Dicer-Like 4 Is Involved in Restricting the Systemic Movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.

Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Ros1 systemic accumulation dramatically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.

Massive integration of large gene libraries in the chromosome of Escherichia coli.

Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in-frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized in vivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 107 VHH genes that encode nanobodies (Nbs). The integration of VHH genes into a custom target locus of the E. coli chromosome enabled stable expression and surface display of the Nbs. Next-generation DNA sequencing confirmed that MAIN preserved the diversity of the gene library after integration. Finally, we screened the integrated library to select Nbs that bind a specific antigen using magnetic and fluorescence-activated cell sorting. This allowed us to identify Nbs binding the epidermal growth factor receptor that were not previously isolated in a similar screening of a multicopy plasmid library. Our results demonstrate that MAIN enables large gene library integration into the E. coli chromosome, creating stably expressed in-frame fusions for functional screening.

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces.

ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest.

High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.