Microsatellite Markers from Peronospora tabacina, the Cause of Blue Mold of Tobacco, Reveal Species Origin, Population Structure, and High Gene Flow.

Peronospora tabacina is an obligate parasite that causes blue mold of tobacco. The pathogen reproduces primarily by sporangia, whereas the sexual oospores are rarely observed. A collection of 122 isolates of P. tabacina was genotyped using nine microsatellites to assess the population structure of individuals from subpopulations collected from central, southern, and western Europe; the Middle East; Central America; North America; and Australia. Genetic variations among the six subpopulations accounted for ∼8% of the total variation, including moderate levels of genetic differentiation, high gene flow among these subpopulations, and a positive correlation between geographic and genetic distance (r = 0.225; P < 0.001). Evidence of linkage disequilibrium (P < 0.001) showed that populations contained partially clonal subpopulations but that subpopulations from Australia and Mediterranean Europe did not. High genetic variation and population structure among samples could be explained by continuous gene flow across continents via infected transplant exchange and/or long-distance dispersal of sporangia via wind currents. This study analyzed the most numerous P. tabacina collection and allowed conclusions regarding the migration, mutation, and evolutionary history of this obligate biotrophic oomycete. The evidence pointed to the species origin in Australia and identified intracontinental and intercontinental migration patterns of this important pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

CYP71BL9, the missing link in costunolide synthesis of sunflower.

Despite intensive research in recent years, the biosynthetic route to costunolide in sunflower so far remained obscured. Additional P450 sequences from public sunflower transcriptomic database were screened to search for candidate enzymes which are able to introduce the 6α-hydroxy-group required for the esterification with the carboxy group of germacarane A acid, the final step in costunolide formation. CYP71BL9, a new P450 enzyme from sunflower was shown to catalyze this hydroxylation, hence being identified as HaCOS. Phylogentically, HaCOS is closer related to HaG8H than to any other known costunolide synthase in Asteraceae.The enzyme was successfully employed to reconstruct the sunflower biosynthesis of costunolide in transformed tobacco. Contrary, in yeast, only minor amounts of sesquiterpene lactone was produced, while 5-hydroxyfarnesylic acid was formed instead. HaCOS in combination with HaG8H produced 8ß-hydroxycostunolide (eupatolide) in transformed plants, thus indicating that sunflower possesses two independent modes of eupatolide synthesis via HaCOS and via HaES. The lack of HaCOS expression and of costunolide in trichomes suggests that the enzyme triggers the costunolied synthesis of the inner tissues of sunflower and might be linked to growth regulation processes.

Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae).

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

Genome Sequence and Architecture of the Tobacco Downy Mildew Pathogen Peronospora tabacina.

Peronospora tabacina is an obligate biotrophic oomycete that causes blue mold or downy mildew on tobacco (Nicotiana tabacum). It is an economically important disease occurring frequently in tobacco-growing regions worldwide. We sequenced and characterized the genomes of two P. tabacina isolates and mined them for pathogenicity-related proteins and effector-encoding genes. De novo assembly of the genomes using Illumina reads resulted in 4,016 (63.1 Mb, N50 = 79 kb) and 3,245 (55.3 Mb, N50 = 61 kb) scaffolds for isolates 968-J2 and 968-S26, respectively, with an estimated genome size of 68 Mb. The mitochondrial genome has a similar size (approximately 43 kb) and structure to those of other oomycetes, plus several minor unique features. Repetitive elements, primarily retrotransposons, make up approximately 24% of the nuclear genome. Approximately 18,000 protein-coding gene models were predicted. Mining the secretome revealed approximately 120 candidate RxLR, six CRN (candidate effectors that elicit crinkling and necrosis), and 61 WY domain-containing proteins. Candidate RxLR effectors were shown to be predominantly undergoing diversifying selection, with approximately 57% located in variable gene-sparse regions of the genome. Aligning the P. tabacina genome to Hyaloperonospora arabidopsidis and Phytophthora spp. revealed a high level of synteny. Blocks of synteny show gene inversions and instances of expansion in intergenic regions. Extensive rearrangements of the gene-rich genomic regions do not appear to have occurred during the evolution of these highly variable pathogens. These assemblies provide the basis for studies of virulence in this and other downy mildew pathogens.

Ten polymorphic microsatellite loci identified from a small insert genomic library for Peronospora tabacina.

Ten polymorphic microsatellite loci for the obligate biotrophic, oomycete pathogen of tobacco, Peronospora tabacina, were identified from a small insert genomic library enriched for GT motifs. Eighty-five percent of the 162 loci identified were composed of dinucleotide repeats, whereas only 4% and 11% were tri-and tetra-nucleotide repeats respectively. About 82% of all the microsatellites were perfect and within the library; only about 7% of the loci were duplicated. Primers were designed for 63 loci; 10 loci were polymorphic, 19 were monomorphic and 34 either failed to amplify or produced ambiguous/inconsistent results. The 10 polymorphic loci were characterized with 44 isolates of P. tabacina collected from tobacco plants growing in Europe, the Near East and North and South America. The number of alleles per locus was either three or four with a mean of 3.2, and the mean number of genotypes per locus was 3.6. Observed heterozygosity was 0.32-0.95, whereas expected heterozygosity was 0.44-0.69 for these loci. All loci except PT054 did not conform to the Hardy-Weinberg distribution. Polymorphic information content (PIC) for the loci was 0.35-0.69 with a mean of 0.50. These microsatellite loci provide a set of markers sufficient to perform genetic diversity and population studies of P. tabacina, and possibly other species of Peronospora.

Different pathotypes of the sunflower downy mildew pathogen Plasmopara halstedii all contain isometric virionsdagger.

Eight pathotypes of Plasmopara halstedii were screened to investigate the occurrence of virions and the potential viral influence on the pathogenicity of the sunflower downy mildew pathogen. In 23 of 26 P. halstedii isolates derived from eight countries in Europe, North America and South America, virions were detected by transmission electron microscopy. By contrast, there were no ultrastructural indications of virus-like particles in eight other related Oomycetes. The virions of representative P. halstedii isolates were morphologically and biochemically characterized and compared among each other. Regardless of their host's pathotypes, the geographical origin of the isolate and the sensitivity towards the fungicide metalaxyl, the viral characters obtained were uniform. The virions were isometric and measured approximately 37 nm in diameter. One polypeptide of c. 36 kDa and two segments of single-stranded RNA (3.0 and 1.6 kb) were detected. Both viral RNA segments were detected by capillary electrophoresis in the three remaining P. halstedii isolates where virions were undetectable by transmission electron microscopy. Virus-specific primers for the 1.6 kb-segment were synthesized and used to determine and compare a partial sequence of the viral coat protein among virions of different P. halstedii pathotypes. In all tested isolates, fragments of 0.7 kb were amplified which were directly sequenced. Sequence variation was insignificant. As both less aggressive and more aggressive P. halstedii isolates contained virions, the presence or absence of virions could not explain the diverse aggressiveness of the downy mildew pathogen towards sunflower. Moreover, the results indicated that pathogenicity of P. halstedii was not related to variation in morphological or biochemical characters of the virions.