A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis.

The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants. However, screening edited alleles, particularly those with multiplex editing, from herbicide- or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes. Current solutions to facilitate these processes rely on different selection markers. Here, by taking advantage of the opposite functions of a d-amino acid oxidase (DAO) in detoxifying d-serine and in metabolizing non-toxic d-valine to a cytotoxic product, we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana. Among five DAOs tested in Escherichia coli, the one encoded by Trigonopsis variabilis (TvDAO) could confer slightly stronger d-serine resistance than other homologs. Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and non-transgenic plants in both d-serine-conditioned positive selection and d-valine-conditioned negative selection. As a proof of concept, we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with d-serine-based positive selection to help identify transgenic plants with multiplex editing, where d-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin. Subsequently, d-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations. Collectively, this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker, which promises more efficient and simplified multiplex CRISPR editing in plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00132-6.

Hidden prevalence of deletion-inversion bi-alleles in CRISPR-mediated deletions of tandemly arrayed genes in plants.

Tandemly arrayed genes (TAGs) with functional redundancy and chromosomal linkage constitute 14 ~ 35% in sequenced plant genomes. The multiplex CRISPR system is the tool of choice for creating targeted TAG deletions. Here, we show that up to ~80% of CRISPR-mediated TAG knockout alleles in Arabidopsis and rice are deletion-inversion (delinver) bi-alleles, which are easily misidentified as homozygous deletion alleles by routine PCR-based genotyping. This can lead to misinterpretation of experimental data and production of progenies with genetic heterogeneity in an unnoticed manner. In ~2,650 transgenic events, delinver mutation frequencies are predominantly correlated with deletion frequencies but unrelated to chromosomal locations or deletion sizes. Delinver mutations also occur frequently at genomic non-TAG loci during multiplexed CRISPR editing. Our work raises the alarm about delinver mutations as common unwanted products of targeted TAG deletions in plants and helps prevent false interpretation of plant TAG functions due to this hidden genotype issue.

WeiTsing: a new face of Ca2+-permeable channels in plant immunity.

Plants employ pattern- and effector-triggered immunity (PTI and ETI) to synergistically defend invading pathogens and insect herbivores. Both PTI and ETI can induce cytosolic Ca2+ spikes, despite in different spatiotemporal patterns, to activate downstream Ca2+-dependent immune signaling cascades. While multiple families of Ca2+-permeable channels at the plasma membrane have been uncovered, the counterparts responsible for Ca2+ release from intracellular stores remain poorly understood. In a groundbreaking paper published recently by Cell, the authors reported that WeiTsing, an Arabidopsis endoplasmic reticulum (ER)-resident protein that was specifically expressed in the pericycle upon Plasmodiophora brassicae (Pb) infection, could form resistosome-like Ca2+-conducting channel and protect the stele of Brassica crops from Pb colonization. As the channel activity of WeiTsing was indispensable for its immune function, the findings highlight a previously underappreciated role of Ca2+ release from intracellular repertoire in promoting plant disease resistance.

Poaceae-specific ß-1,3;1,4-d-glucans link jasmonate signalling to OsLecRK1-mediated defence response during rice-brown planthopper interactions.

Brown planthopper (BPH, Nilaparvata lugens), a highly destructive insect pest, poses a serious threat to rice (Oryza sativa) production worldwide. Jasmonates are key phytohormones that regulate plant defences against BPH; however, the molecular link between jasmonates and BPH responses in rice remains largely unknown. Here, we discovered a Poaceae-specific metabolite, mixed-linkage ß-1,3;1,4-d-glucan (MLG), which contributes to jasmonate-mediated BPH resistance. MLG levels in rice significantly increased upon BPH attack. Overexpressing OsCslF6, which encodes a glucan synthase that catalyses MLG biosynthesis, significantly enhanced BPH resistance and cell wall thickness in vascular bundles, whereas knockout of OsCslF6 reduced BPH resistance and vascular wall thickness. OsMYC2, a master transcription factor of jasmonate signalling, directly controlled the upregulation of OsCslF6 in response to BPH feeding. The AT-rich domain of the OsCslF6 promoter varies in rice varieties from different locations and natural variants in this domain were associated with BPH resistance. MLG-derived oligosaccharides bound to the plasma membrane-anchored LECTIN RECEPTOR KINASE1 OsLecRK1 and modulated its activity. Thus, our findings suggest that the OsMYC2-OsCslF6 module regulates pest resistance by modulating MLG production to enhance vascular wall thickness and OsLecRK1-mediated defence signalling during rice-BPH interactions.

Plasma membrane-nucleo-cytoplasmic coordination of a receptor-like cytoplasmic kinase promotes EDS1-dependent plant immunity.

Plants use cell-surface immune receptors to recognize pathogen-specific patterns to evoke basal immunity. ENHANCED DISEASE SUSCEPTIBILITY (EDS1) is known to be crucial for plant basal immunity, whereas its activation mechanism by pattern recognition remains enigmatic. Here, we show that the fungal pattern chitin induced the plasma membrane-anchored receptor-like cytoplasmic kinase PBS1-LIKE 19 (PBL19) to undergo nuclear translocation in Arabidopsis. The palmitoylation-deficient PBL19C3A variant constantly resided in the nucleus, triggering transcriptional self-amplification mainly through WRKY8 and EDS1-dependent constitutive immunity. Unexpectedly, the metacaspase-cleaved PBL19 lacking the N-terminal nuclear localization sequence specifically interacted with and phosphorylated EDS1 in the cytoplasm. Phosphodeficient EDS1 attenuated PBL19C3A-induced constitutive immunity, while phosphomimetic EDS1 complemented the loss of PBL19 for fungal resistance. Collectively, these findings reveal a compelling model wherein the plasma membrane, nuclear and cytoplasmic pools of PBL19 temporally coordinate distinct roles of immune signal receiver, amplifier and effector to boost plant antifungal immunity via EDS1.

Hide-and-Seek: Chitin-Triggered Plant Immunity and Fungal Counterstrategies.

Fungal pathogens are major destructive microorganisms for land plants and pose growing challenges to global crop production. Chitin is a vital building block for fungal cell walls and also a broadly effective elicitor of plant immunity. Here we review the rapid progress in understanding chitin perception and signaling in plants and highlight similarities and differences of these processes between arabidopsis and rice. We also outline moonlight functions of CERK1, an indispensable chitin coreceptor conserved across the plant kingdom, which imply potential crosstalk between chitin signaling and symbiotic or biotic/abiotic stress signaling in plants via CERK1. Moreover, we summarize current knowledge about fungal counterstrategies for subverting chitin-triggered plant immunity and propose open questions and future directions in this field.

Split Nano luciferase complementation for probing protein-protein interactions in plant cells.

Deciphering protein-protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual-use system enabling the detection of PPIs through the Nluc-based SLC and co-immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA- or FLAG-tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium-transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc-based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.

Enhanced protoplast assay by transfecting PCR-assembled gene expression cassettes with telomeric repeats and thiophosphate modifications.

Transient expression assays are invaluable complements to the stable transgenic assay for studying gene functions by providing desirable time and labor efficiencies and high-throughput potential or circumventing technical difficulties of stable transgenic expression. The protoplast transient expression system is one of the mainstream transient expression assays used in plant research. Here, we developed a PCR amplicon-mediated protoplast transient (PROMPT) assay by using overlapping PCR assembled gene expression cassettes for Arabidopsis protoplast transfection without the need for time- and labor-consuming plasmid construction. When 200 µl of Arabidopsis protoplasts were transfected with 1 µg of PCR amplicons or plasmid DNA, we detected substantially higher gene expression in the former. Moreover, we found that adding telomeric repeats and thiophosphate modifications to the 5' end of the nonsense strand through the reverse primer could further increase the PCR amplicon-mediated gene expression in protoplasts. Importantly, these improvements could also be applied to the protoplast assays in other dicot and monocot species including tobacco, rice and wheat. In addition, the subcellular localization of immune receptor FLS2 could be analyzed by PROMPT method. The PROMPT assay allows an accelerated and robust transient gene expression in protoplasts from diverse plant species.

Autophagy regulates glucose-mediated root meristem activity by modulating ROS production in Arabidopsis.

Glucose produced from photosynthesis is a key nutrient signal regulating root meristem activity in plants; however, the underlying mechanisms remain poorly understood. Here, we show that, by modulating reactive oxygen species (ROS) levels, the conserved macroautophagy/autophagy degradation pathway contributes to glucose-regulated root meristem maintenance. In Arabidopsis thaliana roots, a short exposure to elevated glucose temporarily suppresses constitutive autophagosome formation. The autophagy-defective autophagy-related gene (atg) mutants have enhanced tolerance to glucose, established downstream of the glucose sensors, and accumulate less glucose-induced ROS in the root tips. Moreover, the enhanced root meristem activities in the atg mutants are associated with improved auxin gradients and auxin responses. By acting with AT4G39850/ABCD1 (ATP-binding cassette D1; Formerly PXA1/peroxisomal ABC transporter 1), autophagy plays an indispensable role in the glucose-promoted degradation of root peroxisomes, and the atg mutant phenotype is partially rescued by the overexpression of ABCD1. Together, our findings suggest that autophagy is an essential mechanism for glucose-mediated maintenance of the root meristem. Abbreviation: ABA: abscisic acid; ABCD1: ATP-binding cassette D1; ABO: ABA overly sensitive; AsA: ascorbic acid; ATG: autophagy related; CFP: cyan fluorescent protein; Co-IP: co-immunoprecipitation; DAB: 3',3'-diaininobenzidine; DCFH-DA: 2',7'-dichlorodihydrofluorescin diacetate; DR5: a synthetic auxin response element consists of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element; DZ: differentiation zone; EZ, elongation zone; GFP, green fluorescent protein; GSH, glutathione; GUS: ß-glucuronidase; HXK1: hexokinase 1; H2O2: hydrogen peroxide; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; KIN10/11: SNF1 kinase homolog 10/11; MDC: monodansylcadaverine; MS: Murashige and Skoog; MZ: meristem zone; NBT: nitroblue tetrazolium; NPA: 1-N-naphtylphthalamic acid; OxIAA: 2-oxindole-3-acetic acid; PIN: PIN-FORMED; PLT: PLETHORA; QC: quiescent center; RGS1: Regulator of G-protein signaling 1; ROS: reactive oxygen species; SCR: SCARECROW; SHR, SHORT-ROOT; SKL: Ser-Lys-Leu; SnRK1: SNF1-related kinase 1; TOR: target of rapamycin; UPB1: UPBEAT1; WOX5: WUSCHEL related homeobox 5; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein.

Alternative splicing and translation play important roles in hypoxic germination in rice.

Post-transcriptional mechanisms (PTMs), including alternative splicing (AS) and alternative translation initiation (ATI), may explain the diversity of proteins involved in plant development and stress responses. Transcriptional regulation is important during the hypoxic germination of rice seeds, but the potential roles of PTMs in this process have not been characterized. We used a combination of proteomics and RNA sequencing to discover how AS and ATI contribute to plant responses to hypoxia. In total, 10 253 intron-containing genes were identified. Of these, ~1741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds compared with controls. Over 95% of these were not present in the list of differentially expressed genes. In particular, regulatory pathways such as the spliceosome, ribosome, endoplasmic reticulum protein processing and export, proteasome, phagosome, oxidative phosphorylation, and mRNA surveillance showed substantial AS changes under hypoxia, suggesting that AS responses are largely independent of transcriptional regulation. Considerable AS changes were identified, including the preferential usage of some non-conventional splice sites and enrichment of splicing factors in the DAS data sets. Taken together, these results not only demonstrate that AS and ATI function during hypoxic germination but they have also allowed the identification of numerous novel proteins/peptides produced via ATI.

Natural variation in the promoter of rice calcineurin B-like protein10 (OsCBL10) affects flooding tolerance during seed germination among rice subspecies.

Rice (Oryza sativa L.) has two ecotypes, upland and lowland rice, that have been observed to show different tolerance levels under flooding stress. In this study, two rice cultivars, upland (Up221, flooding-intolerant) and lowland (Low88, flooding-tolerant), were initially used to study their molecular mechanisms in response to flooding germination. We observed that variations in the OsCBL10 promoter sequences in these two cultivars might contribute to this divergence in flooding tolerance. Further analysis using another eight rice cultivars revealed that the OsCBL10 promoter could be classified as either a flooding-tolerant type (T-type) or a flooding-intolerant type (I-type). The OsCBL10 T-type promoter only existed in japonica lowland cultivars, whereas the OsCBL10 I-type promoter existed in japonica upland, indica upland and indica lowland cultivars. Flooding-tolerant rice cultivars containing the OsCBL10 T-type promoter have shown lower Ca2+ flow and higher α-amylase activities in comparison to those in flooding-intolerant cultivars. Furthermore, the OsCBL10 overexpression lines were sensitive to both flooding and hypoxic treatments during rice germination with enhanced Ca2+ flow in comparison to wild-type. Subsequent findings also indicate that OsCBL10 may affect OsCIPK15 protein abundance and its downstream pathways. In summary, our results suggest that the adaptation to flooding stress during rice germination is associated with two different OsCBL10 promoters, which in turn affect OsCBL10 expression in different cultivars and negatively affect OsCIPK15 protein accumulation and its downstream cascade.

OsARM1, an R2R3 MYB Transcription Factor, Is Involved in Regulation of the Response to Arsenic Stress in Rice.

Bioaccumulation of arsenic (As) in rice (Oryza sativa) increases human exposure to this toxic, carcinogenic element. Recent studies identified several As transporters, but the regulation of these transporters remains unclear. Here, we show that the rice R2R3 MYB transcription factor OsARM1 (ARSENITE-RESPONSIVE MYB1) regulates As-associated transporters genes. Treatment with As(III) induced OsARM1 transcript accumulation and an OsARM1-GFP fusion localized to the nucleus. Histochemical analysis of OsARM1pro::GUS lines indicated that OsARM1 was expressed in the phloem of vascular bundles in basal and upper nodes. Knockout of OsARM1 (OsARM1-KO CRISPR/Cas9-generated mutants) improved tolerance to As(III) and overexpression of OsARM1 (OsARM1-OE lines) increased sensitivity to As(III). Measurement of As in As(III)-treated plants showed that under low As(III) conditions (2 µM), more As was transported from the roots to the shoots in OsARM1-KOs. By contrast, more As accumulated in the roots in OsARM1-OEs in response to high As(III) exposure (25 µM). In particular, the As(III) levels in node I were significantly higher in OsARM1-KOs, but significantly lower in OsARM1-OEs, compared to wild-type plants, implying that OsARM1 is important for the regulation of root-to-shoot translocation of As. Moreover, OsLsi1, OsLsi2, and OsLsi6, which encode key As transporters, were significantly downregulated in OsARM1-OEs and upregulated in OsARM1-KOs compared to wild type. Chromatin immunoprecipitation-quantitative PCR of OsARM1-OEs indicated that OsARM1 binds to the conserved MYB-binding sites in the promoters or genomic regions of OsLsi1, OsLsi2, and OsLsi6 in rice. Our findings suggest that the OsARM1 transcription factor has essential functions in regulating As uptake and root-to-shoot translocation in rice.

[Research on fast detecting tomato seedlings nitrogen content based on NIR characteristic spectrum selection].

In order to improve the accuracy and robustness of detecting tomato seedlings nitrogen content based on near-infrared spectroscopy (NIR), 4 kinds of characteristic spectrum selecting methods were studied in the present paper, i. e. competitive adaptive reweighted sampling (CARS), Monte Carlo uninformative variables elimination (MCUVE), backward interval partial least squares (BiPLS) and synergy interval partial least squares (SiPLS). There were totally 60 tomato seedlings cultivated at 10 different nitrogen-treatment levels (urea concentration from 0 to 120 mg . L-1), with 6 samples at each nitrogen-treatment level. They are in different degrees of over nitrogen, moderate nitrogen, lack of nitrogen and no nitrogen status. Each sample leaves were collected to scan near-infrared spectroscopy from 12 500 to 3 600 cm-1. The quantitative models based on the above 4 methods were established. According to the experimental result, the calibration model based on CARS and MCUVE selecting methods show better performance than those based on BiPLS and SiPLS selecting methods, but their prediction ability is much lower than that of the latter. Among them, the model built by BiPLS has the best prediction performance. The correlation coefficient (r), root mean square error of prediction (RMSEP) and ratio of performance to standard derivate (RPD) is 0. 952 7, 0. 118 3 and 3. 291, respectively. Therefore, NIR technology combined with characteristic spectrum selecting methods can improve the model performance. But the characteristic spectrum selecting methods are not universal. For the built model based or single wavelength variables selection is more sensitive, it is more suitable for the uniform object. While the anti-interference ability of the model built based on wavelength interval selection is much stronger, it is more suitable for the uneven and poor reproducibility object. Therefore, the characteristic spectrum selection will only play a better role in building model, combined with the consideration of sample state and the model indexes.

Unsaturation of very-long-chain ceramides protects plant from hypoxia-induced damages by modulating ethylene signaling in Arabidopsis.

Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plants. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxidized lipids, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Disruption of ceramide synthases LOH1, LOH2 and LOH3 enhanced plant sensitivity to dark submergence, but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated very-long-chain (VLC) ceramide species (22:1, 24:1 and 26:1) predominantly declined in the loh1, loh2 and loh3 mutants under dark submergence. In contrast, significant reduction of VLC ceramides in the loh1-1 loh3-1 knockdown double mutant and lacking of VLC unsaturated ceramides in the ads2 mutants impaired plant tolerance to both dark and light submergences. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein and inhibited its kinase activity in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating CTR1-mediated ethylene signaling. The dark submergence-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of VLC ceramides is a protective strategy for hypoxic tolerance in Arabidopsis.

Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism.

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.