Identification of genetic modifiers of ACCELERATED CELL DEATH 6 (ACD6) in natural Arabidopsis thaliana accessions

Plants defend themselves against pathogens by activating responses that can also cause unintended collateral damage to the plant itself. Improved understanding of the evolutionary constraints and molecular mechanisms affecting these responses can provide means to minimize the tradeoff between disease-related losses and hyperimmunity-related yield drag in crops. As a model to investigate this problem, I have exploited natural variation at the ACCELERATED CELL DEATH 6 (ACD6) gene, which controls a major trade-off between growth and disease resistance among natural accessions of Arabidopsis thaliana. The hyperactive allele ACD6-Est-1 is known to confer broad-spectrum immunity, but at the same time to also negatively affect growth in many A. thaliana accessions. Here, I first surveyed a large collection of A. thaliana genomes for the presence of Est-like ACD6 alleles. I confirmed that not all accessions with this allele express overt hyperimmunity. I then demonstrated that Est-like ACD6 alleles from accessions that do not show the typical autoimmune phenotype normally associated with this allele could confer hyperimmunity when transformed into a different genetic background, indicating that the attenuation of the Est-like ACD6 phenotype was likely due to extragenic modifiers. I then investigated pathogen responses of several of these accessions more closely. My experiments revealed that reduced growth and immune responses were partially uncoupled in some of these accessions. These findings dovetailed with genetic results suggesting that different accessions contain genetically distinct modifiers of the typical Est-like ACD6 phenotype. Finally, I demonstrated by quantitative trait loci (QTL) mapping that these modifiers are located in different regions of the genome, with one of the modifiers potentially being a gene in cluster of genes encoding nucleotide-binding domain and leucine-rich repeat (NLR) immune receptors. This is an important finding, as ACD6 had previously been linked only to PAMP-triggered immunity (PTI), but not to effector-triggered immunity (ETI), which predominantly relies on NLR immune receptors. My study thus provides new insights into the complex genetic interactions that affect disease resistance and growth

The WIP6 transcription factor TOO MANY LATERALS specifies vein type in C4 and C3 grass leaves

Grass leaves are invariantly strap shaped with an elongated distal blade and a proximal sheath that wraps around the stem. Underpinning this shape is a scaffold of leaf veins, most of which extend in parallel along the proximo-distal leaf axis. Differences between species are apparent both in the vein types that develop and in the distance between veins across the medio-lateral leaf axis. A prominent engineering goal is to increase vein density in leaves of C3 photosynthesizing species to facilitate the introduction of the more efficient C4 pathway. Here, we discover that the WIP6 transcription factor TOO MANY LATERALS (TML) specifies vein rank in both maize (C4) and rice (C3). Loss-of-function tml mutations cause large lateral veins to develop in positions normally occupied by smaller intermediate veins, and TML transcript localization in wild-type leaves is consistent with a role in suppressing lateral vein development in procambial cells that form intermediate veins. Attempts to manipulate TML function in rice were unsuccessful because transgene expression was silenced, suggesting that precise TML expression is essential for shoot viability. This finding may reflect the need to prevent the inappropriate activation of downstream targets or, given that transcriptome analysis revealed altered cytokinin and auxin signaling profiles in maize tml mutants, the need to prevent local or general hormonal imbalances. Importantly, rice tml mutants display an increased occupancy of veins in the leaf, providing a step toward an anatomical chassis for C4 engineering. Collectively, a conserved mechanism of vein rank specification in grass leaves has been revealed

Flax latitudinal adaptation at LuTFL1 altered architecture and promoted fiber production

After domestication in the Near East around 10,000 years ago several founder crops, flax included, spread to European latitudes. On reaching northerly latitudes the architecture of domesticated flax became more suitable to fiber production over oil, with longer stems, smaller seeds and fewer axillary branches. Latitudinal adaptations in crops typically result in changes in flowering time, often involving the PEBP family of genes that also have the potential to influence plant architecture. Two PEBP family genes in the flax genome, LuTFL1 and LuTFL2, vary in wild and cultivated flax over latitudinal range with cultivated flax receiving LuTFL1 alleles from northerly wild flax populations. Compared to a background of population structure of flaxes over latitude, the LuTFL1 alleles display a level of differentiation that is consistent with selection for an allele III in the north. We demonstrate through heterologous expression in Arabidopsis thaliana that LuTFL1 is a functional homolog of TFL1 in A. thaliana capable of changing both flowering time and plant architecture. We conclude that specialized fiber flax types could have formed as a consequence of a natural adaptation of cultivated flax to higher latitudes

Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1-dependent differences

Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine‐rich repeat receptor kinases (LRR‐RK) FLS2 and EFR, and the LRR receptor protein (LRR‐RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR‐RK and LRR‐RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP‐mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23‐regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2‐signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP‐type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity

Activation of the Arabidopsis thaliana Immune System by Combinations of Common ACD6 Alleles

A fundamental question in biology is how multicellular organisms distinguish self and non-self. The ability to make this distinction allows animals and plants to detect and respond to pathogens without triggering immune reactions directed against their own cells. In plants, inappropriate self-recognition results in the autonomous activation of the immune system, causing affected individuals to grow less well. These plants also suffer from spontaneous cell death, but are at the same time more resistant to pathogens. Known causes for such autonomous activation of the immune system are hyperactive alleles of immune regulators, or epistatic interactions between immune regulators and unlinked genes. We have discovered a third class, in which the Arabidopsis thaliana immune system is activated by interactions between natural alleles at a single locus, ACCELERATED CELL DEATH 6 (ACD6). There are two main types of these interacting alleles, one of which has evolved recently by partial resurrection of a pseudogene, and each type includes multiple functional variants. Most previously studies hybrid necrosis cases involve rare alleles found in geographically unrelated populations. These two types of ACD6 alleles instead occur at low frequency throughout the range of the species, and have risen to high frequency in the Northeast of Spain, suggesting a role in local adaptation. In addition, such hybrids occur in these populations in the wild. The extensive functional variation among ACD6 alleles points to a central role of this locus in fine-tuning pathogen defenses in natural populations

Flax latitudinal adaptation at LuTFL1 altered architecture and promoted fiber production

After domestication in the Near East around 10,000 years ago several founder crops, flax included, spread to European latitudes. On reaching northerly latitudes the architecture of domesticated flax became more suitable to fiber production over oil, with longer stems, smaller seeds and fewer axillary branches. Latitudinal adaptations in crops typically result in changes in flowering time, often involving the PEBP family of genes that also have the potential to influence plant architecture. Two PEBP family genes in the flax genome, LuTFL1 and LuTFL2, vary in wild and cultivated flax over latitudinal range with cultivated flax receiving LuTFL1 alleles from northerly wild flax populations. Compared to a background of population structure of flaxes over latitude, the LuTFL1 alleles display a level of differentiation that is consistent with selection for an allele III in the north. We demonstrate through heterologous expression in Arabidopsis thaliana that LuTFL1 is a functional homolog of TFL1 in A. thaliana capable of changing both flowering time and plant architecture. We conclude that specialized fiber flax types could have formed as a consequence of a natural adaptation of cultivated flax to higher latitudes

Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development

Leaves of flowering plants are characterised by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyse the spatiotemporal localisation of a subset of auxin related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification, and how major and minor vein ranks develop with distinct identities. The localisation of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is pre-patterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarised as differentiation proceeds. Collectively our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves