Defective Mutant Research Articles

The Caenorhabditis elegans cullin-RING ubiquitin ligase CRL4DCAF-1 is required for proper germline nucleolus morphology and male development.

Cullin-RING ubiquitin ligases (CRLs) are the largest class of ubiquitin ligases with diverse functions encompassing hundreds of cellular processes. Inactivation of core components of the CRL4 ubiquitin ligase produces a germ cell defect in Caenorhabditis elegans that is marked by abnormal globular morphology of the nucleolus and fewer germ cells. We identified DDB1 Cullin4 associated factor (DCAF)-1 as the CRL4 substrate receptor that ensures proper germ cell nucleolus morphology. We demonstrate that the dcaf-1 gene is the ncl-2 (abnormal nucleoli) gene, whose molecular identity was not previously known. We also observed that CRL4DCAF-1 is required for male tail development. Additionally, the inactivation of CRL4DCAF-1 results in a male-specific lethality in which a percentage of male progeny arrest as embryos or larvae. Analysis of the germ cell nucleolus defect using transmission electron microscopy revealed that dcaf-1 mutant germ cells possess significantly fewer ribosomes, suggesting a defect in ribosome biogenesis. We discovered that inactivation of the sperm-fate specification gene fog-1 (feminization of the germ line-1) or its protein-interacting partner, fog-3, rescues the dcaf-1 nucleolus morphology defect. Epitope-tagged versions of both FOG-1 and FOG-3 proteins are aberrantly present in adult dcaf-1(RNAi) animals, suggesting that DCAF-1 negatively regulates FOG-1 and FOG-3 expression. Murine CRL4DCAF-1 targets the degradation of the ribosome assembly factor periodic trptophan protein 1 (PWP1). We observed that the inactivation of Caenorhabditis elegansDCAF-1 increases the nucleolar levels of PWP1 in the germ line, intestine, and hypodermis. Reducing the level of PWP-1 rescues the dcaf-1 mutant defects of fewer germ cell numbers and abnormal nucleolus morphology, suggesting that the increase in PWP-1 levels contributes to the dcaf-1 germline defect. Our results suggest that CRL4DCAF-1 has an evolutionarily ancient role in regulating ribosome biogenesis including a conserved target in PWP1.

TONSOKU is required for the maintenance of repressive chromatin modifications in Arabidopsis

The stability of eukaryotic genomes relies on the faithful transmission of DNA sequences and the maintenance of chromatin states through DNA replication. Plant TONSOKU (TSK) and its animal ortholog TONSOKU-like (TONSL) act as readers for newly synthesized histones and preserve DNA integrity via facilitating DNA repair at post-replicative chromatin. However, whether TSK/TONSL regulate the maintenance of chromatin states remains elusive. Here, we show that TSK is dispensable for global histone and nucleosome accumulation but necessary for maintaining repressive chromatin modifications, including H3K9me2, H2A.W, H3K27me3, and DNA methylation. TSK physically interacts with H3K9 methyltransferases and Polycomb proteins. Moreover, TSK mutation strongly enhances defects in Polycomb pathway mutants. TSK is intended to only associate with nascent chromatin until it starts to mature. We propose that TSK ensures the preservation of chromatin states by supporting the recruitment of chromatin modifiers to post-replicative chromatin in a critical short window of time following DNA replication.

Galactosylation of xyloglucan is essential for the stabilization of the actin cytoskeleton and endomembrane system through the proper assembly of cell walls.

Xyloglucan, a major hemicellulose, interacts with cellulose and pectin to assemble primary cell walls in plants. Loss of the xyloglucan galactosyltransferase MURUS3 (MUR3) leads to the deficiency of galactosylated xyloglucan and perturbs plant growth. However, it is unclear whether defects in xyloglucan galactosylation influence the synthesis of other wall polysaccharides, cell wall integrity, cytoskeleton behaviour, and endomembrane homeostasis. Here, we found that in mur3-7 etiolated seedlings cellulose was reduced, CELLULOSE SYNTHASE (CESA) genes were down-regulated, the density and mobility of cellulose synthase complexes (CSCs) were decreased, and cellulose microfibrils become discontinuous. Pectin, rhamnogalacturonan II (RGII), and boron contents were reduced in mur3-7 plants, and B-RGII cross-linking was abnormal. Wall porosity and thickness were significantly increased in mur3-7 seedlings. Endomembrane aggregation was also apparent in the mur3-7 mutant. Furthermore, mutant seedlings and their actin filaments were more sensitive to Latrunculin A (LatA) treatment. However, all defects in mur3-7 mutants were substantially restored by exogenous boric acid application. Our study reveals the importance of MUR3-mediated xyloglucan galactosylation for cell wall structural assembly and homeostasis, which is required for the stabilization of the actin cytoskeleton and the endomembrane system.

Biological and genomic analysis of a symbiotic nitrogen fixation defective mutant in Medicago truncatula.

Medicago truncatula has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes, genetic mutant resources are very useful and necessary tools. Fast Neutron (FN) mutagenesis is effective in inducing deletion mutations in genomes of diverse species. Through this method, we have generated a large mutant resource in M. truncatula. This mutant resources have been used to screen for different mutant using a forward genetics methods. We have isolated and identified a large amount of symbiotic nitrogen fixation (SNF) deficiency mutants. Here, we describe the detail procedures that are being used to characterize symbiotic mutants in M. truncatula. In recent years, whole genome sequencing has been used to speed up and scale up the deletion identification in the mutant. Using this method, we have successfully isolated a SNF defective mutant FN007 and identified that it has a large segment deletion on chromosome 3. The causal deletion in the mutant was confirmed by tail PCR amplication and sequencing. Our results illustrate the utility of whole genome sequencing analysis in the characterization of FN induced deletion mutants for gene discovery and functional studies in the M. truncatula. It is expected to improve our understanding of molecular mechanisms underlying symbiotic nitrogen fixation in legume plants to a great extent.

Arbuscular mycorrhizal fungi enhance phosphate uptake and alter bacterial communities in maize rhizosphere soil.

Arbuscular mycorrhizal fungi (AMF) can symbiose with many plants and improve nutrient uptake for their host plant. Rhizosphere microorganisms have been pointed to play important roles in helping AMF to mobilize soil insoluble nutrients, especially phosphorus. Whether the change in phosphate transport under AMF colonization will affect rhizosphere microorganisms is still unknown. Here, we evaluated the links of interactions among AMF and the rhizosphere bacterial community of maize (Zea mays L.) by using a maize mycorrhizal defective mutant. Loss of mycorrhizal symbiosis function reduced the phosphorus concentration, biomass, and shoot length of maize colonized by AMF. Using 16S rRNA gene amplicon high-throughput sequencing, we found that the mutant material shifted the bacterial community in the rhizosphere under AMF colonization. Further functional prediction based on amplicon sequencing indicated that rhizosphere bacteria involved in sulfur reduction were recruited by the AMF colonized mutant but reduced in the AMF- colonized wild type. These bacteria harbored much abundance of sulfur metabolism-related genes and negatively correlated with biomass and phosphorus concentrations of maize. Collectively, this study shows that AMF symbiosis recruited rhizosphere bacterial communities to improve soil phosphate mobilization, which may also play a potential role in regulating sulfur uptake. This study provides a theoretical basis for improving crop adaptation to nutrient deficiency through soil microbial management practices.

Quorum sensing signal synthases enhance Vibrio parahaemolyticus swarming motility.

Vibrio parahaemolyticus is a significant food-borne pathogen that is found in diverse aquatic habitats. Quorum sensing (QS), a signaling system for cell-cell communication, plays an important role in V. parahaemolyticus persistence. We characterized the function of three V. parahaemolyticus QS signal synthases, CqsAvp , LuxMvp , and LuxSvp , and show that they are essential to activate QS and regulate swarming. We found that CqsAvp , LuxMvp , and LuxSvp activate a QS bioluminescence reporter through OpaR. However, V. parahaemolyticus exhibits swarming defects in the absence of CqsAvp , LuxMvp , and LuxSvp , but not OpaR. The swarming defect of this synthase mutant (termed Δ3AI) was recovered by overexpressing either LuxOvp D47A , a mimic of dephosphorylated LuxOvp mutant, or the scrABC operon. CqsAvp , LuxMvp , and LuxSvp inhibit lateral flagellar (laf) gene expression by inhibiting the phosphorylation of LuxOvp and the expression of scrABC. Phosphorylated LuxOvp enhances laf gene expression in a mechanism that involves modulating c-di-GMP levels. However, enhancing swarming requires phosphorylated and dephosphorylated LuxOvp which is regulated by the QS signals that are synthesized by CqsAvp , LuxMvp , and LuxSvp . The data presented here suggest an important strategy of swarming regulation by the integration of QS and c-di-GMP signaling pathways in V. parahaemolyticus.

HSD17B6 delays type 2 diabetes development via inhibiting SREBP activation

BackgroundThe SREBP/SCAP/INSIG complex plays an essential role in SREBP activation and de novo lipogenesis. Whether the activation process is affected by hydroxysteroid 17-beta dehydrogenase 6 (HSD17B6) remains unknown. MethodsSREBP's transcriptional activities were analyzed using an SRE-luciferase (SRE-luc) reporter in 293T cells, Huh7 hepatoma cells, and primary human hepatocytes following a variety of conditions, including ectopic expression of HSD17B6, HSD17B6 mutants defective in its enzymatic activities, knockdown of HSD17B6, and cholesterol starvation. The interaction between HSD17B6 and SREBP/SCAP/INSIG complex was analyzed in 293T cells, Huh7 cells and mouse liver upon ectopic expression of HSD17B6 and its mutants; the interaction was also analyzed using endogenous proteins. The impacts of HSD17B6 on SREBP target expression, glucose tolerance, diet-induced obesity, and type 2 diabetes (T2D) were examined using Huh7 cells in vitro, and with C57BL/6 and NONcNZO10/LtJ T2D mice in vivo. ResultsHSD17B6 binds to the SREBP/SCAP/INSIG complex and inhibits SREBP signaling in cultured hepatocytes and mouse liver. Although HSD17B6 plays a role in maintaining the equilibrium of 5α-dihydrotestosterone (DHT) in the prostate, a mutant defective in androgen metabolism was as effective as HSD17B6 in inhibiting SREBP signaling. Hepatic expression of both HSD17B6 and the defective mutant improved glucose intolerance and reduced hepatic triglyceride content in diet-induced obese C57BL/6 mice, while hepatic knockdown of HSD17B6 exacerbated glucose intolerance. Consistent with these results, liver-specific expression of HSD17B6 in a polygenic NONcNZO10/LtJ T2D mice reduced T2D development. ConclusionsOur study unveils a novel role of HSD17B6 in inhibiting SREBP maturation via binding to the SREBP/SCAP/INSIG complex; this activity is independent of HSD17B6's sterol oxidase activity. Through this action, HSD17B6 improves glucose tolerance and attenuates the development of obesity-induced T2D. These findings position HSD17B6 as a potential therapeutic target for T2D therapy.

Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis.

The crosstalk between hematopoietic lineages is important for developmental hematopoiesis. However, the role of primitive red blood cells (RBCs) in the formation of definitive hematopoietic stem and progenitor cells (HSPCs) is largely unknown. Primitive RBC deficiencies in mammals always lead to early embryonic lethality, but zebrafish lines with RBC deficiencies can survive to larval stage. By taking advantage of a zebrafish model, we find that the survival of nascent HSPCs is impaired in alas2- or alad-deficient embryos with aberrant heme biosynthesis in RBCs. Heme-deficient primitive RBCs induce ferroptosis of HSPCs through the disruption of iron homeostasis. Mechanistically, heme-deficient primitive RBCs cause blood iron-overload via Slc40a1, and an HSPC iron sensor, Tfr1b, mediates excessive iron absorption. Thus, iron-induced oxidative stress stimulates the lipid peroxidation, which directly leads to HSPC ferroptosis. Anti-ferroptotic treatments efficiently reverse HSPC defects in alas2 or alad mutants. HSPC transplantation assay reveals that the attenuated erythroid reconstitution efficiency may result from the ferroptosis of erythrocyte-biased HSPCs. Together, these results illustrate that heme-deficient primitive RBCs are detrimental to HSPC production and may provide potential implications for iron dysregulation-induced hematological malignancies.

Sall4 restricts glycolytic metabolism in limb buds through transcriptional regulation of glycolytic enzyme genes

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.

RNA binding protein PRRC2B mediates translation of specific mRNAs and regulates cell cycle progression.

Accumulating evidence suggests that posttranscriptional control of gene expression, including RNA splicing, transport, modification, translationand degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and sequencing (PAR-CLIP-seq), we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites near the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by polysome-associated RNA-seq, resulting in reduced G1/S phase transition and cell proliferation. Antisense oligonucleotides blocking PRRC2B interactions with CCND2 mRNA decreased its translation, thus inhibiting G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome analysis revealed RNA-independent interactions with eukaryotic translation initiation factors 3 (eIF3) and 4G2 (eIF4G2). The interaction with translation initiation factors is essential for PRRC2B function since the eIF3/eIF4G2-interacting defective mutant, unlike wild-type PRRC2B, failed to rescue the translation deficiency or cell proliferation inhibition caused by PRRC2B knockdown. Altogether, our findings reveal that PRRC2B is essential for efficiently translating specific proteins required for cell cycle progression and cell proliferation.

The Brucella abortus Type IV Effector BspA Inhibits MARCH6-Dependent ERAD To Promote Intracellular Growth.

Brucella abortus, the intracellular causative agent of brucellosis, relies on type IV secretion system (T4SS) effector-mediated modulation of host cell functions to establish a replicative niche, the Brucella-containing vacuole (BCV). Brucella exploits the host's endocytic, secretory, and autophagic pathways to modulate the nature and function of its vacuole from an endocytic BCV (eBCV) to an endoplasmic reticulum (ER)-derived replicative BCV (rBCV) to an autophagic egress BCV (aBCV). A role for the host ER-associated degradation pathway (ERAD) in the B. abortus intracellular cycle was recently uncovered, as it is enhanced by the T4SS effector BspL to control the timing of aBCV-mediated egress. Here, we show that the T4SS effector BspA also interferes with ERAD, yet to promote B. abortus intracellular proliferation. BspA was required for B. abortus replication in bone marrow-derived macrophages and interacts with membrane-associated RING-CH-type finger 6 (MARCH6), a host E3 ubiquitin ligase involved in ERAD. Pharmacological inhibition of ERAD and small interfering RNA (siRNA) depletion of MARCH6 did not affect the replication of wild-type B. abortus but rescued the replication defect of a bspA deletion mutant, while depletion of the ERAD component UbxD8 affected replication of B. abortus and rescued the replication defect of the bspA mutant. BspA affected the degradation of ERAD substrates and destabilized the MARCH6 E3 ligase complex. Taken together, these findings indicate that BspA inhibits the host ERAD pathway via targeting of MARCH6 to promote B. abortus intracellular growth. Our data reveal that targeting ERAD components by type IV effectors emerges as a multifaceted theme in Brucella pathogenesis.

The origin, evolution and functional divergence of HOOKLESS1 in plants

Apical hooks are functional innovations only observed in angiosperms, which effectively protect the apical meristems out of damage during plant seedlings penetrating soil covers. Acetyltransferase like protein HOOKLESS1 (HLS1) in Arabidopsis thaliana is required for hook formation. However, the origin and evolution of HLS1 in plants are still not solved. Here, we traced the evolution of HLS1 and found that HLS1 originated in embryophytes. Moreover, we found that Arabidopsis HLS1 delayed plant flowering time, in addition to their well-known functions in apical hook development and newly reported roles in thermomorphogenesis. We further revealed that HLS1 interacted with transcription factor CO and repressed the expression of FT to delay flowering. Lastly, we compared the functional divergence of HLS1 among eudicot (A. thaliana), bryophytes (Physcomitrium patens and Marchantia polymorpha) and lycophyte (Selaginella moellendorffii). Although HLS1 from these bryophytes and lycophyte partially rescued the thermomorphogenesis defects in hls1-1 mutants, the apical hook defects and early flowering phenotypes could not be reversed by either P. patens, M. polymorpha or S. moellendorffii orthologs. These results illustrate that HLS1 proteins from bryophytes or lycophyte are able to modulate thermomorphogenesis phenotypes in A. thaliana likely through a conserved gene regulatory network. Our findings shed new light on the understanding of the functional diversity and origin of HLS1, which controls the most attractive innovations in angiosperms.

Exo1 protects DNA nicks from ligation to promote crossover formation during meiosis.

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is essential to produce haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday junction (dHJ) intermediates. This dHJ resolution step involves the actions of Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. Here, we provide genetic evidence in baker's yeast that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 that interact with DNA, such as those required for the bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase reduced the crossover levels of exo1 DNA-binding mutants to levels that approached the exo1 null. In addition, our work identified a role for Exo1 in crossover interference. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.

The rose INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE genes, RbIDL1 and RbIDL4, regulate abscission in an ethylene-responsive manner.

RbIDL1 and RbIDL4 are up-regulated in an ethylene-responsive manner during rose petal abscission and restored the Arabidopsisida-2 mutant abscission defect suggesting functional conservation of the IDA pathway in rose. Abscission is an ethylene-regulated developmental process wherein plants shed unwanted organs in a controlled manner. The INFLORESCENCE DEFICIENT IN ABSCISSION family has been identified as a key regulator of abscission in Arabidopsis, encoding peptides that interact with receptor-like kinases to activate abscission. Loss of function ida mutants show abscission deficiency in Arabidopsis. Functional conservation of the IDA pathway in other plant abscission processes is a matter of interest given the discovery of these genes in several plants. We have identified four members of the INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE family from the ethylene-sensitive, early-abscising fragrant rose, Rosa bourboniana. All four are conserved in sequence and possess well-defined PIP, mIDa and EPIP motifs. Three of these, RbIDL1, RbIDL2 and RbIDL4 show a three-fourfold increase in transcript levels in petal abscission zones (AZ) during ethylene-induced petal abscission as well as natural abscission. The genes are also expressed in other floral tissues but respond differently to ethylene in these tissues. RbIDL1 and RbIDL4, the more prominently expressed IDL genes in rose, can complement the abscission defect of the Arabidopsis ida-2 mutant; while, promoters of both genes can drive AZ-specific expression in an ethylene-responsive manner even in Arabidopsis silique AZs indicating recognition of AZ-specific and ethylene-responsive cis elements in their promoters by the abscission machinery of rose as well as Arabidopsis.

Germline stem cell integrity and quiescence are controlled by an AMPK-dependent neuronal trafficking pathway

During periods of energetic stress, Caenorhabditis elegans can execute a developmentally quiescent stage called "dauer", during which all germline stem cells undergo a G2 cell cycle arrest. In animals that lack AMP-activated protein kinase (AMPK) signalling, the germ cells fail to arrest, undergo uncontrolled proliferation, and lose their reproductive capacity upon recovery from this quiescent stage. These germline defects are accompanied by, and likely result from, an altered chromatin landscape and gene expression program. Through genetic analysis we identified an allele of tbc-7, a predicted RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is modulated by tbc-7 and show that the activity of RAB-7 is critical for the maintenance of germ cell integrity during the dauer stage. We reveal that TBC-7 is regulated by AMPK through two mechanisms when the animals enter the dauer stage. Acutely, the AMPK-mediated phosphorylation of TBC-7 reduces its activity, potentially by autoinhibition, thereby preventing the inactivation of RAB-7. In the more long term, AMPK regulates the miRNAs mir-1 and mir-44 to attenuate tbc-7 expression. Consistent with this, animals lacking mir-1 and mir-44 are post-dauer sterile, phenocopying the germline defects of AMPK mutants. Altogether, we have uncovered an AMPK-dependent and microRNA-regulated cellular trafficking pathway that is initiated in the neurons, and is critical to control germline gene expression cell non-autonomously in response to adverse environmental conditions.

Down-Regulation of Strigolactone Biosynthesis Gene D17 Alters the VOC Content and Increases Sogatella furcifera Infectivity in Rice

DWARF17 (D17/HTD1) is a well-defined rice strigolactone (SL) biosynthesis gene that influences rice tiller development and the production of rice. To investigate whether SLs play a role in the regulation of rice’s defense against the white-backed planthopper (WBPH, Sogatella furcifera), we compared a SL-biosynthetic defective mutant (d17) with WT rice plants. Our olfactory bioassay results revealed that WBPHs are attracted to d17 plants, which may be attributed to changes in rice volatile substances. Hexanal, a volatile substance, was significantly reduced in the d17 plants, and it was demonstrated that it repelled WBPHs at a concentration of 100 μL/L. Compared to the WT plants, WBPH female adults preferred to oviposit on d17 plants, where the egg hatching rate was higher. The transcript level analysis of defense-associated genes in the JA and SA pathways showed that the expression of OsJAmyb, OsJAZ8, OsPR1a and OsWRKY62 were significantly reduced in d17 plants compared to WT plants following WBPH infection. These findings suggest that silencing the strigolactone biosynthesis gene D17 weakens defenses against S. furcifera in rice.

Abstract 3500: A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma

Abstract Amplification of chromosomal material derived from 12q is pathognomonic in a subset of soft tissue sarcomas (STS), including well-differentiated and dedifferentiated liposarcoma. However, it remains unclear which of the amplified genes are oncogenic drivers, obstacles to rational therapeutic development. We defined a minimal 12q amplicon in human liposarcoma, and tested the oncogenic activity of each recurrently amplified gene to accelerate the onset of AKT-induced STS in a genetically engineered zebrafish model of the disease. This revealed several genes whose overexpression accelerated the onset of AKT-induced STS, including METTL1, best-known for its ability to catalyze methylation of tRNAs. Surprisingly, catalytically defective mutants of METTL1 exhibited oncogenic activity that was at least as potent as the wild-type gene in zebrafish and human STS, indicating enzyme-independent oncogenic functions. We found that METTL1 promotes ribosome assembly and oncogenic translation independent of its enzymatic activity. In human liposarcoma cells and mouse embryonic fibroblasts, METTL1 overexpression was sufficient to stimulate polysome formation, and this effect was phenocopied by catalytically dead mutants of METTL1. METTL1 overexpression also robustly stimulated the rate of protein synthesis in cells (P < 0.0001), and in a cell-free biochemical assay for translation of a reporter mRNA (P < 0.001). The ability of METTL1 to stimulate ribosomal function triggered a therapeutically targetable dependency, because small molecule inhibitors of ribosome biogenesis had potent therapeutic activity against METTL1-amplified (P < 0.0001), but not METTL1 wild-type, sarcoma cells. Thus, METTL1 is a soft tissue sarcoma oncogene that stimulates oncogenic translation independent of its enzymatic activity, an oncogenic function that can be targeted therapeutically. Citation Format: Raja Hussain Ali, Brianna Silverman, Esteban A. Orellana, Alyssa Kennedy, Caitlin Bowers, Ashley Gutierrez, David Papke, Christopher D. Fletcher, Antonio P. Atayde, Akiko Shimamura, Richard I. Gregory, Alejandro Gutierrez. A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3500.

Tendril morphogenesis is regulated by a CsaTEN-CsaUFO module in cucumber.

Tendril is a morphological innovation during plant evolution, which provides the plants to obtain climbing ability. However, the tendril morphogenesis is poorly understood. A novel tendril morphogenesis defective mutant (tmd1) was identified in cucumber. The apical part of tendril was replaced by a leaf blade in tmd1 mutant, and it lost the climbing ability. Map-based cloning, qPCR detection, bioinformatic analysis, yeast one-hybrid assay, electrophoretic mobility shift assay, and luciferase assay were used to explore the molecular mechanism of CsaTMD1 in regulating tendril morphogenesis. CsaUFO was the candidate causal gene, and a fragment deletion within promoter impaired CsaUFO expression in tmd1 mutant. A conserved motif 1, which harbored two putative TCP transcription factor binding sites, was located within this deleted fragment. CsaTEN directly bound the motif 1 and positively regulated CsaUFO, and mutation in motif 1 removed this regulation. Our work shows a CsaTEN-CsaUFO module in regulating tendril morphogenesis, indicating that evolution of tendril in cucumber due to simply drive of CsaUFO by CsaTEN in tendril. Additionally, the conserved motif 1 provides a strategy for engineering tendril-less Cucurbitaceae crops.

Multi-copper oxidases SKU5 and SKS1 coordinate cell wall formation using apoplastic redox-based reactions in roots.

The primary cell wall is a fundamental plant constituent that is flexible but sufficiently rigid to support the plant cell shape. Although many studies have demonstrated that reactive oxygen species (ROS) serve as important signaling messengers to modify the cell wall structure and affect cellular growth, the regulatory mechanism underlying the spatial-temporal regulation of ROS activity for cell wall maintenance remains largely unclear. Here, we demonstrate the role of the Arabidopsis (Arabidopsis thaliana) multicopper oxidase-like protein skewed 5 (SKU5) and its homolog SKU5-similar 1 (SKS1) in root cell wall formation through modulating ROS homeostasis. Loss of SKU5 and SKS1 function resulted in aberrant division planes, protruding cell walls, ectopic deposition of iron, and reduced nicotinamide adeninedinucleotide phosphate (NADPH) oxidase-dependent ROS overproduction in the root epidermis-cortex and cortex-endodermis junctions. A decrease in ROS level or inhibition of NADPH oxidase activity rescued the cell wall defects of sku5 sks1 double mutants. SKU5 and SKS1 proteins were activated by iron treatment, and iron over-accumulated in the walls between the root epidermis and cortex cell layers of sku5 sks1. The glycosylphosphatidylinositol-anchored motif was crucial for membrane association and functionality of SKU5 and SKS1. Overall, our results identified SKU5 and SKS1 as regulators of ROS at the cell surface for regulation of cell wall structure and root cell growth.

ASYMMETRIC LEAVES 2 and ASYMMETRIC LEAVES 2-LIKE are partially redundant genes and essential for fruit development in tomato.

Fruit size and shape are controlled by genes expressed during the early developmental stages of fruit. Although the function of ASYMMETRIC LEAVES 2 (AS2) in promoting leaf adaxial cell fates has been well characterized in Arabidopsis thaliana, the molecular mechanisms conferring freshy fruit development as a spatial-temporal expression gene in tomato pericarp remain unclear. In the present study, we verified the transcription of SlAS2 and SlAS2L, two homologs of AS2, in the pericarp during early fruit development. Disruption of SlAS2 or SlAS2L caused a significant decrease in pericarp thickness as a result of a reduction in the number of pericarp cell layers and cell area, leading to smaller tomato fruit size, which revealed their critical roles in tomato fruit development. In addition, leaves and stamens exhibited severe morphological defects in slas2 and slas2l single mutants, as well as in the double mutants. These results demonstrated the redundant and pleiotropic functions of SlAS2 and SlAS2L in tomato fruit development. Yeast two-hybrid and split-luciferase complementation assays showed that both SlAS2 and SlAS2L physically interact with SlAS1. Molecular analyses further indicated that SlAS2 and SlAS2L regulate various downstream genes in leaf and fruit development, and that some genes participating in the regulation of cell division and cell differentiation in the tomato pericarp are affected by these genes. Our findings demonstrate that SlAS2 and SlAS2L are vital transcription factors required for tomato fruit development.