Molecular characterization of cellulose synthase (CesA) genes and impact of mutations on fungicide resistance in oomycetes

Many oomycetes are important pathogens of plants (e.g. arable crops, fruit and forest trees), animals or microbes and, upon attacking their hosts, cause considerable economic damage mainly in agriculture and aquaculture. To minimize yield losses efficient disease control, primarily relying on the application of anti-oomycete compounds with a single-site mode of action, is very important. Some anti-oomycete compounds specifically inhibit mitochondrial respiration, RNA synthesis, microtubule organization or cell wall synthesis. The oomycete cell wall is mainly composed of β-1,3-, β-1,6 glucans and cellulose that provide rigidity to the cell. The synthesis of cellulose was recently shown in Phytophthora infestans to be the target for mandipropamid (MPD), which belongs to the oomycete specific carboxylic acid amide (CAA) fungicides controlling members of the Peronosporales. However, there are several oomycetes that the CAAs cannot control (e.g. the entire genus Pythium), even though cellulose is an important cell wall component of all oomycetes. Furthermore, the mode of action of CAA fungicides is highly specific, thus the resistance risk is assumed to be moderate to high. Consequently, resistant individuals were recovered in Plasmopara viticola and Pseudoperonospora cubensis populations, but the underlying mechanism of resistance remained unknown. This thesis aims to identify and characterize the cellulose synthase (CesA) genes of various oomycete species and thereby to investigate the mechanism(s) of resistance in sensitive species, i.e. species normally affected by CAAs, and tolerance in insensitive species, i.e. species unaffected by CAA fungicides.
A family of four cellulose synthase genes was identified and fully sequenced in the downy mildew pathogens P. viticola and P. cubensis. Phylogenetic analyses of the four genes revealed their close relatedness to cellulose synthase genes of Phytophthora spp. and the red algae Porphyra yezoensis. Sequencing of the CesA genes in CAA- resistant and -sensitive field isolates of P. viticola and P. cubensis uncovered single nucleotide polymorphisms (SNPs) affecting the amino acid structure of CesA proteins. Inheritance of resistance in P. viticola was confirmed to be correlated with one recessive SNP located in the CesA3 gene. This SNP led to an exchange from a highly conserved glycine (encoded by GGC) to serine (AGC) at position 1105 (G1105S), whereas in P. cubensis, mutations causing amino acid substitutions from glycine (GGG) to valine (GTG) or tryptophane (TGG) (G1105V, G1105W) occurred in the same codon. The results demonstrate for the first time that SNPs in codon 1105, when present in both alleles, lead to amino acid exchanges in the CesA3 enzyme causing inheritable and stable resistance to all CAA fungicides.
New insights into the molecular basis of CAA tolerance in Pythiales were provided by characterizing five genes putatively involved in carbohydrate synthesis of the root rot and damping off causative agent Pythium aphanidermatum. Using the CODEHOP PCR strategy, one chitin synthase gene, PaChs, and four cellulose synthase genes, PaCesA1 to PaCesA4, out of which PaCesA3 encodes the MPD target enzyme, were amplified and fully sequenced. These genes were individually upregulated during encystment, germination of cystospores and mycelial growth indicating their relevance for cell wall formation. However, almost no change in PaCesA or PaChs expression was observed when mycelium was treated with MPD concentrations slightly affecting mycelial growth. Detailed analyses of the putative target site in PaCesA3 revealed a specific amino acid configuration (L1109) also present in CAA resistant P. infestans mutants. The affected amino acid residue is located only four amino acids downstream of the G1105 residue, where amino acid exchanges cause inheritable resistance to CAAs in P. viticola and P. cubensis field isolates. This implies that MPD tolerance in P. aphanidermatum, and most likely in other Pythium species, is based on the leucine configuration at position 1109 which may affect the binding of CAAs to the enzyme.
To further examine the results obtained with P. aphanidermatum, the CesA3 gene structure as well as the sensitivity to CAAs of 25 species representing the Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales was investigated. Molecular characterization of the putative target site in CesA3 revealed a conserved glycine at position 1105 (G1105) in all oomycete species. However, at position 1109 the Peronosporales displayed the amino acid valine, whereas all species from the other orders showed either leucine or methionine at this position. The observed amino acid configurations (L1109 and M1109) correlated with MPD tolerance, suggesting that amino acid changes at position 1109 may also affect CAA efficacy in sensitive species. In addition, the full-length nucleotide sequence of the CesA3 gene was used to study phylogenetic relatedness among oomycetes originating from the six distinct orders. The phylogenetic tree constructed with the CesA3 gene sequence was largely in agreement with trees based on other markers (e.g. cox2, SSU, LSU rDNA), implying that this gene represents a promising tool to reconstruct an overall picture of the oomycete phylogeny.
The presented insights into the molecular mechanism(s) of CAA resistance and tolerance significantly contribute to a sound assessment of resistance risk when CAAs are used to control oomycetes. In addition, the results open up novel tools for basic investigations on cellulose biosynthesis in oomycetes.