Claude W. Depamphilis

We present the first chromosome-level genome assembly and annotation for the genus Cuscuta, a twining and leafless parasitic plant of the morning glory family (Convolvulaceae). C. campestris, the study species, is a widely studied model parasite, due in part to its worldwide occurrence as a weed of agricultural and natural plant communities. The species has served as a model parasite for studies of parasite biology, haustorium development, growth responses to chemical and light stimuli, gene content and expression, horizontal gene transfer, and interspecies RNA movement and has a recently developed transformation system. The 505 Mb (1C) genome is assembled into 31 chromosomes and supports annotation of 47,199 protein-coding genes, 214 small RNA loci (including 146 haustoria-specific miRNAs), and 3,238 interspecies mobile mRNA loci. C. campestris is a recent tetraploid with a high retention of duplicated genes and chromosomes, with less than 8% nucleotide divergence between homoeologous chromosomes. We also show that transformation of C. campestris with the RUBY marker system allows visualization of transformed Cuscuta-derived fluorescent mobile molecules that have entered the host stem. This genome, with an associated genome browser and BLAST server, will be of value for scientists performing fundamental research in a wide range of molecular, developmental, population, and evolutionary biology, as well as serve as a research tool for studying interspecies mobile molecules, generating genetic markers for species and genotype identification, and developing highly specific herbicides.

Parasitic plants in Orobanchaceae are noxious agricultural pests that severely impact crops worldwide. These plants acquire water and nutrients from their hosts through a specialized organ called the haustorium. A key step in haustorium development involves cell wall modification. In this study, we identified and analyzed the evolutionary relationships of pectate lyase-like ( PLL ) genes across parasitic plants and other non-parasitic land plant lineages. To support detailed examination of gene models and paralogous gene family members, we used published parasitic plant genomes, as well as a recently generated draft genome assembly and annotation of Triphysaria versicolor . One particular PLL gene, denoted as PLL1 in parasitic Orobanchaceae, emerged as an important candidate gene for parasitism. Our previous comparative transcriptomic analyses showed that PLL1 underwent neofunctionalization via an expression shift from floral tissues in non-parasitic relatives to haustoria in parasitic species. It belongs to the largest sub-clade of the PLL gene family, is highly upregulated in haustoria, and shows signatures of relaxed purifying selection and 15 individual sites with signatures of adaptive evolution. To explore its function in haustorium development, we manipulated PLL1 expression in T. versicolor , a model parasitic species from Orobanchaceae, using direct transformation with the parasite and host-induced-gene-silencing (HIGS). For HIGS, we generated transgenic Medicago hosts expressing hairpin RNAs targeting the PLL1 gene in T. versicolor . An average 60% reduction of PLL1 transcript level was observed in both direct transformation and HIGS treatments, leading to an increased frequency of poorly adhered parasites with fewer xylem connections and a smaller proportion of mature haustoria. These findings demonstrate that PLL1 plays a crucial role in haustorium development and suggest it as a promising target for managing parasitic weeds. Notably, the success of HIGS even before the establishment of a functional haustorium highlights the possibility of early intervention against parasitism.

Mycoheterotrophy is an alternative nutritional strategy whereby plants obtain sugars and other nutrients from soil fungi. Mycoheterotrophy and associated loss of photosynthesis have evolved repeatedly in plants, particularly in monocots. Although reductive evolution of plastomes in mycoheterotrophs is well documented, the dynamics of nuclear genome evolution remains largely unknown. Transcriptome datasets were generated from four mycoheterotrophs in three families (Orchidaceae, Burmanniaceae, Triuridaceae) and related green plants and used for phylogenomic analyses to resolve relationships among the mycoheterotrophs, their relatives, and representatives across the monocots. Phylogenetic trees based on 602 genes were mostly congruent with plastome phylogenies, except for an Asparagales + Liliales clade inferred in the nuclear trees. Reduction and loss of chlorophyll synthesis and photosynthetic gene expression and relaxation of purifying selection on retained genes were progressive, with greater loss in older nonphotosynthetic lineages. One hundred seventy-four of 1375 plant benchmark universally conserved orthologous genes were undetected in any mycoheterotroph transcriptome or the genome of the mycoheterotrophic orchid Gastrodia but were expressed in green relatives, providing evidence for massively convergent gene loss in nonphotosynthetic lineages. We designate this set of deleted or undetected genes Missing in Mycoheterotrophs (MIM). MIM genes encode not only mainly photosynthetic or plastid membrane proteins but also a diverse set of plastid processes, genes of unknown function, mitochondrial, and cellular processes. Transcription of a photosystem II gene (psb29) in all lineages implies a nonphotosynthetic function for this and other genes retained in mycoheterotrophs. Nonphotosynthetic plants enable novel insights into gene function as well as gene expression shifts, gene loss, and convergence in nuclear genomes.

Plant genome-scale resources are being generated at an increasing rate as sequencing technologies continue to improve and raw data costs continue to fall; however, the cost of downstream analyses remains large. This has resulted in a considerable range of genome assembly and annotation qualities across plant genomes due to their varying sizes, complexity, and the technology used for the assembly and annotation. To effectively work across genomes, researchers increasingly rely on comparative genomic approaches that integrate across plant community resources and data types. Such efforts have aided the genome annotation process and yielded novel insights into the evolutionary history of genomes and gene families, including complex non-model organisms. The essential tools to achieve these insights rely on gene family analysis at a genome-scale, but they are not well integrated for rapid analysis of new data, and the learning curve can be steep. Here we present PlantTribes2, a scalable, easily accessible, highly customizable, and broadly applicable gene family analysis framework with multiple entry points including user provided data. It uses objective classifications of annotated protein sequences from existing, high-quality plant genomes for comparative and evolutionary studies. PlantTribes2 can improve transcript models and then sort them, either genome-scale annotations or individual gene coding sequences, into pre-computed orthologous gene family clusters with rich functional annotation information. Then, for gene families of interest, PlantTribes2 performs downstream analyses and customizable visualizations including, (1) multiple sequence alignment, (2) gene family phylogeny, (3) estimation of synonymous and non-synonymous substitution rates among homologous sequences, and (4) inference of large-scale duplication events. We give examples of PlantTribes2 applications in functional genomic studies of economically important plant families, namely transcriptomics in the weedy Orobanchaceae and a core orthogroup analysis (CROG) in Rosaceae. PlantTribes2 is freely available for use within the main public Galaxy instance and can be downloaded from GitHub or Bioconda. Importantly, PlantTribes2 can be readily adapted for use with genomic and transcriptomic data from any kind of organism.

Premise: Parasitic plants and their hosts are model systems for studying genetic variation in species interactions across environments. The parasitic plant Striga hermonthica (witchweed) attacks a range of cereal crop hosts in Africa and exhibits substantial variation in performance on different host species. Some of this variation is due to local adaptation, but the genetic basis of specialization on certain hosts is unknown. Methods: To identify genomic regions that are strongly differentiated between parasites attacking different host species, we present an alignment-free analysis of S. hermonthica population diversity using whole genome sequencing (WGS) data for 68 individuals from western Kenya. We validate our findings with germination experiments and analyses based on a de novo assembled draft genome. Results: Reference-free and reference-based analyses suggest that only a small portion of the S. hermonthica genome is strongly differentiated by host species in populations from western Kenya. Analysis of host-associated k -mers implicated genes involved in development of the parasite haustorium (a specialized structure used to establish vascular connections with host roots) and a potential role of chemocyanins in molecular host-parasitic plant interactions. Conversely, no phenotypic or genomic evidence was observed suggesting host-specific selection on parasite response to strigolactones, hormones exuded by host roots and required for parasite germination. Conclusions: This study demonstrates the utility of WGS for plant species with large, complex genomes and no available reference. Contrasting with theory emphasizing the role of early recognition loci for host specificity, our findings support host-specific selection on later interaction stages, recurring each generation after homogenizing gene flow.

The rapid development of sequencing technologies has led to a deeper understanding of horticultural plant genomes. However, experimental evidence connecting genes to important agronomic traits is still lacking in most non-model organisms. For instance, the genetic mechanisms underlying plant architecture are poorly understood in pome fruit trees, creating a major hurdle in developing new cultivars with desirable architecture, such as dwarfing rootstocks in European pear ( Pyrus communis ). Further, the quality and content of genomes vary widely. Therefore, it can be challenging to curate a list of genes with high-confidence gene models across reference genomes. This is often an important first step towards identifying key genetic factors for important traits. Here we present a draft genome of P. communis ‘d’Anjou’ and an improved assembly of the latest P. communis ‘Bartlett’ genome. To study gene families involved in tree architecture in European pear and other rosaceous species, we developed a workflow using a collection of bioinformatic tools towards curation of gene families of interest across genomes. This lays the groundwork for future functional studies in pear tree architecture. Importantly, our workflow can be easily adopted for other plant genomes and gene families of interest.

Aristolochia, a genus in the magnoliid order Piperales, has been famous for centuries for its highly specialized flowers and wide medicinal applications. Here, we present a new, high-quality genome sequence ofAristolochia fimbriata, a species that, similar toAmborella trichopoda, lacks further whole-genome duplications since the origin of extant angiosperms. As such, theA. fimbriatagenome is an excellent reference for inferences of angiosperm genome evolution, enabling detection of two novel whole-genome duplications in Piperales and dating of previously reported whole-genome duplications in other magnoliids. Genomic comparisons betweenA. fimbriataand other angiosperms facilitated the identification of ancient genomic rearrangements suggesting the placement of magnoliids as sister to monocots, whereas phylogenetic inferences based on sequence data we compiled yielded ambiguous relationships. By identifying associated homologues and investigating their evolutionary histories and expression patterns, we revealed highly conserved floral developmental genes and their distinct downstream regulatory network that may contribute to the complex flower morphology inA. fimbriata. Finally, we elucidated the genetic basis underlying the biosynthesis of terpenoids and aristolochic acids inA. fimbriata.

Fitness responses to environment can shape species distributions, though opposing eco‐evolutionary processes can obscure environmental effects. For example, host specificity influences parasite dynamics, but is unclear how adaptation of parasites to local host communities may scale up to continental distributions. Here, we develop a macroecological framework to determine how host community structure affects the distribution of specialist and generalist populations of Striga hermonthica , an African parasitic plant of cereal crops. Combining data from global crop production and parasite experimental trials, we find that parasites perform best on the host species that is most common in their location of origin. Moreover, niche model contrasts predict parasite specialization on two hosts that evolved alongside Striga during domestication (pearl millet and sorghum), indicating that specialist parasites may be most likely to occur where host niches differ most in multivariate environmental space. Our study demonstrates that patterns of parasite local adaptation to host communities can emerge at continental scales and that differential environmental tolerances of hosts indirectly shape the distribution of specialist and generalist parasites. By predicting spatial dynamics of parasite specialization versus generalization directly from environmental data, our approach may help inform current and future management of pests and disease.

Parasitic plants engage in a complex molecular dialog with potential host plants to identify a host and overcome host defenses to initiate development of the parasitic feeding organ, the haustorium, invade host tissues, and withdraw water and nutrients. While one of two critical signaling events in the parasitic plant life cycle (germination via stimulant chemicals) has been relatively well-studied, the signaling event that triggers haustorium formation remains elusive. Elucidation of this poorly understood molecular dialogue will shed light on plant-plant communication, parasitic plant physiology, and the evolution of parasitism in plants. Here we present an experimental framework that develops easily quantifiable contrasts for the facultative generalist parasitic plant, Triphysaria, as it feeds across a broad range of diverse flowering plants. The contrasts, including variable parasite growth form and mortality when grown with different hosts, suggest a dynamic and host-dependent molecular dialogue between the parasite and host. Finally, by comparing transcriptome datasets from attached versus unattached parasites we gain insight into some of the physiological processes that are altered during parasitic behavior including shifts in photosynthesis-related and stress response genes. This work sheds light on Triphysaria’s parasitic life habit and is an important step towards understanding the mechanisms of haustorium initiation factor perception, a unique form of plant-plant communication.

Horizontal gene transfer (HGT), the movement and genomic integration of DNA across species boundaries, is commonly associated with bacteria and other microorganisms, but functional HGT (fHGT) is increasingly being recognized in heterotrophic parasitic plants that obtain their nutrients and water from their host plants through direct haustorial feeding. Here, in the holoparasitic stem parasite Cuscuta, we identify 108 transcribed and probably functional HGT events in Cuscuta campestris and related species, plus 42 additional regions with host-derived transposon, pseudogene and non-coding sequences. Surprisingly, 18 Cuscuta fHGTs were acquired from the same gene families by independent HGT events in Orobanchaceae parasites, and the majority are highly expressed in the haustorial feeding structures in both lineages. Convergent retention and expression of HGT sequences suggests an adaptive role for specific additional genes in parasite biology. Between 16 and 20 of the transcribed HGT events are inferred as ancestral in Cuscuta based on transcriptome sequences from species across the phylogenetic range of the genus, implicating fHGT in the successful radiation of Cuscuta parasites. Genome sequencing of C. campestris supports transfer of genomic DNA-rather than retroprocessed RNA-as the mechanism of fHGT. Many of the C. campestris genes horizontally acquired are also frequent sources of 24-nucleotide small RNAs that are typically associated with RNA-directed DNA methylation. One HGT encoding a leucine-rich repeat protein kinase overlaps with a microRNA that has been shown to regulate host gene expression, suggesting that HGT-derived parasite small RNAs may function in the parasite-host interaction. This study enriches our understanding of HGT by describing a parasite-host system with unprecedented gene exchange that points to convergent evolution of HGT events and the functional importance of horizontally transferred coding and non-coding sequences.