbonum, HDC1, gives almost the same phenotypes as the ccsnf1 mutation (Baidyaroy et al., 2001). hdc1 mutants have down-regulation of CWDE gene expression and reduced virulence. See histone deacetylases. An outstanding question in the lab at the moment is the regulatory link between HDC1 and ccSNF1.
References on CWDEs from the Walton lab and collaborators:
(1) Walton, J.D. and F. Cervone (1990) Endopolygalacturonase from the maize pathogen Cochliobolus carbonum. Physiol. Mol. Plant Pathol. 36:351-359.
(2) Scott-Craig, J.S., D.G. Panaccione, F. Cervone and J.D. Walton (1990) Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. Plant Cell 2:1191-1200.
(3) Van Hoof, A., J. Leykam, H.J. Schaeffer and J.D. Walton (1991) A single beta1,3-glucanase secreted by the maize pathogen Cochliobolus carbonum acts by an exolytic mechanism. Physiol. Mol. Plant Pathol. 39:259-267.
(4) Holden, F.R. and J.D. Walton (1992) Xylanases from the fungal maize pathogen Cochliobolus carbonum. Physiol. Mol. Plant Pathol. 40:39-47.
(5) Apel, P., D.G. Panaccione, F.R. Holden and J.D. Walton (1993) Cloning and gene disruption of XYL1 encoding the major xylanase in Cochliobolus carbonum. Mol. Plant-Microbe Interact. 6:467-473.
(6) Schaeffer, H.J., J. Leykam and J.D. Walton (1994) Cloning and targeted gene disruption of EXG1 encoding exo-beta1,3-glucanase in the plant pathogenic fungus Cochliobolus carbonum. Appl. Env. Microbiol. 60:594-598.
(7) Walton, J.D. (1994) Deconstructing the cell wall. Plant Physiol. 104: 1113-1118.
(8) Sposato, P., J.-H. Ahn, and J.D. Walton (1995) Characterization and disruption of a gene in the maize pathogen Cochliobolus carbonum encoding a cellulase lacking a cellulose binding domain and hinge region. Mol. Plant-Microbe Interact. 8:602-609.
(9) Murphy, J.M., and J.D. Walton (1996) Three extracellular proteases from Cochliobolus carbonum: cloning and targeted disruption of ALP1. Mol. Plant-Microbe Interact. 9:290-297.
(10) Apel-Birkhold, P.C. and J.D. Walton (1996) Cloning, disruption, and expression of two endo- beta1,4-xylanase genes, XYL2 and XYL3, from the maize pathogen Cochliobolus carbonum. Appl. Env. Microbiol. 62:4129-4135.
(11) Ransom, R.F., and J.D. Walton (1997) Purification and characterization of extracellular beta-xylosidase and alpha-arabinosidase from the plant pathogenic fungus Cochliobolus carbonum. Carbohydr. Res. 297:357-364.
(12) Görlach, J.M., E. Van Der Knaap, and J.D. Walton (1998) Cloning and targeted disruption of MLG1, a gene encoding two of three extracelluar mixed-linked glucanases of Cochliobolus carbonum. Appl. Env. Microbiol. 64:385-391.
(13) Scott-Craig, J.S., Y.-Q. Cheng, F. Cervone, G. DeLorenzo, J.W. Pitkin, and J.D. Walton (1998) Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases. Appl. Env. Microbiol. 64:1497-1503.
(14) Scott-Craig, J.S., P.C. Apel-Birkhold, J.M. Gorlach, A. Nikolskaya, J.W. Pitkin, R.F. Ransom, P. Sposato, J.-H. Ahn, N.J. Tonukari, S. Wegener, and J.D. Walton (1998) Cell wall degrading enzymes in HST-producing fungal pathogens. In: K. Kohmoto and O.C. Yoder, eds., Molecular Genetics of Host-specific Toxins in Plant Disease, Kluwer Academic, Dordrecht, pp. 245-252.
(15) Nikolskaya, A., J.W. Pitkin, H.J. Schaeffer, and J.D. Walton (1998) EXG1p, a novel beta1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides. Biochim. Biophys. Acta 1425:632-636.
(16) Wegener, S., R.F. Ransom, and J.D. Walton (1999) A unique eukaryotic beta-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum. Microbiology 145:1089-1095.
(17) Tonukari, N.J., J.S. Scott-Craig, and J.D. Walton (2000) The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell 12:237-248.
(18) Kim, H., J.-H. Ahn, J. M. Gorlach, C. Caprari, J.S. Scott-Craig, and J.D. Walton (2000) Mutational analysis of two beta-glucanase genes, EXG2 and MLG2, from the plant pathogenic fungus Cochliobolus carbonum. MPMI, in press.
(19) Ahn, J.-H., P. Sposato, S.I. Kim, and J.D. Walton (2001) Molecular cloning and characterization of cel2 from the fungus Cochliobolus carbonum. Biosci. Biotech. Biochem. 65:1406-1411.
(20) Baidyaroy, D., G. Brosch, J.-H. Ahn, S. Graessle, S. Wegener, N.J. Tonoukari, O. Caballero, P. Loidl, and J.D. Walton (2001) A gene related to yeast HOS2 is necessary for extracellular depolymerase expression and virulence in a plant pathogenic fungus. Plant Cell 13:1609-1624.