Rhizogenic Agrobacterium protein RolB interacts with the TOPLESS repressor proteins to reprogram plant immunity and development.

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.

A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system.

Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by-effects, we investigated the impact of GRF4-GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4-GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM-WUS. As agrobacterium  effectors increase the ability of T-DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4-GIF1 with helper plasmid pVS1-VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71-fold of the original method. Compared with BBM-WUS, overexpressing GRF4-GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene-editing tool based on this GRF4-GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Integration of Agrobacterium T-DNA into the Plant Genome.

Agrobacterium strains transfer a single-strand form of T-DNA (T-strands) and Virulence (Vir) effector proteins to plant cells. Following transfer, T-strands likely form complexes with Vir and plant proteins that traffic through the cytoplasm and enter the nucleus. T-strands may subsequently randomly integrate into plant chromosomes and permanently express encoded transgenes, a process known as stable transformation. The molecular processes by which T-strands integrate into the host genome remain unknown. Although integration resembles DNA repair processes, the requirement of known DNA repair pathways for integration is controversial. The configuration and genomic position of integrated T-DNA molecules likely affect transgene expression, and control of integration is consequently important for basic research and agricultural biotechnology applications. This article reviews our current knowledge of the process of T-DNA integration and proposes ways in which this knowledge may be manipulated for genome editing and synthetic biology purposes.

A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19.

Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T-DNA is engineered with the gene(s) of interest. However, gene expression during 'agro-infiltration' is intrinsically and universally impeded by the onset of post-transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co-expression of the tombusvirus-encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side-by-side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co-expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies - an originally unanticipated, yet increasingly popular application - and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits.

GATA transcription factor in common bean: A comprehensive genome-wide functional characterization, identification, and abiotic stress response evaluation.

The GATA transcription factors (TFs) have been extensively studied for its regulatory role in various biological processes in many plant species. The functional and molecular mechanism of GATA TFs in regulating tolerance to abiotic stress has not yet been studied in the common bean. This study analyzed the functional identity of the GATA gene family in the P. vulgaris genome under different abiotic and phytohormonal stress. The GATA gene family was systematically investigated in the P. vulgaris genome, and 31 PvGATA TFs were identified. The study found that 18 out of 31 PvGATA genes had undergone duplication events, emphasizing the role of gene duplication in GATA gene expansion. All the PvGATA genes were classified into four significant subfamilies, with 8, 3, 6, and 13 members in each subfamily (subfamilies I, II, III, and IV), respectively. All PvGATA protein sequences contained a single GATA domain, but subfamily II members had additional domains such as CCT and tify. A total of 799 promoter cis-regulatory elements (CREs) were predicted in the PvGATAs. Additionally, we used qRT-PCR to investigate the expression profiles of five PvGATA genes in the common bean roots under abiotic conditions. The results suggest that PvGATA01/10/25/28 may play crucial roles in regulating plant resistance against salt and drought stress and may be involved in phytohormone-mediated stress signaling pathways. PvGATA28 was selected for overexpression and cloned into N. benthamiana using Agrobacterium-mediated transformation. Transgenic lines were subjected to abiotic stress, and results showed a significant tolerance of transgenic lines to stress conditions compared to wild-type counterparts. The seed germination assay suggested an extended dormancy of transgenic lines compared to wild-type lines. This study provides a comprehensive analysis of the PvGATA gene family, which can serve as a foundation for future research on the function of GATA TFs in abiotic stress tolerance in common bean plants.

Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures.

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.

Ether Bond Cleavage of a Phenylcoumaran ß-5 Lignin Model Compound and Polymeric Lignin Catalysed by a LigE-type Etherase from Agrobacterium sp.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a ß-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.

Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA.

MAIN CONCLUSION: Agrobacterium-mediated transformation of Nicotiana tabacum, using an intragenic T-DNA region derived entirely from the N. tabacum genome, results in the equivalence of micro-translocations within genomes. Intragenic Agrobacterium-mediated gene transfer was achieved in Nicotiana tabacum using a T-DNA composed entirely of N. tabacum DNA, including T-DNA borders and the acetohydroxyacid synthase gene conferring resistance to sulfonylurea herbicides. Genomic analysis of a resulting plant, with single locus inheritance of herbicide resistance, identified a single insertion of the intragenic T-DNA on chromosome 5. The insertion event was composed of three N. tabacum DNA fragments from other chromosomes, as assembled on the T-DNA vector. This validates that intragenic transformation of plants can mimic micro-translocations within genomes, with the absence of foreign DNA.

A method of genetic transformation and gene editing of succulents without tissue culture.

Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.

An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees.

Agrobacterium-mediated transformation of B. juncea reveals that BjuLKP2 functions in plant yellowing.

KEY MESSAGE: A stable Agrobacterium-mediated transformation system was constructed for B. juncea, and BjuLKP2 was overexpressed, leading to plant yellowing. A stable and efficient transformation system is necessary to verify gene functions in plants. To establish an Agrobacterium-mediated transformation system for B. juncea, various factors, including the explant types, hormone combination and concentration, infection time and concentration, were optimized. Eventually, a reliable system was established, and two BjuLKP2 overexpression (OE) lines, which displayed yellowing of cotyledons, shoot tips, leaves and flower buds, as well as a decrease in total chlorophyll content, were generated. qRT-PCR assays revealed significant upregulation of five chlorophyll synthesis genes and downregulation of one gene in the BjuLKP2 OE line. Furthermore, antioxidant capacity assays revealed reduced activities of APX, CAT and SOD, while POD activity increased in the BjuLKP2 OE26. Additionally, the kinetic determination of chlorophyll fluorescence induction suggested a decrease in the photosynthetic ability of BjuLKP2 OE26. GUS assays revealed the expression of BjuLKP2 in various tissues, including the roots, hypocotyls, cotyledons, leaf vasculature, trichomes, sepals, petals, filaments, styles and stigma bases, but not in seeds. Scanning electron revealed alterations in chloroplast ultrastructure in both the sponge and palisade tissue. Collectively, these findings indicate that BjuLKP2 plays a role in plant yellowing through a reduction in chlorophyll content and changes in chloroplasts structure.

Genetic manipulation of Indian mustard genotypes with WRR-gene(s) confers resistance against Albugo candida.

BACKGROUND: Brassica species is the second most important edible oilseed crop in India. Albugo candida (Pers.) Kuntze, a major oomycete disease of oilseed brassica causing white rust, leads to 60% yield loss globally. The prevalence of A. candida race 2 (Ac2V) that specifically infects B. juncea, coupled with limitations of conventional methods has resulted in a dearth of white rust resistance resources in cultivated varieties. METHODS AND RESULTS: In an effort to develop resistant plants, Agrobacterium mediated genetic transformation of three B. juncea genotypes viz., susceptible host var. Varuna, along with its doubled haploid mutant lines C66 and C69 (showing moderate tolerance to field isolates of A. candida) was initiated to transfer resistance genes (WRR8Sf-2 and WRR9Hi-0) identified in Arabidopsis thaliana against race Ac2V, that encode for Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat proteins that recognize effectors of the pathogen races. CONCLUSIONS: Our results demonstrate that introduction of resistance genes from a tertiary gene pool by genetic transformation enhances disease resistance in B. juncea genotypes to a highly virulent Ac2V isolate.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

AIMS: This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). METHODS AND RESULTS: The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. CONCLUSIONS: C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

Robust soybean leaf agroinfiltration.

KEY MESSAGE: 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.

Superior osmotic stress tolerance in oilseed rape transformed with wild-type Rhizobium rhizogenes.

KEY MESSAGE: Natural transformation with R. rhizogenes enhances osmotic stress tolerance in oilseed rape through increasing osmoregulation capacity, enhancing maintenance of hydraulic integrity and total antioxidant capacity. Transformation of plants using wild strains of agrobacteria is termed natural transformation and is not covered by GMO legislation in, e.g., European Union and Japan. In this study, offspring lines of Rhizobium rhizogenes naturally transformed oilseed rape (Brassica napus), i.e., A11 and B3 (termed root-inducing (Ri) lines), were investigated for osmotic stress resilience. Under polyethylene glycol 6000 (PEG) 10% (w/v)-induced osmotic stress, the Ri lines, particularly A11, had less severe leaf wilting, higher stomatal conductance (8.2 times more than WT), and a stable leaf transpiration rate (about 2.9 mmol m-2 s-1). Although the leaf relative water content and leaf water potential responded similarly to PEG treatment between the Ri lines and WT, a significant reduction of the turgid weight to dry weight ratio in A11 and B3 indicated a greater capacity of osmoregulation in the Ri lines. Moreover, the upregulation of plasma membrane intrinsic proteins genes (PIPs) in roots and downregulation of these genes in leaves of the Ri lines implied a better maintenance of hydraulic integrity in relation to the WT. Furthermore, the Ri lines had greater total antioxidant capacity (TAC) than the WT under PEG stress. Collectively, the enhanced tolerance of the Ri lines to PEG-induced osmotic stress could be attributed to the greater osmoregulation capacity, better maintenance of hydraulic integrity, and greater TAC than the WT. In addition, Ri-genes (particularly rolA and rolD) play roles in response to osmotic stress in Ri oilseed rape. This study reveals the potential of R. rhizogenes transformation for application in plant drought resilience.

Targeted DNA insertion in plants.

Conventional methods of DNA sequence insertion into plants, using Agrobacterium-mediated transformation or microprojectile bombardment, result in the integration of the DNA at random sites in the genome. These plants may exhibit altered agronomic traits as a consequence of disruption or silencing of genes that serve a critical function. Also, genes of interest inserted at random sites are often not expressed at the desired level. For these reasons, targeted DNA insertion at suitable genomic sites in plants is a desirable alternative. In this paper we review approaches of targeted DNA insertion in plant genomes, discuss current technical challenges, and describe promising applications of targeted DNA insertion for crop genetic improvement.

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

Investigating the interaction of uranium(VI) with diatoms and their bacterial community: A microscopic and spectroscopic study.

Diatoms and bacteria play a vital role in investigating the ecological effects of heavy metals in the environment. Despite separate studies on metal interactions with diatoms and bacteria, there is a significant gap in research regarding heavy metal interactions within a diatom-bacterium system, which closely mirrors natural conditions. In this study, we aim to address this gap by examining the interaction of uranium(VI) (U(VI)) with Achnanthidium saprophilum freshwater diatoms and their natural bacterial community, primarily consisting of four successfully isolated bacterial strains (Acidovorax facilis, Agrobacterium fabrum, Brevundimonas mediterranea, and Pseudomonas peli) from the diatom culture. Uranium (U) bio-association experiments were performed both on the xenic A. saprophilum culture and on the four bacterial isolates. Scanning electron microscopy and transmission electron microscopy coupled with spectrum imaging analysis based on energy-dispersive X-ray spectroscopy revealed a clear co-localization of U and phosphorus both on the surface and inside A. saprophilum diatoms and the associated bacterial cells. Time-resolved laser-induced fluorescence spectroscopy with parallel factor analysis identified similar U(VI) binding motifs both on A. saprophilum diatoms and the four bacterial isolates. This is the first work providing valuable microscopic and spectroscopic data on U localization and speciation within a diatom-bacterium system, demonstrating the contribution of the co-occurring bacteria to the overall interaction with U, a factor non-negligible for future modeling and assessment of radiological effects on living microorganisms.

The Targeted Regulation of BDUbc and BDSKL1 Enhances Resistance to Blight in Bambusa pervariabilis × Dendrocalamopsis grandis.

Arthrinium phaeospermum is the major pathogen responsible for the significant stem disease "blight" in B. pervariabilis × D. grandis. The interacting proteins of the key pathogenic factor ApCtf1ß, BDUbc and BDSKL1, have previously been obtained by two-hybrid, BiFC, GST pull-down yeast assays. However, the functions of these interacting proteins remain unknown. This study successfully obtained transgenic plants overexpressing BDUbc, BDSKL1, and BDUbc + BDSKL1 via Agrobacterium-mediated gene overexpression. qRT-PCR analysis revealed significantly increased expression levels of BDUbc and BDSKL1 in the transgenic plants. After infection with the pathogenic spore suspension, the disease incidence and severity index significantly decreased across all three transgenic plants, accompanied by a marked increase in defense enzyme levels. Notably, the co-transformed plant, OE-BDUbc + BDSKL1, demonstrated the lowest disease incidence and severity index among the transgenic variants. These results not only indicate that BDUbc and BDSKL1 are disease-resistant genes, but also that these two genes may exhibit a synergistic enhancement effect, which further improves the resistance to blight in Bambusa pervariabilis × Dendrocalamopsis grandis.