Characterization of the cytochrome [beta] gene in plant pathogenic, basidiomycetes and consequences for QoI resistance

Strobilurins are one of the most important classes of agricultural fungicides, which are also known as Qo inhibitors (QoIs) because they inhibit mitochondrial respiration by binding to the Qo (‘Quinone outside’) site of the cytochrome b. Cytochrome b is part of the cytochrome bc1 complex, located in the inner mitochondrial membrane, and it is encoded by the cytochrome b (cyt b) gene. Since the mode of action of QoI fungicides is highly specific, the risk of resistance is also high. Single amino-acid exchanges in the cytochrome b were found conferring resistance to QoI fungicides in different plant pathogens. The major mechanism of resistance is a point mutation, also called Single Nucleotide Polymorphism (SNP), in the cyt b gene leading to a change at amino acid position 143 from glycine to alanine (G143A) or from phenylalanine to leucine at position 129 (F129L). However, other point mutations were described, all located in two highly conserved cytochrome b regions, so called ‘hot spot’ regions (amino acid residues 120-160 and 250-300). A fragment of the cyt b gene of a range of agronomically important plant pathogenic Basidiomycetes was sequenced at cDNA level, including Puccinia recondita f.sp. tritici, P. graminis f.sp. tritici, P. striiformis f.sp. tritici, P. coronata f.sp. avenae, P. hordei, P. recondita f.sp. secalis, P. sorghi, P. arachidis, P. horiana, Uromyces appendiculatus, Phakopsora pachyrhizi, Hemileia vastatrix and Rhizoctonia solani. The sequence data allowed developing specific primers for the cyt b gene of all these species, which easily amplified the fragment of the gene including the ‘hot spot’ regions. Resistance to QoI fungicides has never been reported until now in plant pathogenic Basidiomycetes, and in all tested isolates included in this study, the sequence of the cyt b gene fragment did not contain any point mutations. The deduced amino acid sequences of the cytochrome b were used to study the relatedness of these pathogens as compared to other species of the Basidiomycetes, Ascomycetes and Oomycetes. The relatedness was also studied at nuclear level using the Internal Transcribed Spacers (ITS) in the ribosomal DNA. Our results demonstrated for the first time that the amino acid sequence of the mitochondrial cytochrome b is a valid tool to study phylogenic relatedness among plant pathogenic Basidiomycetes and supports taxonomic grouping based on morphological structures and host specificity. Moreover, the phylogenic clusters generated by analysing the cytochrome b sequence confirmed previous results based on the analysis of rRNA of the Uredinales and more specifically, on the ITS regions of closely related species of Puccinia. The cytochrome b gene fragment (coding sequence about 950 bp long) was sequenced also at genomic DNA level in different Puccinia species and U. appendiculatus. A shorter fragment (coding sequence about 600-700 bp) was sequenced in P. pachyrhizi, H. vastatrix, Alternaria solani, A. alternata and Plasmopara viticola. The cyt b gene structure of these agronomically important plant pathogens was characterized, especially in the two ‘hot spot’ regions and compared with that of other species. The exon/intron organization was characterized and its possible role investigated for the occurrence of point mutations in the cyt b gene, especially the amino acid substitutions G143A and F129. In all rust species included in this study as well as in A. solani, the mutation G143A was not detected, but a group I intron (self-splicing intron) was observed starting exactly after the codon GGT for glycine at position 143. In pathogens such as A. alternata, Blumeria graminis, Pyricularia grisea, Mycosphaerella graminicola, M. fijiensis, Venturia inaequalis and P. viticola, resistance to QoI fungicides is known and the glycine (triplet GGT) is replaced by alanine (triplet GCT) at position 143 in the resistant genotype. In those pathogen species, no intron was observed after the codon 143. In the rust species as well as in A. solani, the codon GGT at position 143 is located exactly at the exon/intron boundary and is likely part of the signal sequences essential for the recognition of the intronic RNA to be excised. We predicted that a nucleotide substitution in the codon 143, which is two nucleotides upstream from the exon/intron junction, may strongly affect the splicing process, leading to a deficient cytochrome b, which is lethal. Therefore, individuals carrying the G143A substitution and an intron at this position will not survive. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in plant pathogens such as Puccinia spp. , U. appendiculatus, P. pachyrhizi, H. vastatrix and A. solani. It cannot be excluded that mutations other than G143A conferring resistance may arise in upcoming rust populations selected by the use of QoI fungicides, as observed for A. solani, in which the mutation F129L was reported in resistant isolates. Our results cannot predict whether such mutations may evolve at all in rusts, but the sequence data produced during this study and the understanding of the cyt b gene structure represent an essential information for easy isolation of the cyt b gene and detection of point mutations conferring resistance to QoI fungicides that eventually may evolve.