Mutant Phenotype Research Articles

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

The blue light-dependent LOV-protein LdaP of Dinoroseobacter shibae acts as antirepressor of the PpsR repressor, regulating photosynthetic gene cluster expression.

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

The occurrence, inheritance, and segregation of complex genomic structural variation in synthetic Brassica napus

“Synthetic” allopolyploids recreated by interspecific hybridization play an important role in providing novel genomic variation for crop improvement. Such synthetic allopolyploids often undergo rapid genomic structural variation (SV). However, how such SV arises, is inherited and fixed, and how it affects important traits, has rarely been comprehensively and quantitively studied in advanced generation synthetic lines. A better understanding of these processes will aid breeders in knowing how to best utilize synthetic allopolyploids in breeding programs. Here, we analyzed three genetic mapping populations (735 DH lines) derived from crosses between advanced synthetic and conventional Brassica napus (rapeseed) lines, using whole-genome sequencing to determine genome composition. We observed high tolerance of large structural variants, particularly toward the telomeres, and preferential selection for balanced homoeologous exchanges (duplication/deletion events between the A and C genomes resulting in retention of gene/chromosome dosage between homoeologous chromosome pairs), including stable events involving whole chromosomes (“pseudoeuploidy”). Given the experimental design (all three populations shared a common parent), we were able to observe that parental SV was regularly inherited, showed genetic hitchhiking effects on segregation, and was one of the major factors inducing adjacent novel and larger SV. Surprisingly, novel SV occurred at low frequencies with no significant impacts on observed fertility and yield-related traits in the advanced generation synthetic lines. However, incorporating genome-wide SV in linkage mapping explained significantly more genetic variance for traits. Our results provide a framework for detecting and understanding the occurrence and inheritance of genomic SV in breeding programs, and support the use of synthetic parents as an important source of novel trait variation.

S1 basic leucine zipper transcription factors shape plant architecture by controlling C/N partitioning to apical and lateral organs

Plants tightly control growth of their lateral organs, which led to the concept of apical dominance. However, outgrowth of the dormant lateral primordia is sensitive to the plant's nutritional status, resulting in an immense plasticity in plant architecture. While the impact of hormonal regulation on apical dominance is well characterized, the prime importance of sugar signaling to unleash lateral organ formation has just recently emerged. Here, we aimed to identify transcriptional regulators, which control the trade-off between growth of apical versus lateral organs. Making use of locally inducible gain-of-function as well as single and higher-order loss-of-function approaches of the sugar-responsive S1-basic-leucine-zipper (S1-bZIP) transcription factors, we disclosed their largely redundant function in establishing apical growth dominance. Consistently, comprehensive phenotypical and analytical studies of S1-bZIP mutants show a clear shift of sugar and organic nitrogen (N) allocation from apical to lateral organs, coinciding with strong lateral organ outgrowth. Tissue-specific transcriptomics reveal specific clade III SWEET sugar transporters, crucial for long-distance sugar transport to apical sinks and the glutaminase GLUTAMINE AMIDO-TRANSFERASE 1_2.1, involved in N homeostasis, as direct S1-bZIP targets, linking the architectural and metabolic mutant phenotypes to downstream gene regulation. Based on these results, we propose that S1-bZIPs control carbohydrate (C) partitioning from source leaves to apical organs and tune systemic N supply to restrict lateral organ formation by C/N depletion. Knowledge of the underlying mechanisms controlling plant C/N partitioning is of pivotal importance for breeding strategies to generate plants with desired architectural and nutritional characteristics.

The maize striate leaves2 (sr2) gene encodes a conserved DUF3732 domain and is homologous to the rice yss1 gene.

Maize striate leaves2 (sr2) is a mutant that causes white stripes on leaves that has been used in mapping studies for decades though the underlying gene has not been identified. The sr2 locus has been previously mapped to small regions of normal chromosome 10 (N10) and a rearranged variant called abnormal chromosome 10 (Ab10). A comparison of assembled genomes carrying N10 and Ab10 revealed only five candidate sr2 genes. Analysis of a stock carrying the sr2 reference allele (sr2-ref) showed that one of the five genes has a transposon insertion that disrupts its protein sequence and has a severe reduction in mRNA. An independent Mutator transposon insertion in the gene (sr2-Mu) failed to complement the sr2-ref mutation, and plants homozygous for sr2-Mu showed white striped leaf margins. The sr2 gene encodes a DUF3732 protein with strong homology to a rice gene with a similar mutant phenotype called young seedling stripe1 (yss1). These and other published data suggest that sr2 may have a function in plastid gene expression.

A natural single‐nucleotide polymorphism in the CAMTA3 transcription factor regulates its function and transcription of its target genes

AbstractCAMTA3, a Ca2+/calmodulin‐binding transcription factor, is a key regulator of plant immunity in Arabidopsis. Here, we identified a novel naturally occurring single‐nucleotide polymorphism that results in a missense nonconservative mutation (CAMTA3H386D) in many Arabidopsis ecotypes. This region of CAMTA3 is not part of any previously characterized regulatory domains. To study the consequence of this change on the function of CAMTA3, we introduced the CAMTA3H386D into camta3, a loss‐of‐function mutant that exhibits a constitutive cell death phenotype, chlorotic lesions on leaves, and reduced plant size. Phenotypic and molecular analysis of these lines indicated that the expression of CAMTA3H386D in the camta3 mutant did not complement the mutant phenotypes. Also, the ecotypes containing the CAMTA3H386D exhibited camta3 phenotypes. Marker genes associated with salicylic acid biosynthesis and pathogen response were upregulated in the CAMTA3H386D lines and the Arabidopsis accessions 7127 (Est‐1) and 9941 (Fei‐0), as in camta3, indicating that H386D mutation alters CAMTA3 activity in regulating the expression of known target genes. In Nicotiana benthamiana transient expression assays, CAMTA3H386D failed to induce the expression of a luciferase reporter gene driven by the rapid stress‐responsive elements (RSRE) that contain the known binding sites of CAMTA3, suggesting that CAMTA3H386D mutation impairs its ability to activate its target genes. Transgenic lines and tested natural accessions expressing CAMTA3H386D showed enhanced levels of H2O2 and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Collectively, our results indicate that the H386D mutation in a previously unknown regulatory region of CAMTA3 is essential for its function.

Sua5 catalyzing universal t6A tRNA modification is responsible for multifaceted functions of the KEOPS complex in Cryptococcus neoformans.

The N6-threonylcarbamoyl adenosine (t6A) tRNA modification is critical for ensuring translation fidelity across three domains of life. Our prior work highlighted the KEOPS complex, organized in a Pcc1-Kae1-Bud32-Cgi121 linear arrangement, not only serves an evolutionarily conserved role in t6A tRNA modification but also exerts diverse functional impacts on pathobiological characteristics in Cryptococcus neoformans, a leading cause of fungal meningitis worldwide. However, the extent to which the pleiotropic functions of the KEOPS complex are specifically tied to tRNA modification remains uncertain. To address this, we undertook a functional characterization of Sua5, responsible for generating the precursor threonylcarbamoyl-adenylate (TC-AMP) for t6A tRNA modification, using a reverse genetics approach. Comparative phenotypic analyses with KEOPS mutants revealed that Sua5 plays a vital role in multiple cellular processes, such as t6A tRNA modification, growth, sexual development, stress response, and virulence factor production, thus reflecting the multifaceted functions of the KEOPS complex. In support of this, sua5Δ bud32Δ double mutants showed phenotypes comparable to those of the corresponding single mutants. Intriguingly, a SUA5 allele lacking a mitochondria targeting sequence (SUA5MTSΔ) was sufficient to restore the wild-type phenotypes in the sua5Δ mutant, suggesting that Sua5's primary functional locus may be cytosolic, akin to the KEOPS complex. Further supporting this, the deletion of Qri7, a mitochondrial paralog of Kae1, had no discernible phenotypic impact on C. neoformans. We concluded that cytosolic t6A tRNA modifications, orchestrated by Sua5 and the KEOPS complex, are central to the regulation of diverse pathobiological functions in C. neoformans.IMPORTANCEUnderstanding cellular functions at the molecular level is crucial for advancing disease treatments. Our research reveals a critical connection between the KEOPS complex and Sua5 in Cryptococcus neoformans, a significant cause of fungal meningitis. While the KEOPS complex is known for its versatile roles in cellular processes, Sua5 is specialized in t6A tRNA modification. Our key finding is that the diverse roles of the KEOPS complex, ranging from cell growth and stress response to virulence, are fundamentally linked to its function in t6A tRNA modification. This conclusion is supported by the remarkable similarities between the impacts of Sua5 and KEOPS on these processes, despite their roles in different steps of the t6A modification pathway. This newfound understanding deepens our insight into fungal biology and opens new avenues for developing potential therapies against dangerous fungal diseases.

Integrated multi-omics and genetic analyses reveal molecular determinants underlying Arabidopsis snap33 mutant phenotype.

The secretory pathway is essential for plant immunity, delivering diverse antimicrobial molecules into the extracellular space. Arabidopsis thaliana soluble N-ethylmaleimide-sensitive-factor attachment protein receptor SNAP33 is a key actor of this process. The snap33 mutant displays dwarfism and necrotic lesions, however the molecular determinants of its macroscopic phenotypes remain elusive. Here, we isolated several new snap33 mutants that exhibited constitutive cell death and H2O2 accumulation, further defining snap33 as an autoimmune mutant. We then carried out quantitative transcriptomic and proteomic analyses showing that numerous defense transcripts and proteins were up-regulated in the snap33 mutant, among which genes/proteins involved in defense hormone, pattern-triggered immunity, and nucleotide-binding domain leucine-rich-repeat receptor signaling. qRT-PCR analyses and hormone dosages supported these results. Furthermore, genetic analyses elucidated the diverse contributions of the main defense hormones and some nucleotide-binding domain leucine-rich-repeat receptor signaling actors in the establishment of the snap33 phenotype, emphasizing the preponderant role of salicylic acid over other defense phytohormones. Moreover, the accumulation of pattern-triggered immunity and nucleotide-binding domain leucine-rich-repeat receptor signaling proteins in the snap33 mutant was confirmed by immunoblotting analyses and further shown to be salicylic acid-dependent. Collectively, this study unveiled molecular determinants underlying the Arabidopsis snap33 mutant phenotype and brought new insights into autoimmunity signaling.

Cloning human Benchwarmer gene (BNCH) harboring E164K in vector pcDNA3.1 by site-directed mutagenesis method

Benchwarmer (BNCH) gene encodes an orphan transmembrane transporter belonging to the Major Facilitator Superfamily (MFS), facilitating the transport of ions, amino acids, simple sugars and recently lysolipids. The loss of BNCH function caused lethality in several animal models with neurodegeneration and senescence. At the cellular level, dysregulation of BNCH leads to adverse phenotypes of lysosome and also autophagy (i.e. dyshomeostasis, accumulation of carbohydrates and sphingolipids, and enlarged lysosome). However, the molecular function and ligand of BNCH protein remain to be unrevealed. This study aims to create a radical substitution change in human BNCH coding gene to knock out the protein functions. More specifically, lysine (K) was used to replace the glutamic acid residue 164 (E164K) which is conserved in many animals (fly, zebrafish, mouse and human) and this E164K mutation recapitulated BNCH mutant phenotype. In conclusion, BNCH harboring E164K (BNCH*) was successfully produced by site-directed mutagenesis and cloned into pcDNA.3.1 vector. The construct was transformed into E. coli OmniMAX and that provides a valuable cell assay to search for the molecular ligand of BNCH.

A polygalacturonase gene OsPG1 modulates water homeostasis in rice

A dynamic plant architecture is the basis of plant adaptation to changing environments. Although many genes regulating leaf rolling have been identified, genes directly associated with water homeostasis are largely unknown. Here, we isolated a rice mutant, dynamic leaf rolling 1 (dlr1), characterized by ‘leaf unfolding in the morning-leaf rolling at noon-leaf unfolding in the evening’ during a sunny day. Water content was decreased in rolled leaves and water sprayed on leaves caused reopening, indicating that in vivo water deficiency induced the leaf rolling. Map-based cloning and expression tests demonstrated that an A1400G single base mutation in Oryza sativa Polygalacturonase 1 (OsPG1)/PHOTO-SENSITIVE LEAF ROLLING 1 (PSL1) was responsible for the dynamic leaf rolling phenotype in the dlr1 mutant. OsPG1 encodes a polygalacturonase, one of the main enzymes that degrade demethylesterified homogalacturonans in plant cell walls. OsPG1 was constitutively expressed in various tissues and was enriched in stomata. Mutants of the OsPG1 gene exhibited defects in stomatal closure and decreased stomatal density, leading to reduced transpiration and excessive water loss under specific conditions, but had normal root development. Further analysis revealed that mutation of OsPG1 led to reduced pectinase activity in the leaves and increased demethylesterified homogalacturonans in guard cells. Our findings reveal a mechanism by which OsPG1 modulates water homeostasis to control dynamic leaf rolling, providing insights for plants to adapt to environmental variation.

Determining Photoreceptor Cell Identity: Rod Versus Cone Fate Governed by tbx2b Opposing nrl.

NRL is an influential transcription factor and central to animal modeling in ophthalmology. Disrupting NRL abrogates rod development and produces an excess of S-cones (also known as "UV cones" or "short-wavelength-sensitive1 [SWS1] cones"). Strikingly, mutations in zebrafish tbx2b produce the exact opposite phenotypes (excess rods and loss of SWS1 cones). We sought to define what genetic relationship exists, if any, between these transcription factors. We also infer whether these two phenotypes (altered rod abundance and altered SWS1 cone abundance) are independent versus inter-related. Zebrafish mutants were bred to disrupt nrl and tbx2b in concert. Rods and SWS1 cones were quantified and characterized at ultrastructural and transcriptional levels. Considering single mutant zebrafish, we confirmed previously established phenotypes and noted that the number of rods lost in nrl-/- mutants is reflected by a concomitant increase in SWS1 cone abundance. The tbx2b-/- mutants present the opposite phenotype(s) but exhibit a similar trade-off in cell abundances, with lots of rods and a concomitant decrease in SWS1 cones. Double mutant nrl-/-;tbx2b-/- zebrafish recapitulate the nrl-/- mutant phenotype(s). The tbx2b is thought to be required for producing SWS1 cones in zebrafish, but this can be over-ridden when nrl is absent. Regarding the altered cell abundances observed in either tbx2b-/- or nrl-/- mutants, the alterations in rod and SWS1 cones appear to not be two separate phenotypes but are instead a single intertwined outcome. The tbx2b and nrl are in an epistatic relationship, with nrl phenotypes dominating, implying that tbx2b is upstream of nrl in photoreceptor cell fate determination.

OsBCAT2, a gene responsible for the degradation of branched-chain amino acids, positively regulates salt tolerance by promoting the synthesis of vitamin B5.

Salt stress negatively affects rice growth, development and yield. Metabolic adjustments contribute to the adaptation of rice under salt stress. Branched-chain amino acids (BCAA) are three essential amino acids that cannot be synthesized by humans or animals. However, little is known about the role of BCAA in response to salt stress in plants. Here, we showed that BCAAs may function as scavengers of reactive oxygen species (ROS) to provide protection against damage caused by salinity. We determined that branched-chain aminotransferase 2 (OsBCAT2), a protein responsible for the degradation of BCAA, positively regulates salt tolerance. Salt significantly induces the expression of OsBCAT2 rather than BCAA synthesis genes, which indicated that salt mainly promotes BCAA degradation and not de novo synthesis. Metabolomics analysis revealed that vitamin B5 (VB5) biosynthesis pathway intermediates were higher in the OsBCAT2-overexpressing plants but lower in osbcat2 mutants under salt stress. The salt stress-sensitive phenotypes of the osbcat2 mutants are rescued by exogenous VB5, indicating that OsBCAT2 affects rice salt tolerance by regulating VB5 synthesis. Our work provides new insights into the enzymes involved in BCAAs degradation and VB5 biosynthesis and sheds light on the molecular mechanism of BCAAs in response to salt stress.

Characterization of the intracellular neurexin interactome by in vivo proximity ligation suggests its involvement in presynaptic actin assembly.

Neurexins are highly spliced transmembrane cell adhesion molecules that bind an array of partners via their extracellular domains. However, much less is known about the signaling pathways downstream of neurexin's largely invariant intracellular domain (ICD). Caenorhabditis elegans contains a single neurexin gene that we have previously shown is required for presynaptic assembly and stabilization. To gain insight into the signaling pathways mediating neurexin's presynaptic functions, we employed a proximity ligation method, endogenously tagging neurexin's intracellular domain with the promiscuous biotin ligase TurboID, allowing us to isolate adjacent biotinylated proteins by streptavidin pull-down and mass spectrometry. We compared our experimental strain to a control strain in which neurexin, endogenously tagged with TurboID, was dispersed from presynaptic active zones by the deletion of its C-terminal PDZ-binding motif. Selection of this control strain, which differs from the experimental strain only in its synaptic localization, was critical to identifying interactions specifically occurring at synapses. Using this approach, we identified both known and novel intracellular interactors of neurexin, including active zone scaffolds, actin-binding proteins (including almost every member of the Arp2/3 complex), signaling molecules, and mediators of RNA trafficking, protein synthesis and degradation, among others. Characterization of mutants for candidate neurexin interactors revealed that they recapitulate aspects of the nrx-1(-) mutant phenotype, suggesting they may be involved in neurexin signaling. Finally, to investigate a possible role for neurexin in local actin assembly, we endogenously tagged its intracellular domain with actin depolymerizing and sequestering peptides (DeActs) and found that this led to defects in active zone assembly. Together, these results suggest neurexin's intracellular domain may be involved in presynaptic actin-assembly, and furthermore highlight a novel approach to achieving high specificity for in vivo proteomics experiments.

MiR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila

miR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. Here, we show that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride content and decreased locomotor activity. In addition, when challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and prolonged survival. We show through genetic epistasis and rescue experiments that this starvation resistance is due to a disruption in insulin signaling. Our studies further show that miR-137 null mutants exhibit a drastic reduction in levels of the phosphorylated/activated insulin receptor, InR (InR-P). We investigated if this is due to the predicted miR-137 target, Protein Tyrosine Phosphatase 61F (PTP61F), ortholog of mammalian TC-PTP/PTP1B, which are known to dephosphorylate InR-P. Indeed, levels of an endogenously tagged GFP-PTP61F are significantly elevated in miR-137 null mutants, and we show that overexpression of PTP61F alone is sufficient to mimic many of the metabolic phenotypes of miR-137 mutants. Finally, we knocked-down elevated levels of PTP61F in the miR-137 null mutant background and show that this rescues levels of InR-P, restores normal body weight and triglyceride content, starvation sensitivity, as well as attenuates locomotor and starvation-induced feeding defects. Our study supports a model in which miR-137 is critical for dampening levels of PTP61F, thereby maintaining normal insulin signaling and energy homeostasis.

IGT/LAZY genes are differentially influenced by light and required for light-induced change to organ angle

BackgroundPlants adjust their growth orientations primarily in response to light and gravity signals. Considering that the gravity vector is fixed and the angle of light incidence is constantly changing, plants must somehow integrate these signals to establish organ orientation, commonly referred to as gravitropic set-point angle (GSA). The IGT gene family contains known regulators of GSA, including the gene clades LAZY, DEEPER ROOTING (DRO), and TILLER ANGLE CONTROL (TAC).ResultsHere, we investigated the influence of light on different aspects of GSA phenotypes in LAZY and DRO mutants, as well as the influence of known light signaling pathways on IGT gene expression. Phenotypic analysis revealed that LAZY and DRO genes are collectively required for changes in the angle of shoot branch tip and root growth in response to light. Single lazy1 mutant branch tips turn upward in the absence of light and in low light, similar to wild-type, and mimic triple and quadruple IGT mutants in constant light and high-light conditions, while triple and quadruple IGT/LAZY mutants show little to no response to changing light regimes. Further, the expression of IGT/LAZY genes is differentially influenced by daylength, circadian clock, and light signaling.ConclusionsCollectively, the data show that differential expression of LAZY and DRO genes are required to enable plants to alter organ angles in response to light-mediated signals.

Insights into the functional role of tomato TM6 as a transcriptional regulator of flower development.

Flower development is a crucial step towards the completion of the plant life cycle. Physiological processes and gene regulatory mechanisms underlying flower formation have been extensively characterized, and the implication of MADS-box transcription factors as primary regulators of flower morphology has been widely described, mainly due to the analysis of loss-of-function mutants in model species. Nevertheless, detailed characterization of allele variation in several MADS-box homologous genes from crop species remains undescribed. Here, we have characterized a tomato mutant with aberrant flower development. Mutant plants exhibit changes in petal cell identity, as well as homeotic transformations of stamens into carpelloid structures, which in most cases result in succulent organs. Molecular analysis proved that a loss-of-function mutation in the TOMATO MADS-BOX 6 (TM6) gene is responsible for this mutant phenotype. Furthermore, as a result of the loss of function of TM6, misregulation of the transcription and mRNA processing of other MADS-box genes involved in reproductive development has been detected. Our findings demonstrate that TM6 is a key player in the complex regulatory network of MADS-box genes controlling flower development and also provide a novel mutant that may be useful for generating male sterile lines in tomatoes.

Upregulation of Wheat Heat Shock Transcription Factor TaHsfC3-4 by ABA Contributes to Drought Tolerance.

Drought stress can seriously affect the yield and quality of wheat (Triticum aestivum). So far, although few wheat heat shock transcription factors (Hsfs) have been found to be involved in the stress response, the biological functions of them, especially the members of the HsfC (heat shock transcription factor C) subclass, remain largely unknown. Here, we identified a class C encoding gene, TaHsfC3-4, based on our previous omics data and analyzed its biological function in transgenic plants. TaHsfC3-4 encodes a protein containing 274 amino acids and shows the basic characteristics of the HsfC class. Gene expression profiles revealed that TaHsfC3-4 was constitutively expressed in many tissues of wheat and was induced during seed maturation. TaHsfC3-4 could be upregulated by PEG and abscisic acid (ABA), suggesting that this Hsf may be involved in the regulation pathway depending on ABA in drought resistance. Further results represented that TaHsfC3-4 was localized in the nucleus but had no transcriptional activation activity. Notably, overexpression of TaHsfC3-4 in Arabidopsis thaliana pyr1pyl1pyl2pyl4 (pyr1pyl124) quadruple mutant plants complemented the ABA-hyposensitive phenotypes of the quadruple mutant including cotyledon greening, root elongation, seedling growth, and increased tolerance to drought, indicating positive roles of TaHsfC3-4 in the ABA signaling pathway and drought tolerance. Furthermore, we identified TaHsfA2-11 as a TaHsfC3-4-interacting protein by yeast two-hybrid (Y2H) screening. The experimental data show that TaHsfC3-4 can indeed interact with TaHsfA2-11 in vitro and in vivo. Moreover, transgenic Arabidopsis TaHsfA2-11 overexpression lines exhibited enhanced drought tolerance, too. In summary, our study confirmed the role of TaHsfC3-4 in response to drought stress and provided a target locus for marker-assisted selection breeding to improve drought tolerance in wheat.

Xenophagy receptors Optn and p62 and autophagy modulator Dram1 independently promote the zebrafish host defense against Mycobacterium marinum.

Anti-bacterial autophagy, also known as xenophagy, is a crucial innate immune process that helps maintain cellular homeostasis by targeting invading microbes. This defense pathway is widely studied in the context of infections with mycobacteria, the causative agents of human tuberculosis and tuberculosis-like disease in animal models. Our previous work in a zebrafish tuberculosis model showed that host defense against Mycobacterium marinum (Mm) is impaired by deficiencies in xenophagy receptors, optineurin (Optn) or sequestome 1 (p62), and Damage-regulated autophagy modulator 1 (Dram1). However, the interdependency of these receptors and their interaction with Dram1 remained unknown. In the present study, we used single and double knockout zebrafish lines in combination with overexpression experiments. We show that Optn and p62 can compensate for the loss of each other's function, as their overexpression restores the infection susceptibility of the mutant phenotypes. Similarly, Dram1 can compensate for deficiencies in Optn and p62, and, vice versa, Optn and p62 compensate for the loss of Dram1, indicating that these xenophagy receptors and Dram1 do not rely on each other for host defense against Mm. In agreement, Dram1 overexpression in optn/p62 double mutants restored the interaction of autophagosome marker Lc3 with Mm. Finally, optn/p62 double mutants displayed more severe infection susceptibility than the single mutants. Taken together, these results suggest that Optn and p62 do not function downstream of each other in the anti-mycobacterial xenophagy pathway, and that the Dram1-mediated defense against Mm infection does not rely on specific xenophagy receptors.

Control of cardiac contractions using Cre-lox and degron strategies in zebrafish

Cardiac contractions and hemodynamic forces are essential for organ development and homeostasis. Control over cardiac contractions can be achieved pharmacologically or optogenetically. However, these approaches lack specificity or require direct access to the heart. Here, we compare two genetic approaches to control cardiac contractions by modulating the levels of the essential sarcomeric protein Tnnt2a in zebrafish. We first recombine a newly generated tnnt2a floxed allele using multiple lines expressing Cre under the control of cardiomyocyte-specific promoters, and show that it does not recapitulate the tnnt2a/silent heart mutant phenotype in embryos. We show that this lack of early cardiac contraction defects is due, at least in part, to the long half-life of tnnt2a mRNA, which masks the gene deletion effects until the early larval stages. We then generate an endogenous Tnnt2a-eGFP fusion line that we use together with the zGRAD system to efficiently degrade Tnnt2a in all cardiomyocytes. Using single-cell transcriptomics, we find that Tnnt2a depletion leads to cardiac phenotypes similar to those observed in tnnt2a mutants, with a loss of blood and pericardial flow-dependent cell types. Furthermore, we achieve conditional degradation of Tnnt2a-eGFP by splitting the zGRAD protein into two fragments that, when combined with the cpFRB2-FKBP system, can be reassembled upon rapamycin treatment. Thus, this Tnnt2a degradation line enables non-invasive control of cardiac contractions with high spatial and temporal specificity and will help further understand how they shape organ development and homeostasis.

SH2 domain protein E and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis.