The Ti-TAN plasmid toolbox for TurboID-based proximity labeling assays in Nicotiana benthamiana.

The Ti-TAN TurboID plasmid toolbox enables proximity labeling applications in transient assays in Nicotiana benthamiana in a fast and cost-efficient manner, making TurboID-based proximity labeling broadly accessible to plant scientists.

Viral Recognition and Evasion in Plants.

Viruses, causal agents of devastating diseases in plants, are obligate intracellular pathogens composed of a nucleic acid genome and a limited number of viral proteins. The diversity of plant viruses, their diminutive molecular nature, and their symplastic localization pose challenges to understanding the interplay between these pathogens and their hosts in the currently accepted framework of plant innate immunity. It is clear, nevertheless, that plants can recognize the presence of a virus and activate antiviral immune responses, although our knowledge of the breadth of invasion signals and the underpinning sensing events is far from complete. Below, I discuss some of the demonstrated or hypothesized mechanisms enabling viral recognition in plants, the step preceding the onset of antiviral immunity, as well as the strategies viruses have evolved to evade or suppress their detection.

Two viral proteins translated from one open reading frame target different layers of plant defense.

Multilayered defense responses are activated upon pathogen attack. Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection, including suppression of host defense. Here, we reveal translation leakage as one of these strategies: two viral effectors encoded by tomato golden mosaic virus, chloroplast-localized C4 (cC4) and membrane-associated C4 (mC4), are translated from two in-frame start codons and function cooperatively to suppress defense. cC4 localizes in chloroplasts, to which it recruits NbPUB4 to induce ubiquitination of the outer membrane; as a result, this organelle is degraded, and chloroplast-mediated defenses are abrogated. However, chloroplast-localized cC4 induces the production of singlet oxygen (1O2), which in turn promotes translocation of the 1O2 sensor NbMBS1 from the cytosol to the nucleus, where it activates expression of the CERK1 gene. Importantly, an antiviral effect exerted by CERK1 is countered by mC4, localized at the plasma membrane. mC4, like cC4, recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1, suppressing membrane-based, receptor-like kinase-dependent defenses. Importantly, this translation leakage strategy seems to be conserved in multiple viral species and is related to host range. This finding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in the field.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

From Metagenomics to Ecogenomics: NGS-Based Approaches for Discovery of New Circular DNA Single-Stranded Viral Species.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.