Knockdown Mutants Research Articles

Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation

Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.

O-GlcNAcylation attenuates ischemia-reperfusion-induced pulmonary epithelial cell ferroptosis via the Nrf2/G6PDH pathway

BackgroundLung ischemia–reperfusion (I/R) injury is a common clinical pathology associated with high mortality. The pathophysiology of lung I/R injury involves ferroptosis and elevated protein O-GlcNAcylation levels, while the effect of O-GlcNAcylation on lung I/R injury remains unclear. This research aimed to explore the effect of O-GlcNAcylation on reducing ferroptosis in pulmonary epithelial cells caused by I/R.ResultsFirst, we identified O-GlcNAc transferase 1 (Ogt1) as a differentially expressed gene in lung epithelial cells of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) patients, using single-cell sequencing, and Gene Ontology analysis (GO analysis) revealed the enrichment of the ferroptosis process. We found a time-dependent dynamic alteration in lung O-GlcNAcylation during I/R injury. Proteomics analysis identified the differentially expressed proteins enriched in ferroptosis and multiple redox-related pathways based on KEGG annotation. Thus, we generated Ogt1-conditional knockout mice and found that Ogt1 deficiency aggravated ferroptosis, as evidenced by lipid reactive oxygen species (lipid ROS), malondialdehyde (MDA), Fe2+, as well as alterations in critical protein expression glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Consistently, we found that elevated O-GlcNAcylation inhibited ferroptosis sensitivity in hypoxia/reoxygenation (H/R) injury-induced TC-1 cells via O-GlcNAcylated NF-E2-related factor-2 (Nrf2). Furthermore, both the chromatin immunoprecipitation (ChIP) assay and the dual-luciferase reporter assay indicated that Nrf2 could bind with translation start site (TSS) of glucose-6-phosphate dehydrogenase (G6PDH) and promote its transcriptional activity. As an important rate-limiting enzyme in the pentose phosphate pathway (PPP), elevated G6PDH provided a mass of nicotinamide adenine dinucleotide phosphate (NADPH) to improve the redox state of glutathione (GSH) and eventually led to ferroptosis resistance. Rescue experiments proved that Nrf2 knockdown or Nrf2-T334A (O-GlcNAcylation site) mutation abolished the protective effect of ferroptosis resistance.ConclusionsIn summary, we revealed that O-GlcNAcylation could protect against I/R lung injury by reducing ferroptosis sensitivity via the Nrf2/G6PDH pathway. Our work will provide a new basis for clinical therapeutic strategies for pulmonary ischemia–reperfusion-induced acute lung injury.

A Novel Gene DUSP8 Missense Mutation Causes Nonsyndromic Hereditary Gingival Fibromatosis by Dysregulating Lysine Lactylation.

The goal of this study was to explore new candidate genes and pathogenesis mechanisms of nonsyndromic hereditary gingival fibromatosis (nsHGF) and to provide an experimental basis for the diagnosis of nsHGF. Whole-exome sequencing (WES) was performed on peripheral blood DNA from three nsHGF family members to screen for new candidate genes, and Sanger sequencing and related databases were used to verify the pathogenicity of this gene deficiency. Moreover, the effects of gene deficiency on the biological characteristics of human gingival fibroblasts (HGFs) were evaluated via cell proliferation assays, extracellular matrix (ECM) deposition detection, cell apoptosis and cell cycle assessment, cell migration and gene expression analyses. A novel missense mutation in dual-specificity phosphatase 8 (DUSP8, c.1348C>T, p.R450C), which is in the nsHGF-related GINGF4 locus, was identified via WES analysis. A functional study revealed that knocking down DUSP8 expression increased cell proliferation, cell migration and the expression of profibrotic factors (particularly COL1A1), inhibited cell apoptosis, and ultimately resulted in nsHGF. Similarly, this DUSP8 mutation inhibited the expression of the encoded protein and promoted cell proliferation and the expression of profibrotic factors. In addition, both DUSP8 knockdown and DUSP8 mutation induced nsHGF by accelerating glycolysis and panlysine lactylation (Kla) to promote cell proliferation and the expression of ECM-related factors. DUSP8 deficiency might be a novel pathogenic factor that contributes to nsHGF.

Phosphorylation of annexin A2 at serine 25 is required for endothelin-1 stimulated cell proliferation and AKT activation in melanoma cells.

Endothelin (ET)-1 contributes to melanoma progression via cell proliferation, invasion, and migration. We previously reported that annexin A2 (AnxA2) binds to ET receptors. In this study, we aimed to further investigate role of AnxA2 in melanoma cell proliferation after ET-1 stimulation. AnxA2 knockdown inhibited ET-1-stimulated cell proliferation and AKT activation in SK-MEL28 melanoma cells. ET-1 stimulation phosphorylated serine on AnxA2, and AnxA2 Ser25 phosphorylation-deficient mutant (AnxA2 S25A) cells showed lower ET-1-stimulated cell proliferation and AKT activation than the rescue AnxA2 knockdown (AnxA2 res) and AnxA2 Ser11 phosphorylation-deficient mutant (AnxA2 S11A) cells. Although AnxA2 S25A was localized to the plasma membrane, it exhibited lower colocalization with ET receptors than AnxA2 res and AnxA2 S11A on the plasma membrane. These results suggest that phosphorylation of AnxA2 Ser25 affects the colocalization of AnxA2 and ETRs and plays an important role in cell proliferation and AKT activation in ET-1 stimulated melanoma cells.

The fungal effector AaAlta1 inhibits PATHOGENESIS-RELATED PROTEIN10-2-mediated callose deposition and defense responses in apple.

Apple leaf spot, caused by Alternaria alternata f. sp mali (ALT), poses a substantial threat to the global apple (Malus × domestica Borkh.) industry. Fungal effectors promote pathogen infestation and survival by interfering with plant immune responses. In our study, we investigated the secretion of effector proteins by the virulent ALT7 strain. Using mass spectrometry, we identified the effector AaAlta1, which belongs to the Alt a 1 protein family (AA1s). Further analysis confirmed that ALT7 secretes AaAlta1. AaAlta1 knockdown mutants displayed reduced pathogenicity in apple tissue culture seedlings, while overexpression strains exhibited enhanced pathogenicity compared to the wild-type ALT7 strain. Using immunoprecipitation followed by mass spectrometry, we isolated pathogenesis-related protein 10-2 (PR10-2) as an interaction partner of AaAlta1 in apple. Knockdown mutants of AaAlta1 showed increased PR10-2-mediated callose deposition in apple, a critical plant defense response. The enhanced defense responses in apple substantially reduced their susceptibility to infection by these ALT7 mutants. Our findings delineate an infection strategy whereby ALT7 secretes AaAlta1 to suppress PR10-2, thereby circumventing the apple defense system.

Generation of viable hypomorphic and null mutant plants via CRISPR-Cas9 targeting mRNA splicing sites.

Genetic analysis is important for modern plant molecular biology, and in this regard, the existence of specific mutants is crucial. While genome editing technologies, particularly CRISPR-Cas9, have revolutionized plant molecular biology by enabling precise gene disruption, knockout methods are ineffective for lethal genes, necessitating alternatives like gene knockdown. This study demonstrates the practical generation of a hypomorphic mutant allele, alongside severe null mutant alleles, via the targeting of mRNA splicing sites using CRISPR-Cas9. The Arabidopsis HIGH PLOIDY 2 (HPY2) encodes a yeast NSE2 ortholog, part of the conserved eukaryotic SMC5/6 complex, with SUMO E3 ligase activity essential for cell cycle progression and plant development. Loss-of-function HPY2 mutants exhibit severe dwarfism and seedling lethality, making functional analysis challenging. To overcome these limitations, we created HPY2 knockdown mutants as novel tools to investigate gene function. Of the three mutant alleles, the hpy2-cr1 and hpy2-cr2 mutants resembled the existing severe hpy2-1 allele, both harboring a single base pair insertion in one exon, causing significant root shortening and seedling lethality. In contrast, the hypomorphic mutant hpy2-cr3, which has a five bp deletion at an intron-exon junction, showed relatively longer root growth and survived until the reproductive stage. RT-PCR analysis of hpy2-cr3 revealed atypical mRNAs producing truncated polypeptides that retained some HPY2 function, explaining the milder phenotype. These results establish the successful generation of novel hypomorphic mutant alleles critical for studying the lethal gene HPY2, and demonstrate the usefulness of CRISPR-Cas9 for producing viable hypomorphic mutants for investigating complex genetic interactions.

A Mycorrhiza-Induced UDP-Glucosyl Transferase Negatively Regulates the Arbuscular Mycorrhizal Symbiosis.

Most terrestrial plants can establish a reciprocal symbiosis with arbuscular mycorrhizal (AM) fungi to cope with adverse environmental stresses. The development of AM symbiosis is energetically costly and needs to be dynamically controlled by plants to maintain the association at mutual beneficial levels. Multiple components involved in the autoregulation of mycorrhiza (AOM) have been recently identified from several plant species; however, the mechanisms underlying the feedback regulation of AM symbiosis remain largely unknown. Here, we report that AM colonization promotes the flavonol biosynthesis pathway in tomato (Solanum lycopersicum), and an AM-specific UDP-glucosyltransferase SlUGT132, which probably has the flavonol glycosylation activity, negatively regulates AM development. SlUGT132 was predominantly expressed in the arbuscule-containing cells, and its knockout or knockdown mutants showed increased soluble sugar content, root colonization level and arbuscule formation. Conversely, overexpression of SlUGT132 resulted in declined soluble sugar content and mycorrhization degree. Metabolomic assay revealed decreased contents of astragalin, tiliroside and cynaroside in slugt132 mycorrhizal roots, but increased accumulation of these flavonoid glycosides in SlUGT132-overexpressing plant roots. Our results highlight the presence of a novel, SlUGT132-mediated AOM mechanism, which enable plants to flexibly control the accumulation of soluble sugars and flavonoid glycosides in mycorrhizal roots and modulate colonization levels.

Investigation of resistance ratios and resistance mechanisms of Cydia pomonella (L.) (lepidoptera: Tortricidae) populations collected from apple orchards in Isparta (Türkiye) against some insecticides

The codling moth, Cydia pomonella is an important pest that causes significant economic losses in apples and walnuts in the world. The aim of this study was to investigate, for the first time in Türkiye, the resistance status and resistance mechanisms of codling moth, Cydia pomonella against indoxacarb, deltamethrin and emamectin benzoate. All apple orchard populations developed resistance ratios ranging from 3.38 to 22.37-fold to deltamethrin, 5.67–29.87-fold to indoxacarb and 1.46–3.05-fold to emamectin benzoate. The interaction of some synergists triphenyl phosphate (TPP), piperonyl butoxide (PBO) and diethyl maleate (DEM) with indoxacarb and deltamethrin was analyzed in the susceptible, MAREM and Tepeli populations with moderate resistance to indoxacarb and deltamethrin. While indoxacarb + TPP showed a significant synergistic effect only in MAREM population, a significant synergistic effect was observed with indoxacarb + TPP and indoxacarb + PBO in Tepeli population. The activities of detoxifying enzymes [esterase, glutathion –S– transferase (GST) and cytochrome P450 monooxygenase (P450)] studied by biochemical methods showed some variation depending on the population. The results of biochemical analyses showed that esterase and GST enzyme activities of all populations was between 0.51 and 0.94, 0.77 and1.29 mOD min−1mg−1 proteins, respectively. The P450 enzyme activities ranged from 0.53 to 0.78, RFU min−1mg−1 proteins. In addition, the L1014F knockdown mutation (CTT to TTT) corresponding to leucine to phenylalanine amino acid substitution of the voltage-gated sodium channel in Cydia pomonella was determined in MAREM and Tepeli populations. It was determined that while MAREM and Tepeli populations developed moderate resistance, the other populations developed a low level resistance to deltamethrin and indoxacarb. On the other hand, all populations developed a low level resistance to emamectin benzoate. The P450 and esterase enzyme activities were significantly higher in MAREM and Tepeli populations which were resistant to deltamethrin and indoxacarb than the susceptible population. In addition, a Kdr point mutation L1014F was detected in the deltamethrin resistant MAREM and Tepeli populations.

Knockdown of β-conglycinin α' and α subunits alters seed protein composition and improves salt tolerance in soybean.

Soybean is an important plant source of protein worldwide. Increasing demands for soybean can be met by improving the quality of its seed protein. In this study, GmCG-1, which encodes the β-conglycinin α' subunit, was identified via combined genome-wide association study and transcriptome analysis. We subsequently knocked down GmCG-1 and its paralogues GmCG-2 and GmCG-3 with CRISPR-Cas9 technology and generated two stable multigene knockdown mutants. As a result, the β-conglycinin content decreased, whereas the 11S/7S ratio, total protein content and sulfur-containing amino acid content significantly increased. Surprisingly, the globulin mutant exhibited salt tolerance in both the germination and seedling stages. Little is known about the relationship between seed protein composition and the salt stress response in soybean. Metabonomics and RNA-seq analysis indicated that compared with the WT, the mutant was formed through a pathway that was more similar to that of active salicylic acid biosynthesis; however, the synthesis of cytokinin exhibited greater defects, which could lead to increased expression of plant dehydrin-related salt tolerance proteins and cell membrane ion transporters. Population evolution analysis suggested that GmCG-1, GmCG-2, and GmCG-3 were selected during soybean domestication. The soybean accessions harboring GmCG-1Hap1 presented relatively high 11S/7S ratios and relatively high salt tolerance. In conclusion, knockdown of the β-conglycinin α and α' subunits can improve the nutritional quality of soybean seeds and increase the salt tolerance of soybean plants, providing a strategy for designing soybean varieties with high nutritional value and high salt tolerance.

Improving Lipid Content in the Diatom Phaeodactylum tricornutum by the Knockdown of the Enoyl-CoA Hydratase Using CRISPR Interference.

The diatom Phaeodactylum tricornutum shows potential as a source for biofuel production because of its considerable lipid content. Fatty acid β-oxidation plays a critical role in lipid breakdown. However, we still have a limited understanding of the role of fatty acid β-oxidation in lipid content in this microalga. In our study, we utilized a CRISPR interference method to reduce the expression of enoyl-CoA hydratase (PtECH), which is involved in the hydration of trans-2-enoyl-CoA to produce 3-hydroxyacyl-CoA during the β-oxidation pathway. Using this method, we developed two transgenic lines, PtECH21 and PtECH1487, which resulted from interference at two different sites of the PtECH gene, respectively. RT-qPCR analysis confirmed that the mRNA levels of PtECH in both mutants were significantly lower compared to the wild type. Surprisingly, the lipid content of both mutants increased notably. Additionally, both knockdown mutants exhibited higher chlorophyll content and improved photosynthetic efficiency of the photosystem II compared to the wild type. This study introduces a new approach for enhancing lipid content in P. tricornutum and expands our knowledge of the functions of enoyl-CoA hydratase in microalgae.

Abstract C119: Exploring the functional role of LRIG1 and WWOX tumor suppressors in breast cancer disparities

Abstract Breast cancer is the most commonly diagnosed cancer among women in the US. Despite a lower rate of breast cancer incidence, Black women in the US suffer higher rates of mortality compared to White women. This disparity poses a major clinical issue for Black women in the US, and we are particularly interested in understanding how molecular and environmental mechanisms may be contributing to these disproportionate outcomes. Our previous study found that in a cohort of women from Baltimore, Maryland (110 Black, 75 White), higher neighborhood deprivation was associated with decreased gene body methylation and gene expression of two tumor suppressor genes, LRIG1 and WWOX. These findings were unique to the tumor, which potentially points to ancestry-specific somatic mutations that may impact gene and protein expression for LRIG1 and WWOX. The goal of this project is to determine gene and protein differences in LRIG1 and WWOX by population group in women with and without breast cancer as it relates to neighborhood-level socioeconomic deprivation. We hypothesize that LRIG1 and WWOX exhibit decreased gene and protein expression in Black women compared to White women. Using the same diverse cohort from Baltimore, Maryland, we identified and conducted DNA genotyping on clinically relevant mutations of LRIG1 and WWOX using amplification by PCR and detection by fluorescent reporters. We found that the genotype frequencies in two single nucleotide polymorphisms (SNPs) for LRIG1, rs1403626 (P=6.3e-1) and rs2306272 (P=0.005967), differ significantly between population groups in these breast tissue samples, an interesting finding that matches our previous predictions. We also identified a dose-dependent relationship between LRIG1 gene expression and rs1403626 SNP genotypes. Additional patient genotyping will be conducted to determine relationships between LRIG1 and WWOX SNPs, differences in DNA methylation, and correlations with neighborhood deprivation measures. To interrogate the impact on protein expression, we will construct LRIG1 and WWOX knockdown mutants in estrogen receptor (ER)-positive and ER-negative commercially available breast cancer cell lines derived from Black and White patients, and determine the functional consequences of LRIG1 and WWOX loss by immunofluorescence and western blot, including how this may differ by population group. These studies will give us a better understanding of how the expression of tumor suppressor genes may contribute to the higher susceptibility of Black women developing more aggressive breast tumors. Citation Format: Angel H. Pajimola, Brittany Jenkins-Lord. Exploring the functional role of LRIG1 and WWOX tumor suppressors in breast cancer disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C119.

The Vacuolar H+-ATPase subunit C is involved in oligogalacturonide (OG) internalization and OG-triggered immunity

In plants, the perception of cell wall fragments initiates signal transduction cascades that activate the immune response. Previous research on early protein dynamics induced by oligogalacturonides (OGs), pectin fragments acting as damage-associated molecular patterns (DAMPs), revealed significant phosphorylation changes in several proteins. Among them, the subunit C of the vacuolar H+-ATPase, known as DE-ETIOLATED 3 (DET3), was selected to elucidate its role in the OG-triggered immune response. The Arabidopsis det3 knockdown mutant exhibited defects in H2O2 accumulation, mitogen-activated protein kinases (MAPKs) activation, and induction of defense marker genes in response to OG treatment. Interestingly, the det3 mutant showed a higher basal resistance to the fungal pathogen Botrytis cinerea that, in turn, was completely reversed by the pre-treatment with OGs. Our results suggest a compromised ability of the det3 mutant to maintain a primed state over time, leading to a weaker defense response when the plant is later exposed to the fungal pathogen. Using fluorescently labelled OGs, we demonstrated that endocytosis of OGs was less efficient in the det3 mutant, implicating DET3 in the internalization process of OGs. This impairment aligns with the observed defect in the priming response in the det3 mutant, underscoring that proper internalization and signaling of OGs are crucial for initiating and maintaining a primed state in plant defense responses.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

The yellow-cotyledon gene (ATYCO) is a crucial factor for thylakoid formation and photosynthesis regulation in Arabidopsis

Chloroplast development underpins plant growth, by facilitating not only photosynthesis but also other essential biochemical processes. Nonetheless, the regulatory mechanisms and functional components of chloroplast development remain largely uncharacterized due to their complexity. In our study, we identified a plastid-targeted gene, ATYCO/RP8/CDB1, as a critical factor in early chloroplast development in Arabidopsis thaliana. YCO knock-out mutant (yco) exhibited a seedling-lethal, albino phenotype, resulting from dysfunctional chloroplasts lacking thylakoid membranes. Conversely, YCO knock-down mutants produced a chlorophyll-deficient cotyledon and normal leaves when supplemented with sucrose. Transcription analysis also revealed that YCO deficiency could be partially compensated by sucrose supplementation, and that YCO played different roles in the cotyledons and the true leaves. In YCO knock-down mutants, the transcript levels of plastid-encoded RNA polymerase (PEP)-dependent genes and nuclear-encoded photosynthetic genes, as well as the accumulation of photosynthetic proteins, were significantly reduced in the cotyledons. Moreover, the chlorophyll-deficient phenotype in YCO knock-down line can be effectively suppressed by inhibition of PSI cyclic electron transport activity, implying an interaction between YCO and PSI cyclic electron transport. Taken together, our findings de underscore the vital role of YCO in early chloroplast development and photosynthesis.

The invertase gene PWIN1 confers chilling tolerance of rice at the booting stage via mediating pollen development.

Pollen fertility is a primary regulator of grain yield and is highly susceptible to cold and other environmental stress. We revealed the roles of rice cell wall invertase gene PWIN1 in pollen development and chilling tolerance. We uncovered its preferential expression in microspores and bicellular pollen and identified its knock-down and knock-out mutants. pwin1 mutants produced a higher proportion of abnormal pollen than wild-type plants. The contents of sucrose, glucose, and fructose were increased, while ATP content and primary metabolism activity were reduced in the mutant pollen. Furthermore, the loss of function of PWIN1 coincided with an increase in SnRK1 activity and a decrease in TOR activity. Under chilling conditions, pwin1 mutants displayed significantly reduced pollen viability and seed-setting rate, while overexpressing PWIN1 notably increased pollen viability and seed-setting rate as compared with the wild-type, indicating that PWIN1 is essential for rice pollen development and grain yield under cold stress. This study provides insights into the molecular mechanisms underlying rice pollen fertility during chilling stress, and a new module to improve chilling tolerance of rice at the booting stage by molecular design.

The origin and insecticide resistance of Aedes albopictus mosquitoes established in southern Mozambique

BackgroundThe Aedes albopictus mosquito is of medical concern due to its ability to transmit viral diseases, such as dengue and chikungunya. Aedes albopictus originated in Asia and is now present on all continents, with the exception of Antarctica. In Mozambique, Ae. albopictus was first reported in 2015 within the capital city of Maputo, and by 2019, it had become established in the surrounding area. It was suspected that the mosquito population originated in Madagascar or islands of the Western Indian Ocean (IWIO). The aim of this study was to determine its origin. Given the risk of spreading insecticide resistance, we also examined relevant mutations in the voltage-sensitive sodium channel (VSSC).MethodsEggs of Ae. albopictus were collected in Matola-Rio, a municipality adjacent to Maputo, and reared to adults in the laboratory. Cytochrome c oxidase subunit I (COI) sequences and microsatellite loci were analyzed to estimate origins. The presence of knockdown resistance (kdr) mutations within domain II and III of the VSSC were examined using Sanger sequencing.ResultsThe COI network analysis denied the hypothesis that the Ae. albopictus population originated in Madagascar or IWIO; rather both the COI network and microsatellites analyses showed that the population was genetically similar to those in continental Southeast Asia and Hangzhou, China. Sanger sequencing determined the presence of the F1534C knockdown mutation, which is widely distributed among Asian populations, with a high allele frequency (46%).ConclusionsThese results do not support the hypothesis that the Mozambique Ae. albopictus population originated in Madagascar or IWIO. Instead, they suggest that the origin is continental Southeast Asia or a coastal town in China.Graphical

Rationally Designed Pooled CRISPRi-Seq Uncovers an Inhibitor of Bacterial Peptidyl-tRNA Hydrolase.

Bacterial mutant libraries in which antibiotic targets are downregulated are useful tools to functionally characterize novel antimicrobials. These libraries are used for chemical-genetic profiling as target-compound interactions can be inferred by differential fitness of mutants during pooled growth. Mutants that are functionally related to the antimicrobial mode of action are usually depleted from the pool upon exposure to the drug. Although powerful, this method can fail when the unequal proliferation of mutant strains before exposure causes mutants to fall below the detection level in the library pool. To address this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi) and analyzed the growth parameters of individual mutant strains. We then modelled depletion levels during pooled growth and used the model to rationally design an optimized CRISPR interference-mediated pooled library of essential genes (CIMPLE). By adjusting the initial inoculum of the knockdown mutants, we achieved coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to a recently discovered antimicrobial of a novel class and discovered it inhibits the peptidyl-tRNA hydrolase, an essential bacterial enzyme. In summary, we demonstrate the utility of CIMPLE and CRISPRi-Seq to uncover the mechanism of action of novel antimicrobial compounds.

PUB40 attenuates Phytophthora capsici resistance by destabilizing the MEK2-SIPK/WIPK cascade in Nicotiana benthamiana

The mitogen-activated protein kinase (MAPK) cascade MEK2-SIPK/WIPK is essential for immunity in Solanaceae plants. This cascade is tightly controlled to prevent harmful hyperactivation. However, the E3 ubiquitin ligases utilized by plants to reduce MEK2- SIPK/WIPK protein levels remain largely elusive. Here, we confirmed the essential role of Nicotiana benthamiana MEK2-SIPK/WIPK in resistance to the oomycete pathogen Phytophthora capsici. Using tobacco rattle virus (TRV)-based gene silencing, we screened prevalent plant U-box protein (PUB)-type E3 ligases with Armadillo (ARM) repeats to characterize those involved in Phytophthora resistance and MEK2-SIPK/WIPK degradation. We found that pub40 knockdown mutants exhibited significantly enhanced resistance to P. capsici. NbPUB40 was under ubiquitination and proteasomal degradation in planta, with two conserved sites (Cys28 and Val41) in the U-box domain being essential for its activity. NbPUB40 was shown to interact with the whole MEK2-SIPK/WIPK cascade and promote their degradation, the ubiquitination levels of which were also notably reduced in the pub40 mutant. Our results reveal a mechanism in which a PUB E3 ubiquitin ligase negatively regulates plant P. capsici resistance by destabilizing the MEK2-SIPK/WIPK cascade.

Overexpression of the plastidial pseudo-protease AtFtsHi3 enhances drought tolerance while sustaining plant growth.

With climate change, droughts are expected to be more frequent and severe, severely impacting plant biomass and quality. Here, we show that overexpressing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phenotypes without compromising plant growth. AtFtsHi3 encodes a chloroplast envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression therefore leads to drought tolerance, while only diminished expression of this gene leads to growth retardation. To understand the underlying mechanisms of the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and drought conditions. Drought-related processes like osmotic stress, water transport, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1 mutants following their enhanced drought response compared to wild-type. The knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93, and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chloroplast pre-protein import. Mathematical modeling was performed to understand how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead to drought tolerance in both overexpression and knock-down lines, yet, on the other hand, affect plant growth so differently. The results led us to hypothesize that AtFtsHi3 may form complexes with at least two other protease subunits, either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9, AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation mechanism for these proteases in the hexamer.

Genetic evidence for functions of Chloroplast CA in Pyropia yezoensis: decreased CCM but increased starch accumulation

In response to the changing intertidal environment, intertidal macroalgae have evolved complicated Ci utilization mechanisms. However, our knowledge regarding the CO2 concentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), a key component of CCM, plays essential roles in many physiological reactions in various organisms. While many genes encode CA in the Pyropia yezoensis genome, the exact function of specific CA in P. yezoensis remains elusive. To explore the particular function of chloroplast CA in intertidal macroalgae, we produced chloroplast-localized βCA1 knockdown mutants of P. yezoensis through RNA interference, and Pyβca1i mutants (hereinafter referred to as ca1i) showed a notable decrease in leaf area and overall biomass, as well as decreased soluble protein and unsaturated fatty acid content under different DIC conditions. However, ca1i mutants showed relatively higher starch content compared to the wild-type. The activity of enzymes involved in the Calvin cycle, photorespiration, Pentose-phosphate pathway, and floridean starch synthesis of P. yezoensis indicated an effective starch accumulation pathway after the interference of βCA1. All results suggest that the decreased activity of PyβCA1 impaired the CCM and development of thalli of P. yezoensis, but stimulated starch accumulation in the cytoplasm through feedback to the photorespiration pathway and pentose phosphate pathway to replenish intermediates for the Calvin cycle. This study is the first to explore the specific function of chloroplast CA in intertidal macroalgae using genomic technology. The results provide valuable insights into the adaption mechanisms of intertidal macroalgae to their environment.