Overexpressing lipid raft protein STOML2 modulates the tumor microenvironment via NF-κB signaling in colorectal cancer.

Colorectal cancer (CRC) is characterized by a complex tumor inflammatory microenvironment, while angiogenesis and immunosuppression frequently occur concomitantly. However, the exact mechanism that controls angiogenesis and immunosuppression in CRC microenvironment remains unclear. Herein, we found that expression levels of lipid raft protein STOML2 were increased in CRC and were associated with advanced disease stage and poor survival outcomes. Intriguingly, we revealed that STOML2 is essential for CRC tumor inflammatory microenvironment, which induces angiogenesis and facilitates tumor immune escape simultaneously both in vitro and in vivo. Moreover, tumors with STOML2 overexpression showed effective response to anti-angiogenesis treatment and immunotherapy in vivo. Mechanistically, STOML2 regulates CRC proliferation, angiogenesis, and immune escape through activated NF-κB signaling pathway via binding to TRADD protein, resulting in upregulation of CCND1, VEGF, and PD-L1. Furthermore, treatment with NF-κB inhibitor dramatically reversed the ability of proliferation and angiogenesis. Clinically, we also observed a strong positive correlation between STOML2 expression and Ki67, CD31, VEGFC and PD-1 of CD8+T cell expression. Taken together, our results provided novel insights into the role of STOML2 in CRC inflammatory microenvironment, which may present a therapeutic opportunity for CRC.

Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity.

Rice (Oryza sativa) is a staple food crop and serves as a model cereal plant. It contains two biosynthetic gene clusters (BGCs) for the production of labdane-related diterpenoids (LRDs), which serve important roles in combating biotic and abiotic stress. While plant BGCs have been subject to genetic analyses, these analyses have been largely confined to the investigation of single genes. CRISPR/Cas9-mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGCs. Deletion of the BGC from chromosome 2 (c2BGC), which is associated with phytocassane biosynthesis, but not that from chromosome 4 (c4BGC), which is associated with momilactone biosynthesis, led to a lesion mimic phenotype. This phenotype is dependent on two closely related genes encoding cytochrome P450 (CYP) mono-oxygenases, CYP76M7 and CYP76M8, from the c2BGC. However, rather than being redundant, CYP76M7 has been associated with the production of phytocassanes, whereas CYP76M8 is associated with momilactone biosynthesis. Intriguingly, the lesion mimic phenotype is not present in a line with both BGCs deleted. These results reveal directional cross-cluster phytotoxicity, presumably arising from the accumulation of LRD intermediates dependent on the c4BGC in the absence of CYP76M7 and CYP76M8, further highlighting their interdependent evolution and the selective pressures driving BGC assembly.

Rice diterpenoid phytoalexins are involved in defence against parasitic nematodes and shape rhizosphere nematode communities.

Rice diterpenoid phytoalexins (DPs) are secondary metabolites with a well known role in resistance to foliar pathogens. As DPs are also known to be produced and exuded by rice roots, we hypothesised that they might play an important role in plant-nematode interactions, and particularly in defence against phytoparasitic nematodes. We used transcriptome analysis on rice roots to analyse the effect of infection by the root-knot nematode Meloidogyne graminicola or treatment with resistance-inducing chemical stimuli on DP biosynthesis genes, and assessed the susceptibility of mutant rice lines impaired in DP biosynthesis to M. graminicola. Moreover, we grew these mutants and their wild-type in field soil and used metabarcoding to assess the effect of impairment in DP biosynthesis on rhizosphere and root nematode communities. We show that M. graminicola suppresses DP biosynthesis genes early in its invasion process and, conversely, that resistance-inducing stimuli transiently induce the biosynthesis of DPs. Moreover, we show that loss of DPs increases susceptibility to M. graminicola. Metabarcoding on wild-type and DP-deficient plants grown in field soil reveals that DPs significantly alter the composition of rhizosphere and root nematode communities. Diterpenoid phytoalexins are important players in basal and inducible defence against nematode pathogens of rice and help shape rice-associated nematode communities.

A (conditional) role for labdane-related diterpenoid natural products in rice stomatal closure.

Rice (Oryza sativa) is the staple food for over half the world's population. Drought stress imposes major constraints on rice yields. Intriguingly, labdane-related diterpenoid (LRD) phytoalexins in maize (Zea mays) affect drought tolerance, as indicated by the increased susceptibility of an insertion mutant of the class II diterpene cyclase ZmCPS2/An2 that initiates such biosynthesis. Rice also produces LRD phytoalexins, utilizing OsCPS2 and OsCPS4 to initiate a complex metabolic network. For genetic studies of rice LRD biosynthesis the fast-growing Kitaake cultivar was selected for targeted mutagenesis via CRISPR/Cas9, with an initial focus on OsCPS2 and OsCPS4. The resulting cps2 and cps4 knockout lines were further crossed to create a cps2x4 double mutant. Both CPSs also were overexpressed. Strikingly, all of the cv Kitaake cps mutants exhibit significantly increased susceptibility to drought, which was associated with reduced stomatal closure that was evident even under well-watered conditions. However, CPS overexpression did not increase drought resistance, and cps mutants in other cultivars did not alter susceptibility to drought, although these also exhibited lesser effects on LRD production. The results suggest that LRDs may act as a regulatory switch that triggers stomatal closure in rice, which might reflect the role of these openings in microbial entry.

Inferring Roles in Defense from Metabolic Allocation of Rice Diterpenoids.

Among their responses to microbial infection, plants deploy an arsenal of natural antibiotic products. Historically these have been identified on the basis of their antibiotic activity in vitro, which leaves open the question of their relevance to defense in planta. The vast majority of such natural products from the important crop plant rice (Oryza sativa) are diterpenoids whose biosynthesis proceeds via either ent- or syn-copalyl diphosphate (CPP) intermediates, which were isolated on the basis of their antibiotic activity against the fungal blast pathogen Magnaporthe oryzae However, rice plants in which the gene for the syn-CPP synthase Os-CPS4 is knocked out do not exhibit increased susceptibility to M. oryzae Here, we show that knocking out or knocking down Os-CPS4 actually decreases susceptibility to the bacterial leaf blight pathogen Xanthomonas oryzae By contrast, genetic manipulation of the gene for the ent-CPP synthase Os-CPS2 alters susceptibility to both M. oryzae and X. oryzae Despite the secretion of diterpenoids dependent on Os-CPS2 or Os-CPS4 from roots, neither knockout exhibited significant changes in the composition of their rhizosphere bacterial communities. Nevertheless, rice plants allocate substantial metabolic resources toward syn- as well as ent-CPP derived diterpenoids upon infection/induction. Further investigation revealed that Os-CPS4 plays a role in fungal non-host disease resistance. Thus, examination of metabolic allocation provides important clues into physiological function.

Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production.

MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator-like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild-type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9-mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double-guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single-guide RNA. The difference between the phenotypic severity of miR160a mutants in Col-0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.

A lipid transfer protein variant with a mutant eight-cysteine motif causes photoperiod- and thermo-sensitive dwarfism in rice.

Plant height is an important trait for architecture patterning and crop yield improvement. Although the pathways involving gibberellins and brassinosteroids have been well studied, there are still many gaps in our knowledge of the networks that control plant height. In this study, we determined that a dominant photoperiod- and thermo-sensitive dwarf mutant is caused by the active role of a mutated gene Photoperiod-thermo-sensitive dwarfism 1 (Ptd1), the wild-type of which encodes a non-specific lipid transfer protein (nsLTP). Ptd1 plants showed severe dwarfism under long-day and low-temperature conditions, but grew almost normal under short-day and high-temperature conditions. These phenotypic variations were associated with Ptd1 mRNA levels and accumulation of the corresponding protein. Furthermore, we found that the growth inhibition in Ptd1 may result from the particular protein conformation of Ptd1 due to loss of two disulfide bonds in the eight-cysteine motif (8-CM) that is conserved among nsLTPs. These results contribute to our understanding of the novel function of disulfide bonds in the 8-CM, and provide a potential new strategy for regulation of cell development and plant height by modifying the amino acid residues involved in protein conformation patterning.

Origins of Chemoselectivity in the Ni-Catalyzed Biaryl and Pd-Catalyzed Acyl Suzuki-Miyaura Cross-Coupling of N-Acetyl-Amides.

The mechanisms and origins of selectivity in the Pd-catalyzed nondecarbonylative and Ni-catalyzed decarbonylative Suzuki-Miyaura cross-coupling of N-acetyl-amides have been explored with density functional theory calculations. The reaction of the two catalysts shares a similar process that contains oxidative addition to break the N-C(O) bond and transmetalation with the Ar'B(OH)2 reagent. Then, the reaction bifurcates at the generated PCy3M(acyl)Ar' (M = Ni/Pd) intermediate. Our results show that the electronegativity of the central metal plays a decisive role in guiding the selectivity of subsequent reactions (acyl reductive coupling versus decarbonylation). Palladium with a higher electronegativity tends to accept electrons for directly C-C reductive elimination, but it is not conducive to the d → π* back donation from the metal to CO to stabilize the decarbonylative process, which restricts the decarbonylation. In contrast, for the nickel-catalyzed system, it prefers to undergo decarbonylation benefiting by the stronger stabilizing effect of d → π* back donation from nickel to CO rather than conduct reductive elimination due to its lower electronegativity. Consequently, the Pd-catalyzed Suzuki-Miyaura cross-coupling of N-acetyl-amides gives biaryl ketones (ArCOAr') but the Ni-catalyzed same reaction generates decarbonylated biaryls (ArAr'). The mechanistic understanding gives useful insight into the further advancement of selective cross-coupling reactions enabled by transition metals.

Characterization and genetic mapping of a Photoperiod-sensitive dwarf 1 locus in rice (Oryza sativa L.).

Plant height is an important agronomic trait for crop architecture and yield. Most known factors determining plant height function in gibberellin or brassinosteroid biosynthesis or signal transduction. Here, we report a japonica rice (Oryza sativa ssp. japonica) dominant dwarf mutant, Photoperiod-sensitive dwarf 1 (Psd1). The Psd1 mutant showed impaired cell division and elongation, and a severe dwarf phenotype under long-day conditions, but nearly normal growth in short-day. The plant height of Psd1 mutant could not be rescued by gibberellin or brassinosteroid treatment. Genetic analysis with R1 and F2 populations determined that Psd1 phenotype was controlled by a single dominant locus. Linkage analysis with 101 tall F2 plants grown in a long-day season, which were derived from a cross between Psd1 and an indica cultivar, located Psd1 locus on chromosome 1. Further fine-mapping with 1017 tall F2 plants determined this locus on an 11.5-kb region. Sequencing analysis of this region detected a mutation site in a gene encoding a putative lipid transfer protein; the mutation produces a truncated C-terminus of the protein. This study establishes the genetic foundation for understanding the molecular mechanisms regulating plant cell division and elongation mediated by interaction between genetic and environmental factors.

An endoplasmic reticulum stress-related signature could robustly predict prognosis and closely associate with response to immunotherapy in pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors. Endoplasmic reticulum stress (ERS) plays an essential role in PDAC progression. Here, we aim to identify the ERS-related genes in PDAC and build reliable risk models for diagnosis, prognosis and immunotherapy response of PDAC patients as well as investigate the potential mechanism. METHODS: We obtained PDAC cohorts with transcriptional profiles and clinical data from the ArrayExpress, The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Univariate Cox regression, LASSO regression and multivariate Cox regression analyses were used to construct an ERS-related prognostic signature. The CIBERSORT and ssGSEA algorithms were applied to explore the correlation between the prognostic signature and immune cell infiltration and immune-related pathways. The GDSC database and TIDE algorithm were used to predict responses to chemotherapy and immunotherapy, identifying potential drugs for treating patients with PDAC. RESULTS: We established and validated an ERS-related prognostic signature comprising eight genes (HMOX1, TGFB1, JSRP1, GAPDH, CAV1, CHRNE, CD74 and ERN2). Patients with higher risk scores displayed worse outcomes than those with lower risk scores. PDAC patients in low-risk groups might benefit from immunotherapy. Dasatinib and lapatinib might have potential therapeutic implications in high-risk PDAC patients. CONCLUSION: We established and validated an ERS-related prognostic signature comprising eight genes to predict the overall survival outcome of PDAC patients, which closely correlating with the response to immunotherapy and sensitivity to anti-tumor drugs, as well as could be beneficial for formulating clinical strategies and administering individualized treatments.

High-yield and sustainable synthesis of quinoidal compounds assisted by keto-enol tautomerism.

The classical synthesis of quinoids, which involves Takahashi coupling and subsequent oxidation, often gives only low to medium yields. Herein, we disclose the keto-enol-tautomerism-assisted spontaneous air oxidation of the coupling products to quinoids. This allows for the synthesis of various indandione-terminated quinoids in high isolated yields (85-95%). The origin of the high yield and the mechanism of the spontaneous air oxidation were ascertained by experiments and theoretical calculations. All the quinoidal compounds displayed unipolar n-type transport behavior, and single crystal field-effect transistors based on the micro-wires of a representative quinoid delivered an electron mobility of up to 0.53 cm2 V-1 s-1, showing the potential of this type of quinoid as an organic semiconductor.

Origins of Unconventional γ Site Selectivity in Palladium-Catalyzed C(sp3)-H Activation and Arylation of Aliphatic Alcohols.

The mechanism of palladium-catalyzed γ-C(sp3)-H arylation of aliphatic alcohols has been explored with density functional theory. In contrast to the common "inner-sphere" C-H activation mode that favors five-membered over six-membered cyclopalladation, the computational results reveal that the "outer-sphere" C-H activation pathway assisted by added base (e.g., Li2CO3) is kinetically favorable and supports six-membered over five-membered cyclopalladation to yield the experimental γ-C(sp3)-H arylation product. Distortion/interaction analysis reveals the origins of the unconventional γ site selectivity.

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

CRISPR/Cas9 for Mutagenesis in Rice.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.

Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9.

Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy.

Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9-based genome editing platform in Tragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted for Tragopogon) was capable of introducing site-specific mutations in Tragopogon protoplasts. Agrobacterium-mediated transformation with Cas9-sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles of TraPDS in albino shoots from Tragopogon porrifolius (2x) and Tragopogon mirus (4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9-based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed in Tragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene-balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems.

A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice.

Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS-WA) has been exploited in the majority of 'three-line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown. Here we report that a new mitochondrial gene, WA352, which originated recently in wild rice, confers CMS-WA because the protein it encodes interacts with the nuclear-encoded mitochondrial protein COX11. In CMS-WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion. WA352-induced sterility can be suppressed by two restorer-of-fertility (Rf) genes, suggesting the existence of different mechanisms to counteract deleterious cytoplasmic factors. Thus, CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene.