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 peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens.

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

Genomic consequences associated with Agrobacterium-mediated transformation of plants.

Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

A glycine zipper motif is required for the translocation of a T6SS toxic effector into target cells.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N-terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39 xxxG43 motif do not affect Tde1-Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39 xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we propose that glycine zipper-mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

A highly conserved ligand-binding site for AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium leads to a different specificity.

Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the l-arabinopyranose moiety in agrocinopines A/B and the d-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the d-glucopyranose-2-phosphate moiety, and with a lower affinity compared with strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.

Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens.

Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: "ori-ori clustering" in which the replication origins of all four replicons interact, and "Ch1-Ch2 alignment" in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.

An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology.

A split GAL4 RUBY assay for visual in planta detection of protein-protein interactions.

Protein-protein interactions (PPIs) are a fundamental process in cellular biogenesis. Here we have developed a split GAL4 RUBY assay that enables macroscopically visual PPI detection in plant leaves in real time. Candidate interacting protein partners are fused to specific domains of the yeast GAL4 and herpes simplex virus VP16 transcription factors and transiently expressed in Nicotiana benthamina leaves by Agrobacterium infiltration. PPI, that may be either direct or indirect, results in transcriptional activation of a RUBY reporter gene leading to the production of the highly visual metabolite, betalain, in leaf tissue of living plants. Samples require no processing for in planta visual qualitative assessment, but with very simple processing steps the assay is quantitative. Its accuracy is demonstrated using a series of known interacting protein partners and mutant derivatives including transcription factors, signalling molecules and plant resistance proteins with cognate pathogen effectors. Using this assay, association between the wheat Sr27 stem rust disease resistance protein and corresponding AvrSr27 avirulence effector family produced by the rust pathogen is detected. Interaction is also observed between this resistance protein and the effector encoded by the corresponding avrSr27-3 virulence allele. However, this association appears weaker in the split GAL4 RUBY assay, which coupled with lower avrSr27-3 expression during stem rust infection, likely enables virulent races of the rust pathogen to avoid Sr27-mediated detection.

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.

Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells.

BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.

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.

Molecular responses of agroinfiltrated Nicotiana benthamiana leaves expressing suppressor of silencing P19 and influenza virus-like particles.

The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co-expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin-related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.

Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up-regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up-regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up-regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5-expressing leaves.

Improving transient protein expression in agroinfiltrated Nicotiana benthamiana.

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.

Nano-delivery platforms for bacterial gene transformation: suitability and challenges.

Nano-scale particles (NPs) have gained increased interest as non-viral vectors for nucleic acid delivery due to their ability to penetrate through unabraded cell membranes. The previous studies performed have evaluated the nanomaterials for their microbial transformation proficiency but have not compared the relative efficacy. The present study aims to identify the most proficient nano-delivery vehicle among the chemically synthesized/functionalized non-metal oxide, metal/metal oxide, and carbon-based (carbon nanotube (CNT), graphene oxide (GO)) nanomaterial(s) (NMs) for the transformation of two gram-negative bacteria, i.e., Escherichia coli and Agrobacterium tumefaciens. The microscopy and spectroscopy studies helped to identify the interaction, adhesion patterns, transformation efficiencies, better delivery, and expression of the target gfp gene by use of NMs. Loading of pgfp on all NMs imparted protection to DNAse I attack except ZnO NPs with maximum by chitosan, layered double hydroxide (LDH), and GO NM-plasmid DNA conjugates. The CNTs and GO significantly enhanced the extra- and intra-cellular protein content, respectively, in both bacteria. However, GO and CNT significantly decreased the cell viability in a time-dependent manner while AuNPs exhibited negligible cell toxicity. Therefore, this study identified the comparative efficiency of metal/metal oxide, non-metal oxide, and carbon nanomaterials with AuNPs as the most biosafe while LDH and chitosan NPs being the most proficient alternative tools for the genetic transformation of gram-negative bacteria by simple incubation method.

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.

Development of activation-tagged gain-of-functional mutants in indica rice line (BPT 5204) for sheath blight resistance.

BACKGROUND: The development of sheath blight (ShB) resistance varieties has been a challenge for scientists for long time in rice. Activation tagging is an efficient gain-of-function mutation approach to create novel phenotypes and to identify their underlying genes. In this study, a mutant population was developed employing activation tagging in the recalcitrant indica rice (Oryza sativa L.) cv. BPT 5204 (Samba Mahsuri) through activation tagging. METHODS AND RESULTS: In this study, we have generated more than 1000 activation tagged lines in indica rice, from these mutant population 38 (GFP- RFP+) stable Ds plants were generated through germinal transposition at T2 generation based on molecular analysis and seeds selected on hygromycin (50 mg/L) containing medium segregation analyses confirmed that the transgene inherited as mendelian segregation ratio of 3:1 (3 resistant: 1 susceptible). Of them, five stable activation tagged Ds lines (M-Ds-1, M-Ds-2, M-Ds-3, M-Ds-4 and M-Ds-5) were selected based on phenotypic observation through screening for sheath blight (ShB) resistance caused by fungal pathogen Rhizoctonia solani (R. solani),. Among them, M-Ds-3 and M-Ds-5 lines showed significant resistance for ShB over other tagged lines and wild type (WT) plants. Furthermore, analysed for launch pad insertion through TAIL-PCR results and mapped on corresponding rice chromosomes. Flanking sequence and gene expression analysis revealed that the upregulation of glycoside hydrolase-OsGH or similar to Class III chitinase homologue (LOC_Os08g40680) in M-Ds-3 and a hypothetical protein gene (LOC_Os01g55000) in M-Ds-5 are potential candidate genes for sheath blight resistance in rice. CONCLUSION: In the present study, we developed Ac-Ds based ShB resistance gain-of-functional mutants through activation tagging in rice. These activation tagged mutant lines can be excellent sources for the development of ShB resistant cultivars in rice.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

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.