Formation and diversification of a paradigm biosynthetic gene cluster in plants

© 2020, The Author(s). Numerous examples of biosynthetic gene clusters (BGCs), including for compounds of agricultural and medicinal importance, have now been discovered in plant genomes. However, little is known about how these complex traits are assembled and diversified. Here, we examine a large number of variants within and between species for a paradigm BGC (the thalianol cluster), which has evolved recently in a common ancestor of the Arabidopsis genus. Comparisons at the species level reveal differences in BGC organization and involvement of auxiliary genes, resulting in production of species-specific triterpenes. Within species, the thalianol cluster is primarily fixed, showing a low frequency of deleterious haplotypes. We further identify chromosomal inversion as a molecular mechanism that may shuffle more distant genes into the cluster, so enabling cluster compaction. Antagonistic natural selection pressures are likely involved in shaping the occurrence and maintenance of this BGC. Our work sheds light on the birth, life and death of complex genetic and metabolic traits in plants

Coevolution between a Family of Parasite Virulence Effectors and a Class of LINE-1 Retrotransposons

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVRk1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVRk1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome

Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of Streptomyces coelicolor by Targeted LC-MS/MS

The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC-MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD00009S

Identification of mendel’s pod colour character in pea: characterisation of the allele conditioning yellow pod colour

International audienceThe inheritance of yellow-podded versus green-podded peas was studied by Mendel more than 150 years ago, but, until now, the allelic variation underlying this difference remained unknown. A report of a novel, spontaneous gp mutant (Pellew and Sverdrup, 1923) suggested that at least two independent gp alleles existed. We undertook genetic complementation crosses with 19 yellow-podded lines present in the John Innes Pisum germplasm collection, one of which is described as “Pellew’s gp”, and we found that all were allelic to gp carried by the type-line, JI0128. This showed that Mendel’s yellow-podded characteris conditioned by variation at a single locus. We used genetic mapping of Axiom markers in a large F2 population to define the position of gp within a 4.4 cM interval, encompassing approximately 8 Mb. We used exome capture to compare genome structures within this interval, in 227 Gp accessions and the 19 gp accessions. We found that all 19 gp lines carry the same large 100 kb deletion, while all 227 Gp accessions do not, indicating that only one gp haplotype exists in the germplasm we studied, and that gp must have been crossed into different backgrounds by breeders and researchers in the past. Three candidate genes associated with the deletion were assessed. After crossing a null TiLLING mutant for one of these candidates with a gp line, yellow-podded F1 progeny were obtained. This genetic complementation test showed that we have identified a new allele of the gp gene. We used backcross lines to gain a better understanding of the mutant phenotype. It was alreadyknown that thylakoid membranes are underdeveloped in gp lines (Price et al., 1988). Additional effects will be described

Identification of mendel’s pod colour character in pea: characterisation of the allele conditioning yellow pod colour

International audienceThe inheritance of yellow-podded versus green-podded peas was studied by Mendel more than 150 years ago, but, until now, the allelic variation underlying this difference remained unknown. A report of a novel, spontaneous gp mutant (Pellew and Sverdrup, 1923) suggested that at least two independent gp alleles existed. We undertook genetic complementation crosses with 19 yellow-podded lines present in the John Innes Pisum germplasm collection, one of which is described as “Pellew’s gp”, and we found that all were allelic to gp carried by the type-line, JI0128. This showed that Mendel’s yellow-podded characteris conditioned by variation at a single locus. We used genetic mapping of Axiom markers in a large F2 population to define the position of gp within a 4.4 cM interval, encompassing approximately 8 Mb. We used exome capture to compare genome structures within this interval, in 227 Gp accessions and the 19 gp accessions. We found that all 19 gp lines carry the same large 100 kb deletion, while all 227 Gp accessions do not, indicating that only one gp haplotype exists in the germplasm we studied, and that gp must have been crossed into different backgrounds by breeders and researchers in the past. Three candidate genes associated with the deletion were assessed. After crossing a null TiLLING mutant for one of these candidates with a gp line, yellow-podded F1 progeny were obtained. This genetic complementation test showed that we have identified a new allele of the gp gene. We used backcross lines to gain a better understanding of the mutant phenotype. It was alreadyknown that thylakoid membranes are underdeveloped in gp lines (Price et al., 1988). Additional effects will be described

Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of <i>Streptomyces coelicolor</i> by Targeted LC–MS/MS

The filamentous bacterium <i>Streptomyces coelicolor</i> modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC–MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC–MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095

PepLine: A Software Pipeline for High-Throughput Direct Mapping of Tandem Mass Spectrometry Data on Genomic Sequences

International audienc

Additional file 2: Table S1. of Evolution of the EKA family of powdery mildew avirulence-effector genes from the ORF 1 of a LINE retrotransposon

Mass spectometric data results for proteomic analysis using the database of predicted EKA proteins merged with the Bgh DH14 protein database. (XLSX 33 kb