Crystallization and preliminary X-ray study of endopolygalacturonase from the pathogenic fungus Stereum purpureum

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The interaction between plant hosts and pathogens are complex systems that consist of a vast multitude of components. These components that contribute to these systems are from host as well as pathogenic origin and compete with one another in an attempt to be more successful than the other party.

There are many different pathogens that infect plants from all five kingdoms of life and each has a different method of infecting the host. There are several traits that are universal requirements for infection such as gaining entry to the host, overcoming the host’s immune response, replicating effectively within the host and spreading to naïve hosts.

Plant cell walls consist of a network of various peptides and polysaccharides. Three categories of polysaccharides exist, namely cellulose, hemicellulose and pectin. Pectin and hemicellulose form the network that the cellulose microfibrils are embedded. The pectin network consists of galacturonic acid (PGA) subunits that are bound by α-1,4-linked glycosidic bonds. This network acts as a primary defence barrier to pathogens that attempt to cross the cell wall to infect the host as well as providing significant structural support.

The biosynthesis of pectin is localised to the Golgi vesicles. It has been estimated that 67 different enzymes are required in the biosynthetic pathway of pectin. 384 The reason for the evolution of pectin is mostly unknown and genome comparisons between different plants would provide a clearer insight into its evolution. What is known about the evolution is that the latest development in angiosperms is the presence of the QUA1 ortholog.

Plant endoPGs are important for the developmental and remodelling processes during their lifecycles.

One of the most prolific enzymes that enable a fungus or bacteria to infect woody plants is the polygalacturonase (PG) enzyme. This enzyme is produced by hosts as well as by the bacteria, fungi and yeasts that infect it. In plants it plays a crucial role in fruit ripening whereas in bacteria and fungi it plays a vital role in the rotting process that occurs upon infection. The group of PG enzymes consist of endoPG (EC 3.2.1.15) and exoPG (EC 3.2.1.67) isozymes. These isozymes both hydrolyse the polygalacturanic acid pectin network, but at different positions. EctoPG cleaves galacturon monomers from the terminals of the polygalacturanic chains whereas endoPG hydrolyses the internal glycosidic bonds within the polygalacturanic acid chain.

EndoPG have been found to be the enzymes that cause tissue maceration due to their ability to degrade the pectin network. This makes them an integral component to the initial rotting process. Once the tissue has been macerated, the plant no longer has the physical barrier to protect its extracellular and intracellular matrices from pathogen intrusion.

When a plant is infected with a pathogen an immune response occurs. One of the compounds that induce an immune response is PGA. PGA induces a conversion of ·OH to ·O2-

In the ascomycete Sclerotinia sclerotiorum there are 7 endoPG enzymes, annotated as pg1-pg7. The endoPG genes are sequentially expressed during the infection process. This leads to the various steps in the infection cycle from no visible symptoms, to mycelia invading the tissue, to the emergence of aerial mycelia to total tissue maceration. The pg1, pg2 and pg3 genes are expressed during the initial tissue invasion and colonisation of the host, pg5 is expressed during the late stages of tissue maceration. pg6 and pg7 are the only endoPG genes that are expressed constitutively throughout the colonisation process.

These function optimally at a low pH and are often secreted with oxalic acid that serves to lower the pH of the cell walls. EndoPGs at this low pH are able to escape PGIP inactivation due to a one of two hypotheses. First, it may be due to the low pH that significantly enhances the enoPG activity by binding to the enzyme’s active site, thereby stabilizing it. Secondly, it allows the cell wall to be more accessible to endoPG enzymes by sequestrating the Ca2+ ions. Third, oxalate may have a direct effect on PGIPs themselves, although this mechanism is still unknown.

Oxalic acid is a chelator of cations such as Ca2+ that are present in the apoplast. This subsequently weakens the cell wall. Along with the lowered pH that it induces, allows some cell wall degrading enzymes such as endoPGs function more effectively.

There are two types of endoPG genes: those that yield endoPG enzymes that function more effectively in basic conditions and those that yield endoPG enzymes that function more effectively under acidic conditions. These genes have very similar sequences which indicate that there has to be another molecule that enables it to function under different conditions.

EndoPG enzymes exist within yeasts, but the expression is limited due to a weak promoter. These enzymes are encoded by the PGL1 gene and are under control of the MAPK regulatory pathway. They are also crucial in pseudohyphae development which is related to the pathogenicity of S. cerevisiae. Experiments have shown the possibility of the endoPG gene being transferred to yeasts from bacteria due to the promoter region of the PGL1 gene showing very high similarity to the promoter of the endoPG genes found in bacteria. The Shine-Dalgarno sequence was missing in the yeast promoter. Different promoter regulation exists in yeasts and bacteria. Under anaerobic conditions PGL1 is downregulated in yeast, but upregulated in bacteria while under aerobic conditions PGL1 is upregulated in yeast, but downregulated in bacteria.

Crystal structures reveal that the fungal endoPG enzymes contain four conserved cysteine residues that form part of disulphide bridges that are structurally important. In yeast, the first two cysteine residues are absent while the last two are present. Bacterial endoPG genes contain the first two cysteine residues but lack the final two cysteine residues. This indicates similar evolution in bacteria and yeast that is separate from the evolution of endoPG genes in fungi. The endoPG genes in fungi contain introns, whereas those in yeasts do not. This also points to closely related evolution between the endoPG genes in bacteria and yeasts. It has been hypothesised that the endoPG genes have been transferred to yeast from bacteria as the gene has many prokaryotic features. Gene transfer between yeast and bacteria is not uncommon and is thus a plausible reason.

A study has shown that S. cerevisiae and Heliobacter pylori contain a great multitude of evolutionary conserved pathways as well as duplications of these pathways that are not under selection and are able to mutate more freely and develop novel functions.

In the oomycete, Phytophthora parasitica a multigene family of endoPG genes titled pppg2-10 exist. (Wu 2007) Pppg2-9, excluding pppg6 contained no introns. All of the pppg genes, except pppg9, are 358-390 amino acid residues in length. The pppg9 gene is 512 amino acid residues in length. The additional amino acid residues represented an additional N-terminal domain. The pppg genes in P. parastica also contain variable amounts of N-glycosylation sites. Analysis of the amino acid sequence of the pppg genes showed that the active site contained a conserved sequence of CXGGHGXSIGSVG. Each of these genes causes specific symptoms when expressed in the host. Phylogenetic analysis of the pppg genes showed that they are more closely related to other fungi than to plants and bacteria.

Within the Phytophthora species there is significant variation between the endoPG genes contained in each genome. In P. cinnamomi contains a 19 member multigene PG family that contains orthologs of some of the pppg that suggest gene duplication occurred before speciation occurred. P. ramorum also contains some of the

The pppg2 and pppg3 are closely related which indicates a recent gene duplication that has also occurred in other genes in Phytophthora that encode secretory peptides.

pppg5 and pppg7 are expressed in a much lower quantity than the other pppg enzymes which indicates that there is a biochemical action acting on their expression such as protein stability or substrate preference. Many future studies will need to be done to determine the full endoPG expression profile.

The Potato virus X was used to infect Nicotiana benthamiana in an attempt to identify the functions of the different endoPG genes of P. parasitica . four different groups emerged from the trial.

The first group contained pppg1 and pppg7. This group showed no significant damage to the leaves of the infected plants. Staining with ruthenium red showed significantly deformed mesophyll cells in the plants that were treated with pppg7 whereas the vectors containing pppg1 showed only slight changes when compared to the control. This may be due to their molecular mass exceeding 50kDa.

The second group contained pppg4 and pppg6 and infection with these genes resulted in dwarf phenotypes. As the leaves grew, they curved towards the back and became wrinkled. There were also shot holes on the leaves that were observable under ultraviolet light. Cracks on the leaf stalks were observed. Cross-sections of infected leaves showed that the palisade and spongy mesophyll cells were severely deformed and that there was very little intercellular space remained in the leaf.

The third group contained pppg2, pppg8 and pppg10. Infection with these genes led to yellowing and dwarfism in the infected leaves. The phenotype was very similar to that of the group two endoPGs. The mesophyll cells were mutated to such an extent that all intercellular space had disappeared.

The fourth group contained pppg5 that resulted in a silvery leaf phenotype with severely deformed mesophyll cells. Cracks on the leaf stalks with increased intercellular space in these cracks were also observed in this group. The epidermal cells were also separated from the wax cuticle.

pppg genes may elicit a defense response from the plant host. This is an avenue of research that still needs to be explored.

The presence and variety of endoPG genes within a genome has been considered an indicator of host range. Phytophthora sojae contains many endoPG homologs but has a characteristically narrow host range. This is a common example to show that correlation does not imply causation.

The presence of endoPG’s is mostly necessary for a fungus to be pathogenic. There have been examples of Claviceps purpurea (Oeser 2002) and Alternaria citri that show that mutations affecting the endoPG genes have led to a reduction or removal of pathogenicity. Cryphonectria parasitica is one of the few fungi that don’t require endoPG genes for virulence.

EndoPGs are usually 40kDa in size. If plant cells are adequately hydrated, molecules smaller than 50kDa can diffuse freely into the cell wall. The target of endoPGs is the middle lamella that resides in the cell wall. This may explain why waterlogged conditions provide such favourable conditions for endoPG expression and action.

Burkholderia cepacia was subjected to a biochemical study of its endoPG gene due to PGIP’s inability to counteract their functions. There are many conserved amino acids that play important roles in the biochemical efficacy of the enzyme. In the BcPeh28A enzymes the conserved amino acids and their functions are Asp222 that acts as a general acid catalyst, Asp201 and Asp223 that act as general base catalysts, His250 that acts as part of the restoration of the acid-base equilibrium of the catalytic aspartates, Arg 287, Lys 289, Tyr323 that are involved in substrate binding, Gly255 and Gly256 that form the cis-peptide motif and define the opposite side of the active site cleft.

BcPeh28A catalytic residues as follows: Asp222 (as the general acid catalyst), Asp201 and Asp223 (as the general base catalysts). The other strictly conserved amino acids are: His250, involved in the regeneration of the acidebase equilibrium of the catalytic aspartates; Arg287, Lys289 and the invariant Tyr323, which participate in the substrate binding; Gly255 and Gly256, forming the so called cis-peptide motif and defining the opposite side of the active site cleft.

The in silico model of the Gram-negative is accurate when compared to what is currently known about the catalytic and substrate binding abilities that have been determined experimentally. There have been future experiments suggested by the bioinformaticists in order to further validate their findings. This paper has been of interest as there have been studies done for fungal endoPG proteins as well.

Crystallization and preliminary X-ray study of endopolygalacturonase from the pathogenic fungus Stereum purpureum

EndoPG gene products also elicit a defense response from the host. In response to the PGs, plants are able to produced polygalacturonase-inhibiting proteins (PGIPs) to protect themselves. PGIPs are able to defend themselves against fungal endoPGs, but not against bacterial endoPGs. This has led to the biochemical and computational study of various endoPG enzymes from various sources.

EndoPG have many commercial applications that include extraction of fruit juice and vegetable oils, bleaching of paper, the treatment of waste water and as additives in poultry feed.

References

Oeser, B., Heidrich, P.M., Muller, U., Tudzynski, P., Tenberge, K.B., 2002, Polygalacturonase is a pathogenicity factor in the Claviceps purpurea/rye interaction, Fungal Genetics and Biology, 36: 176–186

Wu, C., Yan, H., Liu, L., Liou, R., 2007, Functional Characterization of a Gene Family Encoding Polygalacturonases in Phytophthora parasitic, Molecular Plant-Microbe Interactions, 21(4): 480-489

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