SummaryCrop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine whether similar boom-and-bust scenarios hold for wild pathosystems, we carried out a multi-year, multi-site survey of Pseudomonas in its natural host Arabidopsis thaliana. The most common Pseudomonas lineage corresponded to a ubiquitous pathogenic clade. Sequencing of 1,524 genomes revealed this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically identifiable pathogenic sublineages. There is differentiation at the level of both gene content and disease phenotype, although the differentiation may not provide fitness advantages to specific sublineages. The coexistence of sublineages indicates that in contrast to crop systems, no single strain has been able to overtake the studied A. thaliana populations in the recent past. Our results suggest that selective pressures acting on a plant pathogen in wild hosts are likely to be much more complex than those in agricultural systems.

SignificanceWhile evolution has been thought of as playing out over millions of years, adaptation to new environments can occur very rapidly, presenting us with key opportunities to understand evolutionary dynamics. One of the most amazing examples of real-time evolution comes from agriculture, where due to the intense use of a few herbicides, many plant species have evolved herbicide resistance to become aggressive weeds. An important question has been whether herbicide resistance arises only rarely and then spreads quickly, or whether herbicide resistance arises all the time de novo. Our work with glyphosate resistance in US Midwestern and Canadian populations of Amaranthus tuberculatus reveals the answer to be, “it depends,” as we surprisingly find examples for both modes of evolution.

The selection pressure exerted by herbicides has led to the repeated evolution of herbicide resistance in weeds. The evolution of herbicide resistance on contemporary timescales in turn provides an outstanding opportunity to investigate key questions about the genetics of adaptation, in particular, the relative importance of adaptation from new mutations, standing genetic variation, or geographic spread of adaptive alleles through gene flow. Glyphosate-resistant Amaranthus tuberculatus poses one of the most significant threats to crop yields in the midwestern United States (1), with both agricultural populations and herbicide resistance only recently emerging in Canada (2, 3). To understand the evolutionary mechanisms driving the spread of resistance, we sequenced and assembled the A. tuberculatus genome and investigated the origins and population genomics of 163 resequenced glyphosate-resistant and susceptible individuals from Canada and the USA. In Canada, we discovered multiple modes of convergent evolution: in one locality, resistance appears to have evolved through introductions of preadapted US genotypes, while in another, there is evidence for the independent evolution of resistance on genomic backgrounds that are historically nonagricultural. Moreover, resistance on these local, non-agricultural backgrounds appears to have occurred predominantly through the partial sweep of a single haplotype. In contrast, resistant haplotypes arising from the midwestern US show multiple amplification haplotypes segregating both between and within populations. Therefore, while the remarkable species-wide diversity of A. tuberculatus has facilitated geographic parallel adaptation of glyphosate resistance, more recently established agricultural populations are limited to adaptation in a more mutation-limited framework.

Microorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, support the acquisition of nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of the 16S rRNA gene and/or the internal transcribed spacer (ITS) of rRNA genomic loci, which show the relative abundance of the microbes to each other. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S and eukaryotic ITS1 rRNA amplicon data from 176 of these samples. Shotgun data, which unlike the amplicon data capture the ratio of microbe to plant DNA, enable scaling of microbial read abundances to reflect the microbial load on the host. In a more cost-effective hybrid strategy, we show they also allow a similar scaling of amplicon data to overcome compositionality problems. Our wild plants were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. Microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. Critically, considering absolute microbial load led to fundamentally different conclusions about microbiome assembly and the interaction networks among major taxa.

A central goal in microbiome research is to learn what distinguishes a healthy from a dysbiotic microbial community. Shifts in diversity and taxonomic composition are important indicators of dysbiosis, but a full understanding also requires knowledge of absolute microbial biomass. Simultaneous information on both microbiome composition and the quantity of its components can provide insight into microbiome function and disease state. Here we use shotgun metagenomics to simultaneously assess microbiome composition and microbial load in the phyllosphere of wild populations of the plant Arabidopsis thaliana. We find that wild plants vary substantially in the load of colonizing microbes, and that high loads are typically associated with the proliferation of single taxa, with only a few putatively pathogenic taxa achieving high abundances in the field. Our results suggest (i) that the inside of a plant leaf is on average sparsely colonized with an estimated two bacterial genomes per plant genome and an order of magnitude fewer eukaryotic microbial genomes, and (ii) that higher levels of microbial biomass often indicate successful colonization by pathogens. Lastly, our results show that load is a significant explanatory variable for loss of estimated Shannon diversity in phyllosphere microbiomes, implying that reduced diversity may be a significant predictor of microbial dysbiosis in a plant leaf.

SummaryCrop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine 20 whether similar boom-and-bust scenarios hold for wild plant pathogens, we carried out a multi-year multi-site 21 survey of Pseudomonas in the natural host Arabidopsis thaliana. The most common Pseudomonas lineage 22 corresponded to a pathogenic clade present in all sites. Sequencing of 1,524 Pseudomonas genomes revealed 23 this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically distinct 24 pathogenic sublineages. These sublineages have expanded in parallel within the same populations and are 25 differentiated both at the level of gene content and disease phenotype. Such coexistence of diverse sublineages 26indicates that in contrast to crop systems, no single strain has been able to overtake these A. thaliana 27 populations in the recent past. Our results suggest that the selective pressures acting on a plant pathogen in 28 wild hosts may be more complex than those in agricultural systems. 29 Introduction 30In agricultural and clinical settings, pathogenic colonizations are frequently associated with expansions of single 31 or a few genetically identical microbial lineages (Butler et al., 2013; Cai et al., 2011;Kolmer, 2005; Park et al., 32 2015;Stukenbrock and McDonald, 2008; Yoshida et al., 2013). The conditions that lead to such epidemics-such 33 as reduced host genetic diversity (Zhu et al., 2000), absence of competing microbial communities (Brown et al., 34 2013) or high transmission rates (Park et al., 2015)-are, however, by no means a universal feature of 35 pathogenic infections. Instead, many, if not most, pathogens can colonize host populations that are both 36 genetically diverse and that can accommodate a diversity of other microbes (Barrett et al., 2009; Falkinham et 37 al., 2015;Woolhouse et al., 2001). 38Factors that drive pathogen success in such more complex situations are less well understood than for 39 clonal epidemics. For example, if a pathogen species persists at high numbers in non-host environments, does 40 each host become infected by a different pathogen strain? Or does a multitude of genetically distinct pathogens 41 infect each host? And do different colonizing strains use disparate mechanisms to become established even 42 within genetically similar host individuals? The answers to these questions inform on how (and if) a host 43 population can evolve partial or even complete pathogen resistance (Anderson and May, 1982; Barrett et al., 44 2009; Karasov et al., 2014a;Laine et al., 2011). Several studies over the past 20 years have attempted to infer 45 the distributions of non-epidemic pathogens in both host and non-host environments (Falkinham et al., 2015; 46 Wiehlmann et al., 2007). These studies, which have observed a range of different patterns, are unfortunately 47 often limited to the historic strains that are available, and the conclusions vary for different collections, even of 48 the same pathogen ...

Microorganisms from all domains of life establish associations with plants. Although some harm the plant, others antagonize pathogens or prime the plant immune system, acquire nutrients, tune plant hormone levels, or perform additional services. Most culture-independent plant microbiome research has focused on amplicon sequencing of 16S rDNA and/or the internal transcribed spacer (ITS) of rDNA loci, but the decreasing cost of high-throughput sequencing has made shotgun metagenome sequencing increasingly accessible. Here, we describe shotgun sequencing of 275 wild Arabidopsis thaliana leaf microbiomes from southwest Germany, with additional bacterial 16S rDNA and eukaryotic ITS1 amplicon data from 176 of these samples.The shotgun data were dominated by bacterial sequences, with eukaryotes contributing only a minority of reads. For shotgun and amplicon data, microbial membership showed weak associations with both site of origin and plant genotype, both of which were highly confounded in this dataset. There was large variation among microbiomes, with one extreme comprising samples of low complexity and a high load of microorganisms typical of infected plants, and the other extreme being samples of high complexity and a low microbial load. We use the 1 Regalado et al.Combining sequencing methods in plant metagenomics metagenome data, which captures the ratio of bacterial to plant DNA in leaves of wild plants, to scale the 16S rDNA amplicon data such that they reflect absolute bacterial abundance. We show that this cost-effective hybrid strategy overcomes compositionality problems in amplicon data and leads to fundamentally different conclusions about microbiome community assembly.

Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.