Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.

Multitrait engineering of Hassawi red rice for sustainable cultivation.

Sustainable agriculture requires locally adapted varieties that produce nutritious food with limited agricultural inputs. Genome engineering represents a viable approach to develop cultivars that fulfill these criteria. For example, the red Hassawi rice, a native landrace of Saudi Arabia, tolerates local drought and high-salinity conditions and produces grain with diverse health-promoting phytochemicals. However, Hassawi has a long growth cycle, high cultivation costs, low productivity, and susceptibility to lodging. Here, to improve these undesirable traits via genome editing, we established efficient regeneration and Agrobacterium-mediated transformation protocols for Hassawi. In addition, we generated the first high-quality reference genome and targeted the key flowering repressor gene, Hd4, thus shortening the plant's lifecycle and height. Using CRISPR/Cas9 multiplexing, we simultaneously disrupted negative regulators of flowering time (Hd2, Hd4, and Hd5), grain size (GS3), grain number (GN1a), and plant height (Sd1). The resulting homozygous mutant lines flowered extremely early (∼56 days) and had shorter stems (approximately 107 cm), longer grains (by 5.1%), and more grains per plant (by 50.2%), thereby enhancing overall productivity. Furthermore, the awns of grains were 86.4% shorter compared to unedited plants. Moreover, the modified rice grain displayed improved nutritional attributes. As a result, the modified Hassawi rice combines several desirable traits that can incentivize large-scale cultivation and reduce malnutrition.

Function of CAZymes encoded by highly abundant genes in rhizosphere microbiome of Moringa oleifera.

Metagenomic analysis referring to CAZymes (Carbohydrate-Active enZymes) of CAZy classes encoded by the most abundant genes in rhizosphere versus bulk soil microbes of the wild plant Moringa oleifera was conducted. Results indicated that microbiome signatures and corresponding CAZy datasets differ between the two soil types. CAZy class glycoside hydrolases (GH) and its α-amylase family GH13 in rhizobiome were proven to be the most abundant among CAZy classes and families. The most abundant bacteria harboring these CAZymes include phylum Actinobacteria and its genus Streptomyces and phylum Proteobacteria and its genus Microvirga. These CAZymes participate in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway "Starch and sucrose metabolism" and mainly use the "double displacement catalytic mechanism" in their reactions. We assume that microbiome of the wild plant Moringa oleifera is a good source of industrially important enzymes that act on starch hydrolysis and/or biosynthesis. In addition, metabolic engineering and integration of certain microbes of this microbiomes can also be used in improving growth of domestic plants and their ability to tolerate adverse environmental conditions.

Assessing the probiotic potential, antioxidant, and antibacterial activities of oat and soy milk fermented with Lactiplantibacillus plantarum strains isolated from Tibetan Kefir.

Sufficient intake of probiotics has been shown to help in the digestion, protect the body against pathogenic microorganisms and boost the immune system. Recently, due to high prevalence of milk allergies and lactose intolerance in population, the non-dairy based probiotic alternative are becoming increasing popular. In this context, the oat milk and soya milk-based fermented products can be an ideal alternative for the development of Lactic acid bacteria bacteria based probiotics. These bacteria can not only improve the product's flavor and bioavailability but also increases its antibacterial and antioxidant capabilities due to fermentation process. The purpose of the resent work was to assess the antioxidant and probiotic properties of oat and soy milk that had been fermented with three different strains of Lactiplantibacillus plantarum (L. plantarum) including L. plantarum 12-3, L. plantarum K25, and L. plantarum YW11 isolated from Tibetan Kefir. Different validated assays were used to evaluate the probiotic properties, adhesion and survival in the digestive system (stomach, acid and bile salts resistance), antioxidant and antimicrobial activities and safety (ABTS and DPPH scavenging assays) of these strains. Results of the study showed that soya milk and oat milk fermented with L. plantarum strains possess promising probiotic, antibacterial and antioxidant properties. These results can be helpful to produce dairy-free probiotic replacements, which are beneficial for those who are unable to consume dairy products due to dietary or allergic restrictions.

Rapid detection of micronutrient components in infant formula milk powder using near-infrared spectroscopy.

In order to achieve rapid detection of galactooligosaccharides (GOS), fructooligosaccharides (FOS), calcium (Ca), and vitamin C (Vc), four micronutrient components in infant formula milk powder, this study employed four methods, namely Standard Normal Variate (SNV), Multiplicative Scatter Correction (MSC), Normalization (Nor), and Savitzky-Golay Smoothing (SG), to preprocess the acquired original spectra of the milk powder. Then, the Competitive Adaptive Reweighted Sampling (CARS) algorithm and Random Frog (RF) algorithm were used to extract representative characteristic wavelengths. Furthermore, Partial Least Squares Regression (PLSR) and Support Vector Regression (SVR) models were established to predict the contents of GOS, FOS, Ca, and Vc in infant formula milk powder. The results indicated that after SNV preprocessing, the original spectra of GOS and FOS could effectively extract feature wavelengths using the CARS algorithm, leading to favorable predictive results through the CARS-SVR model. Similarly, after MSC preprocessing, the original spectra of Ca and Vc could efficiently extract feature wavelengths using the CARS algorithm, resulting in optimal predictive outcomes via the CARS-SVR model. This study provides insights for the realization of online nutritional component detection and optimization control in the production process of infant formula.

Functional annotation of rhizospheric phageome of the wild plant species Moringa oleifera.

Introduction: The study aims to describe phageome of soil rhizosphere of M.oleifera in terms of the genes encoding CAZymes and other KEGG enzymes. Methods: Genes of the rhizospheric virome of the wild plant species Moringa oleifera were investigated for their ability to encode useful CAZymes and other KEGG (Kyoto Encyclopedia of Genes and Genomes) enzymes and to resist antibiotic resistance genes (ARGs) in the soil. Results: Abundance of these genes was higher in the rhizospheric microbiome than in the bulk soil. Detected viral families include the plant viral family Potyviridae as well as the tailed bacteriophages of class Caudoviricetes that are mainly associated with bacterial genera Pseudomonas, Streptomyces and Mycobacterium. Viral CAZymes in this soil mainly belong to glycoside hydrolase (GH) families GH43 and GH23. Some of these CAZymes participate in a KEGG pathway with actions included debranching and degradation of hemicellulose. Other actions include biosynthesizing biopolymer of the bacterial cell wall and the layered cell wall structure of peptidoglycan. Other CAZymes promote plant physiological activities such as cell-cell recognition, embryogenesis and programmed cell death (PCD). Enzymes of other pathways help reduce the level of soil H2O2 and participate in the biosynthesis of glycine, malate, isoprenoids, as well as isoprene that protects plant from heat stress. Other enzymes act in promoting both the permeability of bacterial peroxisome membrane and carbon fixation in plants. Some enzymes participate in a balanced supply of dNTPs, successful DNA replication and mismatch repair during bacterial cell division. They also catalyze the release of signal peptides from bacterial membrane prolipoproteins. Phages with the most highly abundant antibiotic resistance genes (ARGs) transduce species of bacterial genera Pseudomonas, Streptomyces, and Mycobacterium. Abundant mechanisms of antibiotic resistance in the rhizosphere include "antibiotic efflux pump" for ARGs soxR, OleC, and MuxB, "antibiotic target alteration" for parY mutant, and "antibiotic inactivation" for arr-1. Discussion: These ARGs can act synergistically to inhibit several antibiotics including tetracycline, penam, cephalosporin, rifamycins, aminocoumarin, and oleandomycin. The study highlighted the issue of horizontal transfer of ARGs to clinical isolates and human gut microbiome.

Abundant antibiotic resistance genes in rhizobiome of the human edible Moringa oleifera medicinal plant.

Moringa oleifera (or the miracle tree) is a wild plant species widely grown for its seed pods and leaves, and is used in traditional herbal medicine. The metagenomic whole genome shotgun sequencing (mWGS) approach was used to characterize antibiotic resistance genes (ARGs) of the rhizobiomes of this wild plant and surrounding bulk soil microbiomes and to figure out the chance and consequences for highly abundant ARGs, e.g., mtrA, golS, soxR, oleC, novA, kdpE, vanRO, parY, and rbpA, to horizontally transfer to human gut pathogens via mobile genetic elements (MGEs). The results indicated that abundance of these ARGs, except for golS, was higher in rhizosphere of M. oleifera than that in bulk soil microbiome with no signs of emerging new soil ARGs in either soil type. The most highly abundant metabolic processes of the most abundant ARGs were previously detected in members of phyla Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, and Firmicutes. These processes refer to three resistance mechanisms namely antibiotic efflux pump, antibiotic target alteration and antibiotic target protection. Antibiotic efflux mechanism included resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) antibiotics pumps as well as the two-component regulatory kdpDE system. Antibiotic target alteration included glycopeptide resistance gene cluster (vanRO), aminocoumarin resistance parY, and aminocoumarin self-resistance parY. While, antibiotic target protection mechanism included RbpA bacterial RNA polymerase (rpoB)-binding protein. The study supports the claim of the possible horizontal transfer of these ARGs to human gut and emergence of new multidrug resistant clinical isolates. Thus, careful agricultural practices are required especially for plants used in circles of human nutrition industry or in traditional medicine.

Genomic profiling and network-level understanding uncover the potential genes and the pathways in hepatocellular carcinoma.

Data integration with phenotypes such as gene expression, pathways or function, and protein-protein interactions data has proven to be a highly promising technique for improving human complex diseases, particularly cancer patient outcome prediction. Hepatocellular carcinoma is one of the most prevalent cancers, and the most common cause is chronic HBV and HCV infection, which is linked to the majority of cases, and HBV and HCV play a role in multistep carcinogenesis progression. We examined the list of known hepatocellular carcinoma biomarkers with the publicly available expression profile dataset of hepatocellular carcinoma infected with HCV from day 1 to day 10 in this study. The study covers an overexpression pattern for the selected biomarkers in clinical hepatocellular carcinoma patients, a combined investigation of these biomarkers with the gathered temporal dataset, temporal expression profiling changes, and temporal pathway enrichment following HCV infection. Following a temporal analysis, it was discovered that the early stages of HCV infection tend to be more harmful in terms of expression shifting patterns, and that there is no significant change after that, followed by a set of genes that are consistently altered. PI3K, cAMP, TGF, TNF, Rap1, NF-kB, Apoptosis, Longevity regulating pathway, signaling pathways regulating pluripotency of stem cells, Cytokine-cytokine receptor interaction, p53 signaling, Wnt signaling, Toll-like receptor signaling, and Hippo signaling pathways are just a few of the most commonly enriched pathways. The majority of these pathways are well-known for their roles in the immune system, infection and inflammation, and human illnesses like cancer. We also find that ADCY8, MYC, PTK2, CTNNB1, TP53, RB1, PRKCA, TCF7L2, PAK1, ITPR2, CYP3A4, UGT1A6, GCK, and FGFR2/3 appear to be among the prominent genes based on the networks of genes and pathways based on the copy number alterations, mutations, and structural variants study.

Chemoprotective effects of inositol hexaphosphate against cyclophosphamide-induced testicular damage in rats.

Cyclophosphamide (CP) is commonly used as an anticancer agent but has been associated with high toxicity in several animal organs, including the testes. Inositol hexaphosphate (IP6) is a polyphosphorylated carbohydrate that is present in foods with high fibre contents and has a wide range of essential physiological and pathological activities. Thus, we estimated the defensive effects of IP6 against CP-related testicular toxicity in rats. Sperm counts, motilities, viabilities and abnormalities and levels of testosterone, luteinising hormone and follicle-stimulating hormone were evaluated. Testicle specimens were also processed for histological and biochemical analyses, including determinations of malondialdehyde, nitric oxide, total antioxidant capacity, alkaline phosphatase, acid phosphatase, gamma glutamyl transferase, ß-glucuronidase, c-reactive protein, monocyte chemoattractant protein and leukotriene-4 and in comet assays. CP treatments were associated with deleterious histopathological, biochemical and genetic changes in rat testicles, and these were ameliorated by IP6 supplements in drinking water.

Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System.

Targeted modification of plant genomes is a powerful strategy for investigating and engineering cellular systems, paving the way for the discovery and development of important, novel agricultural traits. Cas9, an RNA-guided DNA endonuclease from the type II adaptive immune CRISPR system of the prokaryote Streptococcus pyogenes, has gained widespread popularity as a genome-editing tool for use in a wide array of cells and organisms, including model and crop plants. Effective genome engineering requires the delivery of the Cas9 protein and guide RNAs into target cells. However, in planta genome modification faces many hurdles, including the difficulty in efficiently delivering genome engineering reagents to the desired tissues. We recently developed a Tobacco rattle virus (TRV)-mediated genome engineering system for Nicotiana benthamiana. Using this platform, genome engineering reagents can be delivered into all plant parts in a simple, efficient manner, facilitating the recovery of progeny plants with the desired genomic modifications, thus bypassing the need for transformation and tissue culture. This platform expands the utility of the CRISPR/Cas9 system for in planta, targeted genome modification. Here, we provide a detailed protocol explaining the methodologies used to develop and implement TRV-mediated genome engineering in N. benthamiana. The protocol described here can be extended to any other plant species susceptible to systemic infection by TRV. However, this approach is not limited to vectors derived from TRV, as other RNA viruses could be used to develop similar delivery platforms.

A Simplified Method to Engineer CRISPR/Cas9-Mediated Geminivirus Resistance in Plants.

Throughout the world, geminiviruses cause devastating losses in economically important crops, including tomato, cotton, cassava, potato, chili, and cucumber; however, control mechanisms such as genetic resistance remain expensive and ineffective. CRISPR/Cas9 is an adaptive immunity mechanism used by prokaryotes to defend against invading nucleic acids of phages and plasmids. The CRISPR/Cas9 system has been harnessed for targeted genome editing in a variety of eukaryotic species, and in plants, CRISPR/Cas9 has been used to modify or introduce many traits, including virus resistance. Recently, we demonstrated that the CRISPR/Cas9 system could be used to engineer plant immunity against geminiviruses by directly targeting the viral genome for degradation. In this chapter, we describe a detailed method for engineering CRISPR/Cas9-mediated resistance against geminiviruses. This method may provide broad, durable viral resistance, as it can target conserved regions of the viral genome and can also be customized to emerging viral variants. Moreover, this method can be used in many crop species, as it requires little or no knowledge of the host plant's genome.

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato.

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

Engineering Molecular Immunity Against Plant Viruses.

Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance.

Plant viruses infect many economically important crops, including wheat, cotton, maize, cassava, and other vegetables. These viruses pose a serious threat to agriculture worldwide, as decreases in cropland area per capita may cause production to fall short of that required to feed the increasing world population. Under these circumstances, conventional strategies can fail to control rapidly evolving and emerging plant viruses. Genome-engineering strategies have recently emerged as promising tools to introduce desirable traits in many eukaryotic species, including plants. Among these genome engineering technologies, the CRISPR (clustered regularly interspaced palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) system has received special interest because of its simplicity, efficiency, and reproducibility. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. Here, we briefly describe the biology of the CRISPR/Cas9 system and plant viruses, and how different genome engineering technologies have been used to target these viruses. We further describe the main findings from recent studies of CRISPR/Cas9-mediated viral interference and discuss how these findings can be applied to improve global agriculture. We conclude by pinpointing the gaps in our knowledge and the outstanding questions regarding CRISPR/Cas9-mediated viral immunity.

Engineering Plants for Geminivirus Resistance with CRISPR/Cas9 System.

The CRISPR/Cas9 system is an efficient genome-editing platform for diverse eukaryotic species, including plants. Recent work harnessed CRISPR/Cas9 technology to engineer resistance to geminiviruses. Here, we discuss opportunities, emerging developments, and potential pitfalls for using this technology to engineer resistance against single and multiple geminivirus infections in plants.

CRISPR/Cas9-Mediated Immunity to Geminiviruses: Differential Interference and Evasion.

The CRISPR/Cas9 system has recently been used to confer molecular immunity against several eukaryotic viruses, including plant DNA geminiviruses. Here, we provide a detailed analysis of the efficiencies of targeting different coding and non-coding sequences in the genomes of multiple geminiviruses. Moreover, we analyze the ability of geminiviruses to evade the CRISPR/Cas9 machinery. Our results demonstrate that the CRISPR/Cas9 machinery can efficiently target coding and non-coding sequences and interfere with various geminiviruses. Furthermore, targeting the coding sequences of different geminiviruses resulted in the generation of viral variants capable of replication and systemic movement. By contrast, targeting the noncoding intergenic region sequences of geminiviruses resulted in interference, but with inefficient recovery of mutated viral variants, which thus limited the generation of variants capable of replication and movement. Taken together, our results indicate that targeting noncoding, intergenic sequences provides viral interference activity and significantly limits the generation of viral variants capable of replication and systemic infection, which is essential for developing durable resistance strategies for long-term virus control.

CRISPR/Cas9-mediated viral interference in plants.

BACKGROUND: The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. RESULTS: In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. CONCLUSIONS: These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.