The pan-NLR'ome of Arabidopsis thaliana

Plants are the major nutritional component of the human diet, provide us with shel- ter, fuel, and enjoyment. Substantial yield loss is caused by plant diseases transmitted by bacteria, fungi, and oomycete pathogens. Plants have an elaborate innate immune system to fight threatening pathogens, relying to a great extend on highly variable re- sistance (R) genes. R genes often encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogens by the presence or the activity of effector proteins in the plants’ cells. NLRs contain variable N-terminal domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The N-terminal domains can be used to distinguish between the evolutionary conserved NLR classes TNL (with a toll/interleucin-1 receptor homology (TIR) domain), CNL (with a coiled-coil (CC) domain), and RNL (with an RPW8 domain). The archi- tectural diversity is increased by additional integrated domains (IDs) found in different positions. Plant species have between a few dozen and several hundred NLRs. The intraspecific R gene diversity is also high, and the still few known NLRs responsible for long-term resistance are often accession-specific. Intraspecific NLR studies to date suffer from several shortcomings: The pan-NLR’omes (the collection of all NLR genes and alleles occurring in a species) can often not be comprehensively described because too few accessions are analyzed, and NLR detection is essentially always guided by reference genomes, which biases the detection of novel genes and alleles. In addition, inappropriate or immature bioinformatics analysis pipelines may miss NLRs during the assembly or annotation phase, or result in erroneous NLR annotations. Knowing the pan-NLR’ome of a plant species is key to obtain novel resistant plants in the future. I created an extensive and reliable database that defines the near-complete pan-NLR’ome of the model plant Arabidopsis thaliana. Efforts were focused on a panel of 65 diverse accessions and applied state-of-the-art targeted long read sequencing (SMRT RenSeq). My analysis pipeline was designed to include optimized methods that could be applied to any SMRT RenSeq project. In the first part of my thesis I set quality control standards for the assembly of NLR-coding genomic fragments. I further introduce a novel and thorough gene annotation pipeline, supported by careful manual curation. In the second part, I present the manuscript reporting the saturated near-complete A. thaliana pan- NLR’ome. The species-wide high NLR diversity is revealed on the domain architecture level, and the usage of novel IDs is highlighted. The core NLR complement is defined and presence-absence polymorphisms in non-core NLRs are described. Furthermore, haplotype saturation is shown, selective forces are quantified, and evolutionary coupled co-evolving NLRs are detected. The method optimization results show that final NLR assembly quality is mainly influenced by the amount and the quality of input sequencing data. The results further show that manual curation of automated NLR predictions are crucial to prevent frequently occurring misannotations. The saturation of an NLR’ome has not been shown in any plant species so far, thus this study provides an unprecedented view on intraspecific NLR variation, the core NLR complement, and the evolutionary trajectories of NLRs. IDs are more frequently used than known before, suggesting a pivotal role of noncanonical NLRs in plant-pathogen interactions. This work sets new standards for the analysis of gene families at the species level. Future NLR’ome projects applied to important crop species will profit from my results and the easy-to-adopt anal- ysis pipeline. Ultimately, this will extend our knowledge of intraspecific NLR diversity beyond few reference species or genomes, and will facilitate the detection of functional NLRs, to be used in disease resistance breeding programs

A species-wide inventory of NLR genes and alleles in Arabidopsis thaliana

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes

miRNA Profiles of Canine Intestinal Carcinomas, Lymphomas and Enteritis Analysed by Digital Droplet PCR from FFPE Material

Most canine intestinal tumours are B-cell or T-cell lymphomas or carcinomas. They have to be distinguished from cases of enteritis. Non-invasive biomarkers such as miRNAs would be a step towards faster diagnosis. The aim of this study was to investigate shifts in miRNA expression in tissue samples collected from cases of enteritis, carcinoma and lymphoma of the small and large intestine to better understand the potential of miRNA as biomarkers for tumour diagnosis and classification. We selected two oncogenic miRNAs (miR-18b and 20b), two tumour suppressive miRNAs (miR-192 and 194) and two potential biomarkers for neoplasms (miR-126 and 214). They were isolated from FFPE material, quantified by ddPCR, normalised with RNU6B and compared with normal tissue values. Our results confirmed that ddPCR is a suitable method for quantifying miRNA from FFPE material. Expression of miR-18b and miR-192 was higher in carcinomas of the small intestine than in those of the large intestine. Specific miRNA patterns were observed in cases of enteritis, B-cell and T-cell lymphoma and carcinoma. However, oncogenic miR-18b and 20b were not elevated in any group and miR-126 and 214 were down-regulated in T-cell and B-cell lymphoma, as well as in carcinomas and lymphoplasmacytic enteritis of the small intestine

Mutual Facilitation Among Invading Nuttall’s Waterweed and Quagga Mussels

Nuttall’s waterweed (Elodea nuttallii) is the most abundant invasive aquatic plant species in several European countries. Elodea populations often follow a boom-bust cycle, but the causes and consequences of this dynamics are yet unknown. We hypothesize that both boom and bust periods can be affected by dreissenid mussel invasions. While mutual facilitations between these invaders could explain their rapid parallel expansion, subsequent competition for space might occur. To test this hypothesis, we use data on temporal changes in the water quality and the abundance of E. nuttallii and the quagga mussel Dreissena rostriformis bugensis in a temperate shallow lake. Lake Müggel(see Germany) was turbid and devoid of submerged macrophytes for 20 years (1970–1989), but re-colonization with macrophytes started in 1990 upon reductions in nutrient loading. We mapped macrophyte abundance from 1999 and mussel abundance from 2011 onwards. E. nuttallii was first detected in 2011, spread rapidly, and was the most abundant macrophyte species by 2017. Native macrophyte species were not replaced, but spread more slowly, resulting in an overall increase in macrophyte coverage to 25% of the lake surface. The increased abundance of E. nuttallii was paralleled by increasing water clarity and decreasing total phosphorus concentrations in the water. These changes were attributed to a rapid invasion by quagga mussels in 2012. In 2017, they covered about one-third of the lake area, with mean abundances of 3,600 mussels m−2, filtering up to twice the lake’s volume every day. The increasing light availability in deeper littoral areas supported the rapid spread of waterweed, while in turn waterweed provided surface for mussel colonization. Quantities of dreissenid mussels and E. nuttallii measured at 24 locations were significantly correlated in 2016, and yearly means of E. nuttallii quantities increased with increasing mean dreissenid mussel quantities between 2011 and 2018. In 2018, both E. nuttallii and dreissenid abundances declined. These data imply that invasive waterweed and quagga mussels initially facilitated their establishment, supporting the invasional meltdown hypothesis, while subsequently competition for space may have occurred. Such temporal changes in invasive species interaction might contribute to the boom-bust dynamics that have been observed in Elodea populations

A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function

As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS-but not a truncated version lacking the F-box domain-display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission ("skirt") phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function

Modulation of ACD6 dependent hyperimmunity by natural alleles of an Arabidopsis thaliana NLR resistance gene

Plants defend themselves against pathogens by activating an array of immune responses. Unfortunately, immunity programs may also cause unintended collateral damage to the plant itself. The quantitative disease resistance gene ACCELERATED CELL DEATH 6 (ACD6) serves to balance growth and pathogen resistance in natural populations of Arabidopsis thaliana. An autoimmune allele, ACD6-Est, which strongly reduces growth under specific laboratory conditions, is found in over 10% of wild strains. There is, however, extensive variation in the strength of the autoimmune phenotype expressed by strains with an ACD6-Est allele, indicative of genetic modifiers. Quantitative genetic analysis suggests that ACD6 activity can be modulated in diverse ways, with different strains often carrying different large-effect modifiers. One modifier is SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1), located in a highly polymorphic cluster of nucleotide-binding domain and leucinerich repeat (NLR) immune receptor genes, which are prototypes for qualitative disease resistance genes. Allelic variation at SNC1 correlates with ACD6-Est activity in multiple accessions, and a common structural variant affecting the NL linker sequence can explain differences in SNC1 activity. Taken together, we find that an NLR gene can mask the activity of an ACD6 autoimmune allele in natural A. thaliana populations, thereby linking different arms of the plant immune system. © 2018 Zhu et al

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes. In plants, NLR proteins are important intracellular receptors with roles in innate immunity and disease resistance. This work provides a panoramic view of this diverse and complicated gene family in the model species A. thaliana and provides a foundation for the identification and functional study of disease-resistance genes in agronomically important species with complex genomes

Modulation of ACD6 dependent hyperimmunity by natural alleles of an Arabidopsis thaliana NLR resistance gene.

Plants defend themselves against pathogens by activating an array of immune responses. Unfortunately, immunity programs may also cause unintended collateral damage to the plant itself. The quantitative disease resistance gene ACCELERATED CELL DEATH 6 (ACD6) serves to balance growth and pathogen resistance in natural populations of Arabidopsis thaliana. An autoimmune allele, ACD6-Est, which strongly reduces growth under specific laboratory conditions, is found in over 10% of wild strains. There is, however, extensive variation in the strength of the autoimmune phenotype expressed by strains with an ACD6-Est allele, indicative of genetic modifiers. Quantitative genetic analysis suggests that ACD6 activity can be modulated in diverse ways, with different strains often carrying different large-effect modifiers. One modifier is SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1), located in a highly polymorphic cluster of nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes, which are prototypes for qualitative disease resistance genes. Allelic variation at SNC1 correlates with ACD6-Est activity in multiple accessions, and a common structural variant affecting the NL linker sequence can explain differences in SNC1 activity. Taken together, we find that an NLR gene can mask the activity of an ACD6 autoimmune allele in natural A. thaliana populations, thereby linking different arms of the plant immune system