Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest, enabling the covalent biotin labelling of proteins and subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report 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 which 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 test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.

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.

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.

35Proximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a 36 peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and 37 allows subsequent capture and identification of interacting and neighbouring proteins without 38 the need for the protein complex to remain intact. To date, only few papers report on the use of 39 PDL in plants. Here we present the results of a systematic study applying a variety of PDL 40 approaches in several plant systems using various conditions and bait proteins. We show that 41TurboID is the most promiscuous variant in several plant model systems and establish protocols 42 which combine Mass Spectrometry-based analysis with harsh extraction and washing 43 conditions. We demonstrate the applicability of TurboID in capturing membrane-associated 44 protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. 45We further benchmark the efficiency of various PBLs in comparison with one-step affinity 46 purification approaches. We identified both known as well as novel interactors of the endocytic 47Protein-protein interaction (PPI) studies often fail to capture low-affinity interactions as these 53 are usually not maintained following cell lysis, protein extraction and protein complex 54 purification. Particularly, this is the case for PPI's of integral membrane proteins because of 55 the harsh conditions during protein extraction and purification. Proximity-dependent biotin 56 labelling (PDL) on the contrary, uses covalent biotinylation of proteins that are interactors or 57 near-neighbours of a bait protein of interest in vivo (Varnaite and MacNeill, 2016). Hence, to 58 identify interactions, they do not need to remain intact during purification. Although biotin is 59 an essential cofactor for a small number of omnipresent biotin-dependent enzymes involved 60 mainly in the transfer of CO2 during HCO3 --dependent carboxylation reactions, biotinylation 61 is a relatively rare in vivo protein modification. Moreover, biotinylated proteins can be 62 selectively isolated with high affinity using streptavidin-biotin pairing. PDL, therefore, permits 63

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, H. (2019). Updated inventory, evolutionary and expression analyses of G (PDR) type ABC transporter genes of rice.

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