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Microbial Biotechnology Jun 2024Pathogens resistant to classical control strategies pose a significant threat to crop yield, with seeds being a major transmission route. Bacteriophages, viruses...
Pathogens resistant to classical control strategies pose a significant threat to crop yield, with seeds being a major transmission route. Bacteriophages, viruses targeting bacteria, offer an environmentally sustainable biocontrol solution. In this study, we isolated and characterized two novel phages, Athelas and Alfirin, which infect Pseudomonas syringae and Agrobacterium fabrum, respectively, and included the recently published Pfeifenkraut phage infecting Xanthomonas translucens. Using a simple immersion method, phages coated onto seeds successfully lysed bacteria post air-drying. The seed coat mucilage (SCM), a polysaccharide-polymer matrix exuded by seeds, plays a critical role in phage binding. Seeds with removed mucilage formed five to 10 times less lysis zones compared to those with mucilage. The podovirus Athelas showed the highest mucilage dependency. Phages from the Autographiviridae family also depended on mucilage for seed adhesion. Comparative analysis of Arabidopsis SCM mutants suggested the diffusible cellulose as a key component for phage binding. Long-term activity tests demonstrated high phage stability on seed surfaces and significantly increasing seedling survival rates in the presence of pathogens. Using non-virulent host strains enhanced phage presence on seeds but also has potential limitations. These findings highlight phage-based interventions as promising, sustainable strategies for combating pathogen resistance and improving crop yield.
Topics: Seeds; Pseudomonas syringae; Plant Diseases; Bacteriophages; Arabidopsis; Xanthomonas; Plant Mucilage; Biological Control Agents; Virus Attachment
PubMed: 38884488
DOI: 10.1111/1751-7915.14507 -
Genes & Genomics Jun 2024Verticillium wilt, causes mainly by the soilborne pathogen Verticillium dahliae, is a devastated vascular disease resulting in huge financial losses in cotton, so...
BACKGROUND
Verticillium wilt, causes mainly by the soilborne pathogen Verticillium dahliae, is a devastated vascular disease resulting in huge financial losses in cotton, so research on improving V. dahliae stress tolerance in cotton is the utmost importance. Calcium as the second messenger acts as a crucial role in plant innate immunity. Cytosolic Caduring the pathogen infection is a significant increase in plant immune responses. Calcineurin B-like (CBL) proteins are widely known calcium sensors that regulate abiotic stress responses. However, the role of cotton CBLs in response to V. dahliae stress remains unclear.
OBJECTIVE
To discover and utilize the gene to Verticillium wilt resistance and defense response mechanism of cotton.
METHODS
Through screening the gene to Verticillium wilt resistance in cotton, four GhCBL3 copies were obtained from the current common cotton genome sequences. The protein domain and phylogenetic analyses of GhCBL3 were performed using NCBI Blast, DNAMAN, and MotifScan programs. Real-time RT-PCR was used to detect the expression of GhCBL3 gene in cotton seedlings under various stress treatments. The expression construct including GhCBL3 cDNA was transduced into Agrobacterium tumefaciens (GV3101) by heat shock method and transformed into cotton plants by Virus-Induced Gene Silencing (VIGS) method. The results of silencing of GhCBl3 on ROS accumulation and plant disease resistance in cotton plants were assessed.
RESULTS
A member of calcineurin B-like proteins (defined as GhCBL3) in cotton was obtained. The expression of GhCBL3 was significantly induced and raised by various stressors, including dahliae, jasmonic acid (JA) and HO stresses. Knockdown GhCBL3 in cotton by Virus-Induced Gene Silencing analysis enhanced Verticillium wilt tolerance and changed the occurrence of reactive oxygen species. Some disease-resistant genes were increased in GhCBL3-silencing cotton lines.
CONCLUSION
GhCBL3 may function on regulating the Verticillium dahliae stress response of plants.
PubMed: 38879677
DOI: 10.1007/s13258-024-01528-4 -
Proceedings of the National Academy of... Jun 2024Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which...
Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in and potentially acts more widely in multiple proteobacterial lineages.
Topics: Biofilms; Agrobacterium tumefaciens; Pterins; Cyclic GMP; Bacterial Proteins; Proteobacteria; Molybdenum Cofactors; Periplasm; Periplasmic Proteins; Periplasmic Binding Proteins; Gene Expression Regulation, Bacterial
PubMed: 38870058
DOI: 10.1073/pnas.2319903121 -
Methods in Molecular Biology (Clifton,... 2024CRISPR/Cas9 system is one of the most often utilized engineering tools for genome editing in many organisms including crop plants and presents great value in both basic...
CRISPR/Cas9 system is one of the most often utilized engineering tools for genome editing in many organisms including crop plants and presents great value in both basic and applied research. This is a preferred method because of its relative simplicity, cost-effectiveness, and reliability. The Cas9 nuclease guided by a short single guide RNA (gRNA) can generate double-strand DNA breaks (DSB) at the specific sites in chromosomal DNA. The DSB site is repaired by error-prone repair methods. During repair, some nucleotides are deleted or added at the target site. Here, we present a simplified protocol for generating mutants in gene of interest in rice using CRISPR/Cas9.
Topics: Oryza; CRISPR-Cas Systems; Gene Editing; RNA, Guide, CRISPR-Cas Systems; Mutation; Plants, Genetically Modified; DNA Breaks, Double-Stranded; Stress, Physiological
PubMed: 38869803
DOI: 10.1007/978-1-0716-3973-3_20 -
Plant Direct Jun 2024-mediated transient expression methods are widely used to study gene function in both model and non-model plants. Using a dual-luciferase assay, we quantified the effect...
-mediated transient expression methods are widely used to study gene function in both model and non-model plants. Using a dual-luciferase assay, we quantified the effect of -infiltration parameters on the transient transformation efficiency of seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre- and post-infiltration dark incubation and is less sensitive to the growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven- to eight-fold while a dark incubation pre- and post-infiltration increased transformation efficiency by five- to 13-fold. in exponential compared with stationary phase increased transformation efficiency by two-fold. Finally, we quantified the variation in our -infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6-fold in raw firefly luciferase () and raw luciferase () luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of infiltration in seedlings will facilitate the study of this important medicinal plant and will expand the application of -mediated transformation methods in other plant species.
PubMed: 38855128
DOI: 10.1002/pld3.596 -
BMC Plant Biology Jun 2024One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and...
BACKGROUND
One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah.
METHODS
The elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control.
RESULTS
Elicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3'-hydroxylase (F3'H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds.
CONCLUSIONS
This study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.
Topics: Oxylipins; Cyclopentanes; Acetates; Plant Roots; Phenols; Ficus; Flavonoids; Gene Expression Regulation, Plant; Antioxidants; Basidiomycota; Plant Growth Regulators; Agrobacterium
PubMed: 38853268
DOI: 10.1186/s12870-024-05178-2 -
The New Phytologist Jun 2024Agroinfiltration of Nicotiana benthamiana is routinely used in plant science and molecular pharming to transiently express proteins of interest. Here, we discuss four...
Agroinfiltration of Nicotiana benthamiana is routinely used in plant science and molecular pharming to transiently express proteins of interest. Here, we discuss four phenomena that should be avoided to improve transient expression. Immune responses can be avoided by depleting immune receptors and employing pathogen-derived effectors; transcript degradation by using silencing inhibitors or RNA interference machinery mutants; endoplasmic reticulum stress by co-expressing chaperones; and protein degradation can be avoided with subcellular targeting, protease mutants and co-expressing protease inhibitors. We summarise the reported increased yields for various recombinant proteins achieved with these approaches and highlight remaining challenges to further improve the efficiency of this versatile protein expression platform.
PubMed: 38849321
DOI: 10.1111/nph.19894 -
The New Phytologist Jun 2024
PubMed: 38837421
DOI: 10.1111/nph.19881 -
Plant Cell Reports Jun 2024A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and...
A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration.
Topics: Glycine max; Plant Leaves; Plants, Genetically Modified; Agrobacterium; Gene Expression Regulation, Plant; Nicotiana; Genetic Vectors
PubMed: 38837057
DOI: 10.1007/s00299-024-03245-4 -
Planta Jun 2024We have developed and optimized a rapid, versatile Agrobacterium-mediated transient expression system for cannabis seedlings that can be used in functional genomics...
We have developed and optimized a rapid, versatile Agrobacterium-mediated transient expression system for cannabis seedlings that can be used in functional genomics studies of both hemp-type and drug-type cannabis. Cannabis (Cannabis sativa L.) holds great promise in the medical and food industries due to its diverse chemical composition, including specialized cannabinoids. However, the study of key genes involved in various biological processes, including secondary metabolite biosynthesis, has been hampered by the lack of efficient in vivo functional analysis methods. Here, we present a novel, short-cycle, high-efficiency transformation method for cannabis seedlings using Agrobacterium tumefaciens. We used the RUBY reporter system to monitor transformation results without the need for chemical treatments or specialized equipment. Four strains of A. tumefaciens (GV3101, EHA105, LBA4404, and AGL1) were evaluated for transformation efficiency, with LBA4404 and AGL1 showing superior performance. The versatility of the system was further demonstrated by successful transformation with GFP and GUS reporter genes. In addition, syringe infiltration was explored as an alternative to vacuum infiltration, offering simplicity and efficiency for high-throughput applications. Our method allows rapid and efficient in vivo transformation of cannabis seedlings, facilitating large-scale protein expression and high-throughput characterization studies.
Topics: Agrobacterium tumefaciens; Seedlings; Genomics; Cannabis; Transformation, Genetic; Plants, Genetically Modified; Genes, Reporter; Green Fluorescent Proteins
PubMed: 38837044
DOI: 10.1007/s00425-024-04448-5