Division Zone Activity Determines the Potential of Drought-Stressed Maize Leaves to Resume Growth after Rehydration.

Drought is one of the most devastating causes of yield losses in crops like maize, and the anticipated increases in severity and duration of drought spells due to climate change pose an imminent threat to agricultural productivity. To understand the drought response, phenotypic and molecular studies are typically performed at a given time point after drought onset, representing a steady-state adaptation response. Because growth is a dynamic process, we monitored the drought response with high temporal resolution and examined cellular and transcriptomic changes after rehydration at 4 and 6 days after leaf four appearance. These data showed that division zone activity is a determinant for full organ growth recovery upon rehydration. Moreover, a prolonged maintenance of cell division by the ectopic expression of PLASTOCHRON1 extends the ability to resume growth after rehydration. The transcriptome analysis indicated that GROWTH-REGULATING FACTORS (GRFs) affect leaf growth by impacting cell division duration, which was confirmed by a prolonged recovery potential of the GRF1-overexpression line after rehydration. Finally, we used a multiplex genome editing approach to evaluate the most promising differentially expressed genes from the transcriptome study and as such narrowed down the gene space from 40 to seven genes for future functional characterization.

Antimicrobial activity of NK cells to Trypanosoma cruzi infected human primary Keratinocytes.

Infection with the protozoan parasite Trypanosoma cruzi is causative for Chagas disease, which is a highly neglected tropical disease prevalent in Latin America. Humans are primary infected through vectorial transmission by blood-sucking triatomine bugs. The parasite enters the human host through mucous membranes or small skin lesions. Since keratinocytes are the predominant cell type in the epidermis, they play a critical role in detecting disruptions in homeostasis and aiding in pathogen elimination by the immune system in the human skin as alternative antigen-presenting cells. Interestingly, keratinocytes also act as a reservoir for T. cruzi, as the skin has been identified as a major site of persistent infection in mice with chronic Chagas disease. Moreover, there are reports of the emergence of T. cruzi amastigote nests in the skin of immunocompromised individuals who are experiencing reactivation of Chagas disease. This observation implies that the skin may serve as a site for persistent parasite presence during chronic human infection too and underscores the significance of investigating the interactions between T. cruzi and skin cells. Consequently, the primary objective of this study was to establish and characterize the infection kinetics in human primary epidermal keratinocytes (hPEK). Our investigation focused on surface molecules that either facilitated or hindered the activation of natural killer (NK) cells, which play a crucial role in controlling the infection. To simulate the in vivo situation in humans, an autologous co-culture model was developed to examine the interactions between T. cruzi infected keratinocytes and NK cells. We evaluated the degranulation, cytokine production, and cytotoxicity of NK cells in response to the infected keratinocytes. We observed a strong activation of NK cells by infected keratinocytes, despite minimal alterations in the expression of activating or inhibitory ligands on NK cell receptors. However, stimulation with recombinant interferon-gamma (IFN-γ), a cytokine known to be present in significant quantities during chronic T. cruzi infections in the host, resulted in a substantial upregulation of these ligands on primary keratinocytes. Overall, our findings suggest the crucial role of NK cells in controlling acute T. cruzi infection in the upper layer of the skin and shed light on keratinocytes as potential initial targets of infection.

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.

Continual improvement of CRISPR-induced multiplex mutagenesis in Arabidopsis.

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.

Different Methods to Modify the Hydrophilicity of Titanium Implants with Biomimetic Surface Topography to Induce Variable Responses in Bone Marrow Stromal Cells.

The osteoblastic differentiation of bone marrow stromal cells (bMSCs), critical to the osseointegration of titanium implants, is enhanced on titanium surfaces with biomimetic topography, and this is further enhanced when the surfaces are hydrophilic. This is a result of changing the surface free energy to change protein adsorption, improving cell attachment and differentiation, and improving bone-to-implant contact in patients. In this study, we examined different methods of plasma treatment, a well-accepted method of increasing hydrophilicity, and evaluated changes in surface properties as well as the response of bMSCs in vitro. Commercially pure Ti and titanium-aluminum-vanadium (Ti6Al4V) disks were sand-blasted and acid-etched to impart microscale and nanoscale roughness, followed by treatment with various post-processing surface modification methods, including ultraviolet light (UV), dielectric barrier discharge (DBD)-generated plasma, and plasma treatment under an argon or oxygen atmosphere. Surface wettability was based on a sessile water drop measurement of contact angle; the elemental composition was analyzed using XPS, and changes in topography were characterized using scanning electron microscopy (SEM) and confocal imaging. The cell response was evaluated using bMSCs; outcome measures included the production of osteogenic markers, paracrine signaling factors, and immunomodulatory cytokines. All plasma treatments were effective in inducing superhydrophilic surfaces. Small but significant increases in surface roughness were observed following UV, DBD and argon plasma treatment. No other modifications to surface topography were noted. However, the relative composition of Ti, O, and C varied with the treatment method. The cell response to these hydrophilic surfaces depended on the plasma treatment method used. DBD plasma treatment significantly enhanced the osteogenic response of the bMSCs. In contrast, the bMSC response to argon plasma-treated surfaces was varied, with an increase in OPG production but a decrease in OCN production. These results indicate that post-packaging methods that increased hydrophilicity as measured by contact angle did not change the surface free energy in the same way, and accordingly, cells responded differently. Wettability and surface chemistry alone are not enough to declare whether an implant has an improved osteogenic effect and do not fully explain how surface free energy affects cell response.

Apoptotic cell identity induces distinct functional responses to IL-4 in efferocytic macrophages.

Macrophages are functionally heterogeneous cells essential for apoptotic cell clearance. Apoptotic cells are defined by homogeneous characteristics, ignoring their original cell lineage identity. We found that in an interleukin-4 (IL-4)-enriched environment, the sensing of apoptotic neutrophils by macrophages triggered their tissue remodeling signature. Engulfment of apoptotic hepatocytes promoted a tolerogenic phenotype, whereas phagocytosis of T cells had little effect on IL-4-induced gene expression. In a mouse model of parasite-induced pathology, the transfer of macrophages conditioned with IL-4 and apoptotic neutrophils promoted parasitic egg clearance. Knockout of phagocytic receptors required for the uptake of apoptotic neutrophils and partially T cells, but not hepatocytes, exacerbated helminth infection. These findings suggest that the identity of apoptotic cells may contribute to the development of distinct IL-4-driven immune programs in macrophages.

Wnt16 Increases Bone-to-Implant Contact in an Osteopenic Rat Model by Increasing Proliferation and Regulating the Differentiation of Bone Marrow Stromal Cells.

Osseointegration is a complex biological cascade that regulates bone regeneration after implant placement. Implants possessing complex multiscale surface topographies augment this regenerative process through the regulation of bone marrow stromal cells (MSCs) that are in contact with the implant surface. One pathway regulating osteoblastic differentiation is Wnt signaling, and upregulation of non-canonical Wnts increases differentiation of MSCs on these titanium substrates. Wnt16 is a non-canonical Wnt shown to regulate bone morphology in mouse models. This study evaluated the role of Wnt16 during surface-mediated osteoblastic differentiation of MSCs in vitro and osseointegration in vivo. MSCs were cultured on Ti substrates with different surface properties and non-canonical Wnt expression was determined. Subsequently, MSCs were cultured on Ti substrates +/-Wnt16 (100 ng/mL) and anti-Wnt16 antibodies (2 µg/mL). Wnt16 expression was increased in cells grown on microrough surfaces that were processed to be hydrophilic and have nanoscale roughness. However, treatment MSCs on these surfaces with exogenous rhWnt16b increased total DNA content and osteoprotegerin production, but reduced osteoblastic differentiation and production of local factors necessary for osteogenesis. Addition of anti-Wnt16 antibodies blocked the inhibitor effects of Wnt16. The response to Wnt16 was likely independent of other osteogenic pathways like Wnt11-Wnt5a signaling and semaphorin 3a signaling. We used an established rat model of cortical and trabecular femoral bone impairment following botox injections (2 injections of 8 units/leg each, starting and maintenance doses) to assess Wnt16 effects on whole bone morphology and implant osseointegration. Wnt16 injections did not alter whole bone morphology significantly (BV/TV, cortical thickness, restoration of trabecular bone) but were effective at increasing cortical bone-to-implant contact during impaired osseointegration in the botox model. The mechanical quality of the increased bone was not sufficient to rescue the deleterious effects of botox. Clinically, these results are important to understand the interaction of cortical and trabecular bone during implant integration. They suggest a role for Wnt16 in modulating bone remodeling by reducing osteoclastic activity. Targeted strategies to temporally regulate Wnt16 after implant placement could be used to improve osseointegration by increasing the net pool of osteoprogenitor cells.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Nmes1 is a novel regulator of mucosal response influencing intestinal healing potential.

The initiation of tissue remodeling following damage is a critical step in preventing the development of immune-mediated diseases. Several factors contribute to mucosal healing, leading to innovative therapeutic approaches for managing intestinal disorders. However, uncovering alternative targets and gaining mechanistic insights are imperative to enhance therapy efficacy and broaden its applicability across different intestinal diseases. Here we demonstrate that Nmes1, encoding for Normal Mucosa of Esophagus-Specific gene 1, also known as Aa467197, is a novel regulator of mucosal healing. Nmes1 influences the macrophage response to the tissue remodeling cytokine IL-4 in vitro. In addition, using two murine models of intestinal damage, each characterized by a type 2-dominated environment with contrasting functions, the ablation of Nmes1 results in decreased intestinal regeneration during the recovery phase of colitis, while enhancing parasitic egg clearance and reducing fibrosis during the advanced stages of Schistosoma mansoni infection. These outcomes are associated with alterations in CX3CR1+ macrophages, cells known for their wound-healing potential in the inflamed colon, hence promising candidates for cell therapies. All in all, our data indicate Nmes1 as a novel contributor to mucosal healing, setting the basis for further investigation into its potential as a new target for the treatment of colon-associated inflammation.

Phosphoproteome analyses pinpoint the F-box protein SLOW MOTION as a regulator of warm temperature-mediated hypocotyl growth in Arabidopsis.

Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation.

Whisker kinematics in the cerebellum.

The whisker system is widely used as a model system for understanding sensorimotor integration. Purkinje cells in the crus regions of the cerebellum have been reported to linearly encode whisker midpoint, but it is unknown whether the paramedian and simplex lobules as well as their target neurons in the cerebellar nuclei also encode whisker kinematics and if so which ones. Elucidating how these kinematics are represented throughout the cerebellar hemisphere is essential for understanding how the cerebellum coordinates multiple sensorimotor modalities. Exploring the cerebellar hemisphere of mice using optogenetic stimulation, we found that whisker movements can be elicited by stimulation of Purkinje cells in not only crus1 and crus2, but also in the paramedian lobule and lobule simplex; activation of cells in the medial paramedian lobule had on average the shortest latency, whereas that of cells in lobule simplex elicited similar kinematics as those in crus1 and crus2. During spontaneous whisking behaviour, simple spike activity correlated in general better with velocity than position of the whiskers, but it varied between protraction and retraction as well as per lobule. The cerebellar nuclei neurons targeted by the Purkinje cells showed similar activity patterns characterized by a wide variety of kinematic signals, yet with a dominance for velocity. Taken together, our data indicate that whisker movements are much more prominently and diversely represented in the cerebellar cortex and nuclei than assumed, highlighting the rich repertoire of cerebellar control in the kinematics of movements that can be engaged during coordination. KEY POINTS: Excitation of Purkinje cells throughout the cerebellar hemispheres induces whisker movement, with the shortest latency and longest duration within the paramedian lobe. Purkinje cells have differential encoding for the fast and slow components of whisking. Purkinje cells encode not only the position but also the velocity of whiskers. Purkinje cells with high sensitivity for whisker velocity are preferentially located in the medial part of lobule simplex, crus1 and lateral paramedian. In the downstream cerebellar nuclei, neurons with high sensitivity for whisker velocity are located at the intersection between the medial and interposed nucleus.

Pathogenetic mechanisms and treatment targets in cerebral malaria.

Malaria, the most prevalent mosquito-borne infectious disease worldwide, has accompanied humanity for millennia and remains an important public health issue despite advances in its prevention and treatment. Most infections are asymptomatic, but a small percentage of individuals with a heavy parasite burden develop severe malaria, a group of clinical syndromes attributable to organ dysfunction. Cerebral malaria is an infrequent but life-threatening complication of severe malaria that presents as an acute cerebrovascular encephalopathy characterized by unarousable coma. Despite effective antiparasite drug treatment, 20% of patients with cerebral malaria die from this disease, and many survivors of cerebral malaria have neurocognitive impairment. Thus, an important unmet clinical need is to rapidly identify people with malaria who are at risk of developing cerebral malaria and to develop preventive, adjunctive and neuroprotective treatments for cerebral malaria. This Review describes important advances in the understanding of cerebral malaria over the past two decades and discusses how these mechanistic insights could be translated into new therapies.

Plasmodium falciparum infection reshapes the human microRNA profiles of red blood cells and their extracellular vesicles.

Plasmodium falciparum, a human malaria parasite, develops in red blood cells (RBCs), which represent approximately 70% of all human blood cells. Additionally, RBC-derived extracellular vesicles (RBC-EVs) represent 7.3% of the total EV population. The roles of microRNAs (miRNAs) in the consequences of P. falciparum infection are unclear. Here, we analyzed the miRNA profiles of non-infected human RBCs (niRBCs), ring-infected RBCs (riRBCs), and trophozoite-infected RBCs (trRBCs), as well as those of EVs secreted from these cells. Hsa-miR-451a was the most abundant miRNA in all RBC and RBC-EV populations, but its expression level was not affected by P. falciparum infection. Overall, the miRNA profiles of RBCs and their EVs were altered significantly after infection. Most of the differentially expressed miRNAs were shared between RBCs and their EVs. A target prediction analysis of the miRNAs revealed the possible identity of the genes targeted by these miRNAs (CXCL10, OAS1, IL7, and CCL5) involved in immunomodulation.

Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus.

Industrial chicory (Cichorium intybus var. sativum) and witloof (C. intybus var. foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway are GERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO), COSTUNOLIDE SYNTHASE (COS) and KAUNIOLIDE SYNTHASE (KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found that C. intybus SL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybus CYP71 enzymes transiently produced in Nicotiana benthamiana and identified several functional paralogs for each of the GAO, COS and KLS genes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing in C. intybus. Metabolite profiling of mutant C. intybus lines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into the C. intybus SL biosynthetic pathway and paves the way for the engineering of C. intybus bitterness.

Combining multiplex gene editing and doubled haploid technology in maize.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Hallmarks of the relationship between host and Trypanosoma cruzi sulfated glycoconjugates along the course of Chagas disease.

American Trypanosomiasis or Chagas disease (ChD), a major problem that is still endemic in large areas of Latin America, is caused by Trypanosoma cruzi. This agent holds a major antigen, cruzipain (Cz). Its C-terminal domain (C-T) is retained in the glycoprotein mature form and bears several post-translational modifications. Glycoproteins containing sulfated N-linked oligosaccharides have been mostly implicated in numerous specific procedures of molecular recognition. The presence of sulfated oligosaccharides was demonstrated in Cz, also in a minor abundant antigen with serine-carboxypeptidase (SCP) activity, as well as in parasite sulfatides. Sulfate-bearing glycoproteins in Trypanosomatids are targets of specific immune responses. T. cruzi chronically infected subjects mount specific humoral immune responses to sulfated Cz. Unexpectedly, in the absence of infection, mice immunized with C-T, but not with sulfate-depleted C-T, showed ultrastructural heart anomalous pathological effects. Moreover, the synthetic anionic sugar conjugate GlcNAc6SO3-BSA showed to mimic the N-glycan-linked sulfated epitope (sulfotope) humoral responses that natural Cz elicits. Furthermore, it has been reported that sulfotopes participate via the binding of sialic acid Ig-like-specific lectins (Siglecs) to sulfosialylated glycoproteins in the immunomodulation by host-parasite interaction as well as in the parasite infection process. Strikingly, recent evidence involved Cz-sulfotope-specific antibodies in the immunopathogenesis and infection processes during the experimental ChD. Remarkably, sera from chronically T. cruzi-infected individuals with mild disease displayed higher levels of IgG2 antibodies specific for sulfated glycoproteins and sulfatides than those with more severe forms of the disease, evidencing that T. cruzi sulfotopes are antigenic independently of the sulfated glycoconjugate type. Ongoing assays indicate that antibodies specific for sulfotopes might be considered biomarkers of human cardiac ChD progression, playing a role as predictors of stability from the early mild stages of chronic ChD.

OMIP-93: A 41-color high parameter panel to characterize various co-inhibitory molecules and their ligands in the lymphoid and myeloid compartment in mice.

This 41-color panel has been designed to characterize both the lymphoid and the myeloid compartments in mice. The number of immune cells isolated from organs is often low, whilst an increasing number of factors need to be analyzed to gain a deeper understanding of the complexity of an immune response. With a focus on T cells, their activation and differentiation status, as well as their expression of several co-inhibitory and effector molecules, this panel also allows the analysis of ligands to these co-inhibitory molecules on antigen-presenting cells. This panel enables deep phenotypic characterization of CD4+ and CD8+ T cells, regulatory T cells, γδ T cells, NK T cells, B cells, NK cells, monocytes, macrophages, dendritic cells, and neutrophils. Whilst previous panels have focused on these topics individually, this is the first panel to enable simultaneous analysis of these compartments, thus enabling a comprehensive analysis with a limited number of immune cells/sample size. This panel is designed to analyze and compare the immune response in different mouse models of infectious diseases, but can also be extended to other disease models, for example tumors or autoimmune diseases. Here, we apply this panel to C57BL/6 mice infected with Plasmodium berghei ANKA, a mouse model of cerebral malaria.

Ulva: An emerging green seaweed model for systems biology.

Green seaweeds exhibit a wide range of morphologies and occupy various ecological niches, spanning from freshwater to marine and terrestrial habitats. These organisms, which predominantly belong to the class Ulvophyceae, showcase a remarkable instance of parallel evolution toward complex multicellularity and macroscopic thalli in the Viridiplantae lineage. Within the green seaweeds, several Ulva species ("sea lettuce") are model organisms for studying carbon assimilation, interactions with bacteria, life cycle progression, and morphogenesis. Ulva species are also notorious for their fast growth and capacity to dominate nutrient-rich, anthropogenically disturbed coastal ecosystems during "green tide" blooms. From an economic perspective, Ulva has garnered increasing attention as a promising feedstock for the production of food, feed, and biobased products, also as a means of removing excess nutrients from the environment. We propose that Ulva is poised to further develop as a model in green seaweed research. In this perspective, we focus explicitly on Ulva mutabilis/compressa as a model species and highlight the molecular data and tools that are currently available or in development. We discuss several areas that will benefit from future research or where exciting new developments have been reported in other Ulva species.

SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.

Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.