Background: Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix). Results: We detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others’ previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC₄(3). Conclusions: Level of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels–Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.

Two sets of bioorthogonally applicable, double fluorogenic probes, capable of sensing DNA–protein interactions, were prepared by installing an azide or tetrazine motif onto structurally fluorogenic, DNA sensitive frames. Installation of these bioorthogonal functions onto DNA intercalating dyes furnished these scaffolds with reactivity based fluorogenicity, rendering these probes double-fluorogenic, AND-type logic switches that require the simultaneous occurrence of a bioorthogonal reaction and interaction with DNA to trigger high intensity fluorescence. The probes were evaluated for double fluorogenic behavior in the presence/absence of DNA and a complementary bioorthogonal function. Our studies revealed that azide and tetrazine appending thiazole orange frames show remarkable double fluorogenic features. One of these probes, a membrane permeable tetrazine modified thiazole orange derivative was further tested in live cell labeling studies. Cells expressing bioorthogonalized DNA-binding proteins showed intensive fluorescence characteristics of the localization of the proteins upon treatment with our double fluorogenic probe. On the contrary, labeling similarly bioorthogonalized cytosolic proteins did not result in the appearance of the fluorescence signal. These studies suggest that such double-fluorogenic probes are indeed capable of sensing DNA–protein interactions in cells.

Pepper (Capsicum annuum L.) carrying the gds (corresponding to bs5) gene can prevent the development of bacterial leaf spot disease without HR. However, little is known regarding the development of the resistance mechanism encoded by gds, especially its influence on the bacterium. Here, the effect of gds was compared with pattern-triggered immunity (PTI), another form of asymptomatic resistance, to reveal the interactions and differences between these two defense mechanisms. The level of resistance was examined by its effect on the bacterial growth and in planta expression of the stress and pathogenicity genes of Xanthomonas euvesicatoria. PTI, which was activated with a Pseudomonas syringae hrcC mutant pretreatment, inhibited the growth of Xanthomonas euvesicatoria to a greater extent than gds, and the effect was additive when PTI was activated in gds plants. The stronger influence of PTI was further supported by the expression pattern of the dpsA bacterial stress gene, which reached its highest expression level in PTI-induced plants. PTI inhibited the hrp/hrc expression, but unexpectedly, in gds plant leaves, the hrp/hrc genes were generally expressed at a higher level than in the susceptible one. These results imply that different mechanisms underlie the gds and PTI to perform the symptomless defense reaction.

Fluorogen-activating proteins (FAPs) provide renewable fluorescence signals through continuous fluorogen exchange, resulting in imaging that is virtually resistant to photobleaching, an especially appealing feature for deterministic super-resolution microscopy techniques. Furthermore, careful adjustment of the fluorogen concentration may lead to reversible, exchange-driven stochastic blinking, allowing the use of FAPs in stochastic super-resolution microscopy methods. The green-light–excitable, far-red–emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) is particularly well suited for biological imaging due to its favorable spectral properties and live-cell compatibility, however, its performance across different super-resolution microscopy (SRM) modalities has not been evaluated. Here, we assess frFAST for single-molecule localization microscopy (SMLM), fluctuation-based computational approaches (super-resolution optical fluctuation imaging - SOFI and extended super-resolution radial fluctuations - eSRRF), and stimulated emission depletion (STED) microscopy. By tuning fluorogen concentration, we induced reversible stochastic blinking in fixed and live cells, enabling SMLM imaging of cytoskeletal structures and filopodia in mammalian and neuroblastoma cells, without the need for harsh reducing agents. Additionally, we successfully labeled and imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. While the renewable nature of the frFAST:HPAR-3OM interaction supports extended live-cell imaging for up to 40 minutes with limited photobleaching, we found that its performance is strongly modality dependent. In particular, intrinsic photophysical properties limit its suitability for classical SMLM, whereas fluctuation-based methods and live-cell STED microscopy are more compatible with frFAST.

Live cell super-resolution microscopy (SRM) imaging is often limited by the lack of optimal fluorescent labelling approaches. Here, we report two new chemogenetic approaches allowing far-red fluorescence detection to label intracellular protein targets to achieve background free SRM images in fixed and live mammalian cells. These methods are based on Fluorescence-activating and Absorption Shifting Tag (FAST), a small monomeric protein tag that forms reversible fluorescent assembly with various rhodanine fluorophores. We developed a Förster resonance energy transfer based (FRET-FAST) method that allows single molecule localization microscopy (SMLM) imaging of proteins in fixed mammalian cells. Additionally, we applied a far-red emitting variant (frFAST) to conduct fixed and live cell SMLM and stimulated emission depletion (STED) microscopy. Time-lapse SMLM up to 40 minutes revealed cytoskeletal dynamics without significant photobleaching. Additionally, we imaged filopodia in living neuroblastoma cells. Moreover, we succesfully imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. It is envisioned that the excellent photostability achieved by the presented far-red emitting FAST labelling techniques provides a new alternative for stochastic and deterministic SRM imaging of living cells, even for longer imaging sessions.