Pinus patula is the single most important commercial plantation forest tree species in South Africa. It accounts for 52% approximately (700,000 ha) of total commercial plantation area in the country and is utilised for saw logs and pulp and paper production. P. patula is a tropical pine species indigenous to Mexico. Excellent ex situ conservation and range-wide provenance trials have been established for P. patula in South Africa and South America. These highly organised trials provide the opportunity to perform association genetic studies with the long term aim to identify trait linked markers for future molecular improvement of P. patula. In this study, the first gene-based assessment of allelic diversity in P. patula was performed. This pilot study focused on two cellulose biosynthetic genes as representatives of wood formation genes and assayed molecular evolution parameters such as nucleotide diversity, allelic diversity and linkage disequilibrium (LD) in a species-wide reference population of P. patula. Two novel cellulose synthase (CesA) genes were isolated and characterised in P. patula. One of these genes, PpCesA1, is putatively involved in the biosynthesis of secondary cell walls of tissues such as xylem (wood), while the other, PpCesA2 is proposed to be associated with primary cell wall formation in rapidly growing tissue types. The genomic DNA copies of PpCesA1 and PpCesA2 were 6025 bp and 6365 bp in length, respectively. The corresponding cDNA sequences encoded 1083 and 1058 amino acids, respectively, and differed considerably from each other (73% amino acid identity). Both amino acid sequences contained the key domains and motifs characteristic of functional CESA proteins isolated in other higher plants. Phylogenetic analysis revealed that PpCesA1 was most similar (99%) to its putative ortholog in Pinus taeda, PtCesA3, and PpCesA2 was highly similar to a putative ortholog in Pinus radiata, PrCesA2 (99% identity). This phylogenetic analysis supported previous findings that the divergence between the primary and secondary cell wall associated CESA proteins occurred before the divergence of angiosperms and gymnosperms approximately 300 million years ago. A fragment of a putative paralogous gene copy of PpCesA1, named PpCesA1-B was also isolated. The PpCesA1-B gene fragment was found to differ from PpCesA1 by 22 nucleotide polymorphisms and its non-allelic (paralogous) status was confirmed by segregation analysis in P. patula. In order to gain an understanding of molecular genetic variation that might affect wood formation in P. patula, we sequenced multiple allelic variants of PpCesA1, PpCesA1-B and PpCesA2, which we sampled from a species-wide reference population of P. patula. The average levels of nucleotide diversity were found to be low for all three genes (π ≈ 0.0015), which may be a property of functional members of the CesA gene family. As a result of the low nucleotide diversity, only small numbers of pair-wise informative sites were available for LD analysis and the decay in LD could only be studied in PpCesA2 where it was found to decay very rapidly (within 200 bp). Tests of neutrality suggested that the exon sequences of PpCesA1 and PpCesA2 were under significant positive (adaptive) selection. Comparison of levels of nucleotide diversity and selection in different parts of the two genes indicated that the highest levels of adaptive selection occurred in areas where amino acid substitutions could alter protein structure or function. This study provides valuable insights for designing future allele discovery efforts in P. patula with the ultimate goal of developing gene-based markers for the molecular improvement of wood formation in this tree species. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

<p>Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus <em>Phytophthora </em>are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus <em>Saprolegnia</em>, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen <em>Phytophthora infestans</em> and the fish pathogen <em>Saprolegnia monoica</em>.</p><p> </p><p>A new Oomycete <em>CesA</em> gene family was identified. It contains four homologues designated as <em>CesA1, CesA2, CesA3</em> and <em>CesA4</em>. The gene products of <em>CesA1, 2</em> and <em>4 </em>contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete <em>CesA </em>genes. <em>CesA3</em> is the dominantly expressed <em>CesA </em>homologue in the mycelium of both <em>S. monoica</em> and <em>P. infestans</em>, while <em>CesA1</em> and<em> CesA2</em> are up-regulated in virulent life stages of <em>P. infestans</em>. <em>CesA4</em> was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole <em>CesA</em> gene family in <em>P. infestans</em> lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on <em>SmCesA</em> gene expression, which was accompanied by an increased b-glucan synthase activity <em>in vitro</em>. In addition, these inhibitors slowed down the growth of the mycelium from <em>S. monoica</em>. Zoospores from <em>P. infestans</em> treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the <em>CesA</em> gene family.</p><p>Altogether these results show that at least some of the <em>CesA1-4</em> genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by <em>P. infestans</em>.</p><p> </p>

Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans. / QC 20101110

Some oomycetes species are severe pathogens of fish or crops. As such, they are responsible for important losses in the aquaculture industry as well as in agriculture. Saprolegnia parasitica is a major concern in aquaculture as there is currently no method available for controlling the diseases caused by this microorganism. The cell wall is an extracellular matrix composed essentially of polysaccharides, whose integrity is required for oomycete viability. Thus, the enzymes involved in the biosynthesis of cell wall components, such as cellulose and chitin synthases, represent ideal targets for disease control. However, the biochemical properties of these enzymes are poorly understood, which limits our capacity to develop specific inhibitors that can be used for blocking the growth of pathogenic oomycetes. In our work, we have used Saprolegnia monoica as a model species for oomycetes to characterize two types of domains that occur specifically in oomycete carbohydrate synthases: the Pleckstrin Homology (PH) domain of a cellulose synthase and the so-called ‘Microtubule Interacting and Trafficking’ (MIT) domain of chitin synthases. In addition, the chitin synthase activity of the oomycete phytopathogen Aphanomyces euteiches was characterized in vitro using biochemical approaches. The results from our in vitro investigations revealed that the PH domain of the oomycete cellulose synthase binds to phosphoinositides, microtubules and F-actin. In addition, cell biology approaches were used to demonstrate that the PH domain co-localize with F-actin in vivo. The structure of the MIT domain of chitin synthase (CHS) 1 was solved by NMR. In vitro binding assays performed on recombinant MIT domains from CHS 1 and CHS 2 demonstrated that both proteins strongly interact with phosphatidic acid in vitro. These results were further supported by in silico data where biomimetic membranes composed of different phospholipids were designed for interaction studies. The use of a yeast-two-hybrid approach suggested that the MIT domain of CHS 2 interacts with the delta subunit of Adaptor Protein 3, which is involved in protein trafficking. These data support a role of the MIT domains in the cellular targeting of CHS proteins. Our biochemical data on the characterization of the chitin synthase activity of A. euteiches suggest the existence of two distinct enzymes responsible for the formation of water soluble and insoluble chitosaccharides, which is consistent with the existence of two putative CHS genes in the genome of this species. Altogether our data support a role of the PH domain of cellulose synthase and MIT domains of CHS in membrane trafficking and cellular location. / <p>QC 20151014</p>