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Heinlein, M.

Publications and source records attributed to Heinlein, M..

4 recordsLinked to original sources

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated with in situ sequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF{kappa}B signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in vivo screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

systems biology↗

Designer antisense circRNAGFP reduces GFP protein abundance in transgenic Arabidopsis protoplasts in a sequence-specific manner, independent of RNAi pathways

Circular RNAs (circRNAs) are single-stranded RNA molecules characterised by their covalently closed structure and are emerging as key regulators of cellular processes in mammals, including gene expression, protein function and immune responses. Recent evidence suggests that circRNAs also play significant roles in plants, influencing development, nutrition, biotic stress resistance, and abiotic stress tolerance. However, the potential of circRNAs to modulate target protein abundance in plants remains largely unexplored. In this study, we investigated the potential of designer circRNAs to modulate target protein abundance in plants using Arabidopsis as a model system. We demonstrate that treatment with a 50 nt circRNAGFP, containing a 30 nt GFP antisense sequence stretch, results in reduced GFP reporter target protein abundance in a dose- and sequence-dependent manner. Notably, a single-stranded open isoform of circRNAGFP had little effect on protein abundance, indicating the importance of the closed circular structure. Additionally, circRNAGFP also reduced GFP abundance in Arabidopsis mutants defective in RNA interference (RNAi), suggesting that circRNA activity is independent of the RNAi pathway. We also show that circRNA, unlike dsRNA, does not induce pattern-triggered immunity (PTI) in plants. Findings of this proof-of-principle study together are crucial first steps in understanding the potential of circRNAs as versatile tools for modulating gene expression and offer exciting prospects for their application in agronomy, particularly for enhancing crop traits through metabolic pathway manipulation. HighlightsWe demonstrate the potential of non-immunogenic circRNA as a tool for targeted gene regulation in plants, where circRNA acts in an isoform- and sequence-specific manner, paving the way for future agronomic applications.

plant biology↗

dsRNA-induced immunity targets plasmodesmata and is suppressed by viral movement proteins

Emerging evidence indicates that in addition to the well-recognized antiviral RNA silencing, dsRNA elicits responses of pattern-triggered immunity (PTI), likely contributing plant resistance against virus infections. However, compared to bacterial and fungal elicitor-mediated PTI, the mode-of-action and signaling pathway of dsRNA-induced defense remain poorly characterized. Here, using multi-color in vivo imaging by GFP mobility, staining of callose and plasmodesmal marker lines, we show that dsRNA-induced PTI restricts the progression of virus infection by triggering callose deposition at plasmodesmata, thereby likely limiting the macromolecular transport through these cell-to-cell communication channels. The plasma membrane-resident kinase module of SERK1 and BIK1/PBL1, plasmodesmata-localized proteins PDLP1/2/3 and calmodulin-like CML41, and Ca2+ signals are involved in the dsRNA-induced signaling leading to callose deposition at plasmodesmata and antiviral defense. In addition, unlike classical bacterial elicitor flagellin, dsRNA does not trigger detectable reactive oxygen species (ROS) burst, further substantiating a partially shared immune signaling framework with distinct features triggered by different microbial patterns. Likely as a counteract strategy, viral movement proteins from different viruses suppress the dsRNA-induced host response leading to callose deposition to achieve infection. Thus, our data support the new model of how plant immune signaling constrains the virus movement by inducing callose deposition at plasmodesmata and how viruses counteract this layer of immunity. One-sentence summarydsRNA-induced antiviral PTI targets plasmodesmata for callose deposition and is suppressed by virus-encoded movement proteins. IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSPlants use different defense mechanisms pathogens. The major mechanism that plants use for defense against viruses is known as RNA silencing. This mechanism is triggered by the presence of viral double-stranded (ds)RNA and uses small RNAs to inhibit viral replication by targeting the viral genome for degradation. Recently, it was found that dsRNA elicits antiviral defense also through a protein-mediated mechanism known as pattern-triggered immunity (PTI). However, the underlying mechanism of antiviral PTI and how viruses overcome this plant defense mechanism to cause infection is unknown. QuestionIn this study we asked how dsRNA-induced PTI acts to inhibit virus infection and whether we can identify components of the PTI signaling pathway. Moreover, we wanted to know how viruses overcome this plant host defense response in order to cause infection. FindingsWe demonstrate that dsRNA-induced PTI targets plasmodesmata (PD), the intercellular communication conduits in plant cell walls that viruses use to spread infection from cell to cell. By inducing the deposition of callose, dsRNA-induced PTI reduces PD permeability, thus restricting virus movement. We identified PTI signaling components required for dsRNA-induced PD callose deposition and delineate a PTI pathway showing important difference to PTI pathways triggered by microbial elicitors. Moreover, viral movement proteins (MPs) suppress the dsRNA-induced callose deposition response at PD. This leads to a new model of how plant immune signaling constrains virus movement and how viruses counteract this layer of immunity. Next stepsThis study calls upon the identification of the PTI dsRNA receptor and the mechanisms of PTI signaling (involving identified components such as SERK1, BIK1, calcium channels, CML41, PDLP1/2/3) and PTI suppression by MPs, and how dsRNA-induced PTI and RNA silencing are controlled during the spread of infection.

plant biology↗

Suppression of a dsRNA-induced plant immunity pathway by viral movement protein

The virome of plants is dominated by RNA viruses 1 and several of these cause devastating diseases in cultivated plants leading to global crop losses 2. To infect plants, RNA viruses engage in complex interactions with compatible plant hosts. In cells at the spreading infection front, RNA viruses replicate their genome through double-stranded RNA (dsRNA) intermediates and interact with cellular transport processes to achieve cell-to-cell movement of replicated genome copies through cell wall channels called plasmodesmata (PD) 3. In order to propagate, viruses also must overcome host defense responses. In addition to triggering the antiviral RNA silencing response, RNA virus infection also elicits pattern-triggered immunity (PTI) 4 whereby dsRNA, a hallmark of virus replication, acts as an important elicitor 5. This innate antiviral immune response is also triggered when dsRNA is applied externally and does not require sequence homology to the virus 5. However, the mechanism by which PTI restricts virus infection is not known. Here, we show that dsRNA inhibits the progression of virus infection by triggering callose deposition at plasmodesmata and the inhibition of transport through these cell-to-cell communication channels. The dsRNA-induced signaling pathway leading to callose deposition is independent of ROS production and thus distinguished from pathways triggered by bacterial and fungal elicitors. The dsRNA-induced host response at plasmodesmata is suppressed by the Tobacco mosaic virus movement protein (MP). Thus, the virus uses MP to inhibit innate dsRNA-induced immunity at plasmodesmata, which could be a general strategy of phytoviruses to overcome plant defenses and spread infection.

plant biology↗