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Methods in Molecular Biology (Clifton,... 2024Transformation of foreign DNA into Cryptococcus species is a powerful tool for exploring gene functions in these human pathogens. Agrobacterium tumefaciens-mediated...
Transformation of foreign DNA into Cryptococcus species is a powerful tool for exploring gene functions in these human pathogens. Agrobacterium tumefaciens-mediated transformation (AtMT) has been used for the stable introduction of exogenous DNA into Cryptococcus for over two decades, being particularly impactful for insertional mutagenesis screens to discover new genes involved in fungal biology. A detailed protocol to conduct this transformation method is provided in the chapter. Scope for modifications and the benefits and disadvantages of using AtMT in Cryptococcus species are also presented.
Topics: Transformation, Genetic; Cryptococcus; Agrobacterium tumefaciens; DNA, Bacterial; Genetic Vectors; Gene Transfer Techniques
PubMed: 38758312
DOI: 10.1007/978-1-0716-3722-7_6 -
The New Phytologist Jul 2024Nucleotide-binding domain and leucine-rich repeat (NLR) proteins with pathogen sensor activities have evolved to initiate immune signaling by activating helper NLRs....
Nucleotide-binding domain and leucine-rich repeat (NLR) proteins with pathogen sensor activities have evolved to initiate immune signaling by activating helper NLRs. However, the mechanisms underpinning helper NLR activation by sensor NLRs remain poorly understood. Although coiled coil (CC) type sensor NLRs such as the Potato virus X disease resistance protein Rx have been shown to activate the oligomerization of their downstream helpers NRC2, NRC3 and NRC4, the domains involved in sensor-helper signaling are not known. Here, we used Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana to show that the nucleotide-binding (NB) domain within the NB-ARC of Rx is necessary and sufficient for oligomerization and immune signaling of downstream helper NLRs. In addition, the NB domains of the disease resistance proteins Gpa2 (cyst nematode resistance), Rpi-amr1, Rpi-amr3 (oomycete resistance) and Sw-5b (virus resistance) are also sufficient to activate their respective downstream NRC helpers. Using transient expression in the lettuce (Lactuca sativa), we show that Rx (both as full length or as NB domain truncation) and its helper NRC2 form a minimal functional unit that can be transferred from solanaceous plants (lamiids) to Campanulid species. Our results challenge the prevailing paradigm that NLR proteins exclusively signal via their N-terminal domains and reveal a signaling activity for the NB domain of NRC-dependent sensor NLRs. We propose a model in which helper NLRs can perceive the status of the NB domain of their upstream sensors.
Topics: Signal Transduction; Nicotiana; NLR Proteins; Disease Resistance; Protein Domains; Plant Proteins; Lactuca; Protein Multimerization; Nucleotides; Plant Diseases; Plants, Genetically Modified; Plant Immunity
PubMed: 38757730
DOI: 10.1111/nph.19818 -
Frontiers in Microbiology 2024Alterations in the microbial community significantly impact the yield and quality of ginseng. Yet, the dynamics of microbial community shifts within the root endophytes...
Alterations in the microbial community significantly impact the yield and quality of ginseng. Yet, the dynamics of microbial community shifts within the root endophytes of ginseng across varying cultivation periods remain inadequately understood. This study zeroes in on the microbial community variations within the xylem (M), phloem (R), and fibrous roots (X) of ginseng during the fourth (F4) and fifth (F5) years of cultivation, aiming to bridge this research gap. We assessed soil physicochemical properties, enzyme activities, and nine individual saponins, complemented by high-throughput sequencing techniques (16S rDNA and ITS) to determine their profiles. The results showed that cultivation years mainly affected the microbial diversity of endophytic bacteria in ginseng fibrous roots compartment: the ASVs number and α-diversity Chao1 index of bacteria and fungi in F5X compartment with higher cultivation years were significantly higher than those in F4X compartment with lower cultivation years. It is speculated that the changes of fibrous roots bacterial groups may be related to the regulation of amino acid metabolic pathway. Such as D-glutamine and D-glutamate metabolism D-glutamine, cysteine and methionine metabolism regulation. The dominant bacteria in ginseng root are Proteobacteria (relative abundance 52.07-80.35%), Cyanobacteria (1.97-42.52%) and Bacteroidota (1.11-5.08%). Firmicutes (1.28-3.76%). There were two dominant phyla: Ascomycota (60.10-93.71%) and Basidiomycota (2.25-30.57%). Endophytic fungi were more closely related to soil physicochemical properties and enzyme activities. AN, TK, OP, SWC and EC were the main driving factors of endophytic flora of ginseng root. decreased with the increase of cultivation years, and the decrease was more significant in phloem (F4R: 33.36%, F5R: 16.48%). The relative abundance of and in each ecological niche increased with the increase of cultivation years. The relative abundance of and in F5X increased by 8.35 and 9.29 times, respectively, and in F5M increased by 5.57 times. We found a variety of potential beneficial bacteria and pathogen antagonists related to ginseng biomass and saponins, such as , , and , which have good potential for practical application and development.
PubMed: 38756733
DOI: 10.3389/fmicb.2024.1402921 -
Plant Signaling & Behavior Dec 2024The purpose of this study was to analyze the role of transcription factor in , proving that the transcription factor was related to the plant phenotypes of - cv....
The purpose of this study was to analyze the role of transcription factor in , proving that the transcription factor was related to the plant phenotypes of - cv. 'GuangYaoDa1' and it could be used in molecular-assisted breeding. 'GuangYaoDa1' was used as the material and its DNA was the template to clone DsWRKY6, the transgenic line was constructed by agrobacterium tumefaciens‑mediated transformation. Transgenic was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that transgenic had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of increased and the abscisic acid content significantly increased in transgenic compared with the control group. According to the above results, could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the transcription factor might be the cause of the upright growth of 'GuangYaoDa1'.
Topics: Arabidopsis; Plants, Genetically Modified; Cloning, Molecular; Plant Proteins; Transcription Factors; Gene Expression Regulation, Plant; Fabaceae; Phenotype; Abscisic Acid; Genes, Plant
PubMed: 38743594
DOI: 10.1080/15592324.2024.2349868 -
BMC Plant Biology May 2024'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11...
'Candidatus Phytoplasma mali' SAP11-Like protein modulates expression of genes involved in energy production, photosynthesis, and defense in Nicotiana occidentalis leaves.
BACKGROUND
'Candidatus Phytoplasma mali', the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11 which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11 and changes in the transcriptome were recorded until 5 days post infiltration.
RESULTS
The RNA-seq analysis revealed that presence of SAP11 in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced.
CONCLUSIONS
The results indicate that early SAP11 expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.
Topics: Nicotiana; Phytoplasma; Plant Leaves; Photosynthesis; Gene Expression Regulation, Plant; Plant Diseases; Bacterial Proteins; Energy Metabolism
PubMed: 38741080
DOI: 10.1186/s12870-024-05087-4 -
Plants (Basel, Switzerland) Apr 2024Increasing the ultraviolet radiation (UV) level, particularly UV-B due to damage to the stratospheric ozone layer by human activities, has huge negative effects on plant...
Increasing the ultraviolet radiation (UV) level, particularly UV-B due to damage to the stratospheric ozone layer by human activities, has huge negative effects on plant and animal metabolism. As a widely grown cool-season forage grass and turfgrass in the world, perennial ryegrass () is UV-B-sensitive. To study the effects of miR164, a highly conserved microRNA in plants, on perennial ryegrass under UV stress, both Osa overexpression (OE164) and target mimicry (MIM164) transgenic perennial ryegrass plants were generated using agrobacterium-mediated transformation, and UV-B treatment (~600 μw cm) of 7 days was imposed. Morphological and physiological analysis showed that the gene affected perennial ryegrass UV tolerance negatively, demonstrated by the more scorching leaves, higher leaf electrolyte leakage, and lower relative water content in OE164 than the WT and MIM164 plants after UV stress. The increased UV sensitivity could be partially due to the reduction in antioxidative capacity and the accumulation of anthocyanins. This study indicated the potential of targeting and/or its targeted genes for the genetic manipulation of UV responses in forage grasses/turfgrasses; further research to reveal the molecular mechanism underlying how miR164 affects plant UV responses is needed.
PubMed: 38732457
DOI: 10.3390/plants13091242 -
Microbial Pathogenesis Jul 2024Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in...
The novel bacteriocin BacYB1 produced by Leuconostoc mesenteroides YB1: From recent analytical characterization to biocontrol Verticillium dahliae and Agrobacterium tumefaciens.
Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases.
Topics: Agrobacterium tumefaciens; Bacteriocins; Olea; Plant Diseases; RNA, Ribosomal, 16S; Leuconostoc mesenteroides; Biological Control Agents; Verticillium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Antibiosis; Phylogeny; Anti-Bacterial Agents
PubMed: 38729380
DOI: 10.1016/j.micpath.2024.106680 -
PloS One 2024Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle,...
Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
Topics: Helianthus; Transformation, Genetic; Agrobacterium tumefaciens; Plants, Genetically Modified; Tissue Culture Techniques; Plant Roots; Plant Breeding; Crops, Agricultural
PubMed: 38722945
DOI: 10.1371/journal.pone.0298299 -
ACS Synthetic Biology May 2024Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for...
Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular -based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, DFL 12 and B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; 2011 and "" C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales ( S1) as well as in one member of Caulobacterales ( CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in .
Topics: Plasmids; Genetic Vectors; Alphaproteobacteria; Host Specificity
PubMed: 38718218
DOI: 10.1021/acssynbio.4c00062 -
Biotechnology Letters May 2024Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic...
OBJECTIVE
Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences.
RESULTS
The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 μmol g s, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 μmol g s and 18 ± 4 μmol g s, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides.
CONCLUSION
Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.
PubMed: 38717662
DOI: 10.1007/s10529-024-03489-w