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In the face of carbon, nitrogen, and phosphorus starvation, microorganisms have evolved adaptive mechanisms to maintain growth. In a previous study, we identified a protein predicted to contain acyl-CoA-binding domains in the plant pathogenic fungus Verticillium dahliae. The predicted protein, designated VdAcb1, possesses an atypical signal peptide. However, the functions of this acyl-CoA-binding protein in V. dahliae are not clear. In this research, in vivo or in vitro assays confirmed that VdAcb1 is secreted extracellularly from V. dahliae, although it does not have the typical signal peptide. Furthermore, the unconventional secretion of VdAcb1 was dependent on VdGRASP, a member of the compartment for unconventional protein secretion (CUPS). The deletion mutant strain of VdAcb1 (ΔVdAcb1) exhibited significant sensitivity to carbon starvation. RNA-seq revealed that the expression of genes related to filamentous growth (MSB2 pathway) and sugar transport were regulated by VdAcb1 under conditions of carbon starvation. Yeast one-hybrid experiments further showed that the expression of VdAcb1 was positively regulated by the transcription factor VdMsn4. The ΔVdAcb1 strain showed significantly reduced virulence on Gossypium hirsutum and Nicotiana benthamiana. We hypothesize that under conditions of carbon starvation, the expression of VdAcb1 is activated by VdMsn4 and VdAcb1 is secreted into the extracellular space. In turn, this activates the downstream MAPK pathway to enhance filamentous growth and virulence of V. dahliae.

Robust genome editing technologies are becoming part of the crop breeding toolbox. Currently, genome editing is usually conducted either at a single locus, or multiple loci, in a variety at one time. Massively parallel genomics platforms, multifaceted genome editing capabilities, and flexible transformation systems enable targeted variation at nearly any locus, across the spectrum of genotypes within a species. We demonstrate here the simultaneous transformation and editing of many genotypes, by targeting mixed seed embryo explants with genome editing machinery, followed by re-identification through genotyping after plant regeneration. Transformation and Editing of Mixed Lines (TREDMIL) produced transformed individuals representing 101 of 104 (97%) mixed elite genotypes in soybean; and 22 of 40 (55%) and 9 of 36 (25%) mixed maize female and male elite inbred genotypes, respectively. Characterization of edited genotypes for the regenerated individuals identified over 800 distinct edits at the Determinate1 (Dt1) locus in samples from 101 soybean genotypes and 95 distinct Brown midrib3 (Bm3) edits in samples from 17 maize genotypes. These results illustrate how TREDMIL can help accelerate the development and deployment of customized crop varieties for future precision breeding.

The widely used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is thought to have evolved from IS200/IS605 transposons. TnpB proteins, encoded by one type of IS200/IS605 transposon, are considered to be the evolutionary ancestors of Cas12 nucleases, which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells. TnpB nucleases, which are smaller than Cas nucleases, have been engineered for use in genome editing in animal systems, but the feasibility of this approach in plants remained unknown. Here, we obtained stably transformed genome-edited mutants in rice (Oryza sativa) by adapting three recently identified TnpB genome editing vectors, encoding distinct TnpB nucleases (ISAam1, ISDra2, and ISYmu1), for use in plants, demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes. ISDra2 and ISYmu1 precisely edited their target sequences, with no off-target mutations detected, showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants. Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs.

Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from Acidibacillus sulfuroxidans (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored. In this study, through a systematic investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10 and 11 bp, suggesting significant potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture.

The diversity of plant natural products presents a rich resource for accelerating drug discovery and addressing pressing human health issues. However, the challenges in accessing and cultivating source species, as well as metabolite structural complexity, and general low abundance present considerable hurdles in developing plant-derived therapeutics. Advances in high-throughput sequencing, genome assembly, gene synthesis, analytical technologies, and synthetic biology approaches, now enable us to efficiently identify and engineer enzymes and metabolic pathways for producing natural and new-to-nature therapeutics and drug candidates. This review highlights challenges and progress in plant natural product discovery and engineering by example of recent breakthroughs in identifying the missing enzymes involved in the biosynthesis of the anti-cancer agent Taxol^®. These enzyme resources offer new avenues for the bio-manufacture and semi-synthesis of an old blockbuster drug.

Salicylic acid (SA) is a phytohormone required for plant growth and defense signaling. There are two major SA biosynthesis pathways in plants: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia-lyase (PAL) pathway. It has been demonstrated in several plant species, including the model plant Arabidopsis, that SA is derived predominantly from the ICS pathway. Here, we employed the CRISPR/Cas9 system to generate ICS knockout mutants in rice (Oryza sativa L.). The Osics mutants display severe growth defects, and are completely devoid of phylloquinone, an isochorismate-derived product. The growth defects of Osics can be rescued through exogenous application of 1,4-dihydroxy-2-naphthoic acid (NA), a precursor of phylloquinone. Remarkably, the basal SA levels are not altered in the Osics mutants. Our findings support a role of OsICS in the biosynthesis of phylloquinone, and imply that SA biosynthesis in rice may occur through an alternative route other than the ICS pathway.

Phytic acid (PA) in grain seeds reduces the bioavailability of nutrient elements in monogastric animals, and an important objective for crop seed biofortification is to decrease the seed PA content. Here, we employed CRISPR/Cas9 to generate a PA mutant population targeting PA biosynthesis and transport genes, including two multi-drug-resistant protein 5 (MRP5) and three inositol pentose-phosphate kinases (IPK1). We characterized a variety of lines containing mutations on multiple IPK and MRP5 genes. The seed PA was more significantly decreased in higher-order mutant lines with multiplex mutations. However, such mutants also exhibited poor agronomic performance. In the population, we identified  two lines carrying single mutations in ipk1b and ipk1c, respectively. These mutants exhibited moderately reduced PA content, and regular agronomic performance compared to the wild type. Our study indicates that moderately decreasing PA by targeting single GmIPK1 genes, rather than multiplex mutagenesis toward ultra-low PA, is an optimal strategy for low-PA soybean with a minimal trade-off in yield performance.

Bakanae disease, caused by Fusarium fujikuroi, poses a significant threat to rice production and has been observed in most rice-growing regions. The disease symptoms caused by different pathogens may vary, including elongated and weak stems, slender and yellow leaves, and dwarfism, as example. Bakanae disease is likely to cause necrosis of diseased seedlings, and it may cause a large area of infection in the field through the transmission of conidia. Therefore, early disease surveillance plays a crucial role in securing rice production. Traditional monitoring methods are both time-consuming and labor-intensive and cannot be broadly applied. In this study, a combination of hyperspectral imaging technology and deep learning algorithms were used to achieve in situ detection of rice seedlings infected with bakanae disease. Phenotypic data were obtained on the 9th, 15th, and 21st day after rice infection to explore the physiological and biochemical performance, which helps to deepen the research on the disease mechanism. Hyperspectral data were obtained over these same periods of infection, and a deep learning model, named Rice Bakanae Disease-Visual Geometry Group (RBD-VGG), was established by leveraging hyperspectral imaging technology and deep learning algorithms. Based on this model, an average accuracy of 92.2% was achieved on the 21st day of infection. It also achieved an accuracy of 79.4% as early as the 9th day. Universal characteristic wavelengths were extracted to increase the feasibility of using portable spectral equipment for field surveillance. Collectively, the model offers an efficient and non-destructive surveillance methodology for monitoring bakanae disease, thereby providing an efficient avenue for disease prevention and control.

Rice (Oryza sativa) produces numerous diterpenoid phytoalexins that are important in defense against pathogens. Surprisingly, despite extensive previous investigations, a major group of such phytoalexins, the abietoryzins, were only recently reported. These aromatic abietanes are presumably derived from ent-miltiradiene, but such biosynthetic capacity has not yet been reported in O. sativa. While wild rice has been reported to contain such an enzyme, specifically ent-kaurene synthase-like 10 (KSL10), the only characterized ortholog from O. sativa (OsKSL10), specifically from the well-studied cultivar (cv.) Nipponbare, instead has been shown to make ent-sandaracopimaradiene, precursor to the oryzalexins. Notably, in many other cultivars, OsKSL10 is accompanied by a tandem duplicate, termed here OsKSL14. Biochemical characterization of OsKLS14 from cv. Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene. Strikingly, phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv. Nipponbare is an outlier, whereas the alleles from most other cultivars group with those from wild rice, suggesting that these also might produce ent-miltiradiene. Indeed, OsKSL10 from cv. Kitaake exhibits such activity as well, consistent with its production of abietoryzins but not oryzalexins. Similarly consistent with these results is the lack of abietoryzin production by cv. Nipponbare. Although their equivalent product outcome might suggest redundancy, OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns, indicating such differences may underlie retention of these duplicated genes. Regardless, the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.