Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.

Motif-based endomembrane trafficking.

Endomembrane trafficking, which allows proteins and lipids to flow between the different endomembrane compartments, largely occurs by vesicle-mediated transport. Transmembrane proteins intended for transport are concentrated into a vesicle or carrier by undulation of a donor membrane. This is followed by vesicle scission, uncoating, and finally, fusion at the target membrane. Three major trafficking pathways operate inside eukaryotic cells: anterograde, retrograde, and endocytic. Each pathway involves a unique set of machinery and coat proteins that pack the transmembrane proteins, along with their associated lipids, into specific carriers. Adaptor and coatomer complexes are major facilitators that function in anterograde transport and in endocytosis. These complexes recognize the transmembrane cargoes destined for transport and recruit the coat proteins that help form the carriers. These complexes use either linear motifs or posttranslational modifications to recognize the cargoes, which are then packaged and delivered along the trafficking pathways. In this review, we focus on the different trafficking complexes that share a common evolutionary branch in Arabidopsis (Arabidopsis thaliana), and we discuss up-to-date knowledge about the cargo recognition motifs they use.

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.

Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator.

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

Updated inventory, evolutionary and expression analyses of G (PDR) type ABC transporter genes of rice.

ABC transporters constitute the largest family of transporter proteins in living organisms and divided into eight subfamilies, from A-H. ABCG members, specific to plants and fungi, belong to subfamily G. In this study, we provide updated inventory, detailed account of phylogeny, gene structure characteristics, and expression profiling during reproductive development, abiotic and biotic stresses of members of ABCG gene family in rice along with reannotation and cloning of FL-cDNA of OsABCG50/PDR23. We observed that of the 22 ABCGs/PDRs, four genes evolved as a result of gene duplication events and their expression pattern changed after duplication. Analysis of expression revealed seed and developmental stage preferential expression of five ABCG/PDR members. Transcript levels of eight ABCGs/PDRs were affected by abiotic and biotic stresses. Expression of seven ABCG/PDR genes was also altered by hormonal elicitors. The modulated expression is nicely correlated with the presence of tissue/stress specific cis-acting elements present in putative promoter region.