To characterize the mechanism through which myosin XI-K attaches to its principal endomembrane cargo, a yeast two-hybrid library of Arabidopsis thaliana cDNAs was screened using the myosin cargo binding domain as bait. This screen identified two previously uncharacterized transmembrane proteins (hereinafter myosin binding proteins or MyoB1/2) that share a myosin binding, conserved domain of unknown function 593 (DUF593). Additional screens revealed that MyoB1/2 also bind myosin XI-1, whereas myosin XI-I interacts with the distantly related MyoB7. The in vivo interactions of MyoB1/2 with myosin XI-K were confirmed by immunoprecipitation and colocalization analyses. In epidermal cells, the yellow fluorescent protein–tagged MyoB1/2 localize to vesicles that traffic in a myosin XI–dependent manner. Similar to myosin XI-K, MyoB1/2 accumulate in the tip-growing domain of elongating root hairs. Gene knockout analysis demonstrated that functional cooperation between myosin XI-K and MyoB proteins is required for proper plant development. Unexpectedly, the MyoB1-containing vesicles did not correspond to brefeldin A–sensitive Golgi and post-Golgi or prevacuolar compartments and did not colocalize with known exocytic or endosomal compartments. Phylogenomic analysis suggests that DUF593 emerged in primitive land plants and founded a multigene family that is conserved in all flowering plants. Collectively, these findings indicate that MyoB are membrane-anchored myosin receptors that define a distinct, plant-specific transport vesicle compartment.

bThe improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

We investigate the myosin XI-driven transport network in Arabidopsis using protein-protein interaction, subcellular localization, gene knockout, and bioinformatics analyses. The two major groups of nodes in this network are myosins XI and their membrane-anchored receptors (MyoB) that, together, drive endomembrane trafficking and cytoplasmic streaming in the plant cells. The network shows high node connectivity and is dominated by generalists, with a smaller fraction of more specialized myosins and receptors. We show that interaction with myosins and association with motile vesicles are common properties of the MyoB family receptors. We identify previously uncharacterized myosin-binding proteins, putative myosin adaptors that belong to two unrelated families, with four members each (MadA and MadB). Surprisingly, MadA1 localizes to the nucleus and is rapidly transported to the cytoplasm, suggesting the existence of myosin XI-driven nucleocytoplasmic trafficking. In contrast, MadA2 and MadA3, as well as MadB1, partition between the cytosolic pools of motile endomembrane vesicles that colocalize with myosin XI-K and diffuse material that does not. Gene knockout analysis shows that MadB1-4 contribute to polarized root hair growth, phenocopying myosins, whereas MadA1-4 are redundant for this process. Phylogenetic analysis reveals congruent evolutionary histories of the myosin XI, MyoB, MadA, and MadB families. All these gene families emerged in green algae and show concurrent expansions via serial duplication in flowering plants. Thus, the myosin XI transport network increased in complexity and robustness concomitantly with the land colonization by flowering plants and, by inference, could have been a major contributor to this process.myosins | receptors | adaptors | cytoplasmic streaming | nuclear transport F or half of a century, it had been assumed that rapid organelle trafficking and cytoplasmic streaming in plant cells rely on myosin motors (1) and, in particular, class XI myosins that are homologous to fungal and animal class V myosins (2). Over the past decade, a massive amount of information on specific functions of myosins and mechanisms of myosin XI-dependent processes in plants has been obtained (3-5). The first genetic evidence of myosin function in cell expansion involved the demonstration of a dramatic reduction of the polarized root hair growth upon inactivation of the myosin XI-K and XI-2 genes (6, 7). Subsequently, it has been shown that myosindependent trafficking is required for the growth of multiple cell types, as well as normal plant growth and development (4,(8)(9)(10). Among the 13 paralogous myosins XI encoded in the Arabidopsis thaliana genome, the highly expressed myosins XI-K, XI-1, and XI-2 have been shown to provide the most pronounced, functionally redundant contributions to each of these processes. In contrast, another highly expressed paralog, myosin XI-I, plays only limited roles in cell and plant growth but has a specialized function in nuclear repositioning through binding nuclear e...

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Mercury is a redox-active heavy metal that reacts with active thiols and depletes cellular antioxidants. Active resistance to the mercuric ion is a widely distributed trait among bacteria and results from the action of mercuric reductase (MerA). Protein phylogenetic analysis of MerA in bacteria indicated the occurrence of a second distinctive form of MerA among the archaea, which lacked an N-terminal metal recruitment domain and a C-terminal active tyrosine. To assess the distribution of the forms of MerA in an interacting community comprising members of both prokaryotic domains, studies were conducted at a naturally occurring mercuryrich geothermal environment. Geochemical analyses of Coso Hot Springs indicated that mercury ore (cinnabar) was present at concentrations of parts per thousand. Under high-temperature and acid conditions, cinnabar may be oxidized to the toxic form Hg 2؉ , necessitating mercury resistance in resident prokaryotes. Culture-independent analysis combined with culture-based methods indicated the presence of thermophilic crenarchaeal and gram-positive bacterial taxa. Fluorescence in situ hybridization analysis provided quantitative data for community composition. DNA sequence analysis of archaeal and bacterial merA sequences derived from cultured pool isolates and from community DNA supported the hypothesis that both forms of MerA were present. Competition experiments were performed to assess the role of archaeal merA in biological fitness. An essential role for this protein was evident during growth in a mercury-contaminated environment. Despite environmental selection for mercury resistance and the proximity of community members, MerA retains the two distinct prokaryotic forms and avoids genetic homogenization.The separation of prokaryotes into bacterial and archaeal domains (or superkingdoms) has gained considerable support from the ongoing input of genome sequencing efforts and their identification of proteins separately categorized in this organizational structure. The exchange of genes between domains, however, means that this finding is not absolute because it can result in genomes having mixed compositions. Lateral gene transfer (LGT) has been reported within the archaeal domain (22) and between the bacterial and archaeal domains (4,12,13,23). In addition, some authors have noted that there is preferential gene transfer of housekeeping genes, also called operational genes, rather than genes concerned with information processing (16).LGT also plays a role in the evolution of mercury resistance encoded in the mer operon, which makes it suitable for analysis of evolutionary processes. Recent studies of microbes residing at marine hydrothermal vents support such efforts (38). The mer genes have been well studied in bacteria and usually are plasmid encoded and therefore mobile (5). This may explain why some bacteria possess mer genes even if they do not live in environments rich in mercury. However, mer genes appear to be absent in anaerobic organisms, which perhaps reflects a reduced n...