Cell Wall Degrading Enzymes

Last Updated on Fri, 01/07/2011 - 16:52

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, lignin, etc., even 'inert' substrates like rubber and 'man-made' substrates like plastic.

Only certain fungi can degrade lignin. An interesting theory attributes the rise of oxygen levels during the Carboniferous and the deposition of modern day coal beds to the 50-million year lag between the evolution of lignin in early land plants and the evolution of a biological mechanism to degrade it. Lignin-degrading fungi include Phanerochaete chrysosporium.

Consistent with their mode of living, fungi make and secrete a huge variety of extracellular depolymerases.

Originally, we were interested in cell wall degrading enzymes (CWDEs) for their role in plant pathogenesis. Now we study them for their utility in biomass conversion (see Enzymes for Bioenergy).

We have identified and characterized 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 verifiedby gene disruption.

CWDE and related genes from the Walton lab: All are from C. carbonum.

 

GENE

GENE PRODUCT

REFERENCE 
(see below)

Glycosyl 
Hydrolyase 
Familya

GENBANK
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unpublished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

 

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 experimentally determined N-terminal amino acid sequences, some of them (e.g., PGN1) are predicted to undergo a second processing event that removes additional amino acids. The average signal peptide for the C. carbonum CWDEs is  17-19 amino acids.

Beta-xylosidase, the product of XYP1, is the only secreted C. carbonum CWDE that lacks a predicted signal peptide (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 these enzymes, like many extracellular enzymes, are post-translationally modified, particularly by glycosylation. The different peaks represent different glycosylation isoforms.

Few of the CWDE mutants we have made to date show any decrease in virulence. Possible explanations for this are:

(1) CWDEs have no role in the disease process. However, much indirect evidence indicates that C. carbonumpenetrates maize epidermal cells by enzymatic and 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 virulence

Another 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. As expected, the ccSNF1 mutant has reduced growth on alternate carbon sources and down-regulation of CWDE genes. Gratifyingly, the ccSNF1 mutant also has reduced virulence due to reduced penetration efficiency. Thus, these studies support a role for CWDE in virulence (without, however, indicating which CWDE genes are important).

 

From Tonukari et al. (2000).

 

Tzima et al. (2011) also found that an ortholog of SNF1 is essential for virulence and CWDE expression inVerticillium dahliae (MPMI 24:129).

Unexpectedly, disruption of one of the histone deacetylase genes of C. carbonumHDC1, 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 under Research. An interesting question 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, andexpression 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 14:1436-1443.

(19) Ahn, J.-H., P. Sposato, S.I. Kim, and J.D. Walton (2001) Molecular cloning and characterization of cel2from 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.