In vitro binding of the tomato bZIP transcriptional activator VSF-1 to a regulatory element that controls xylem-specific gene expression

The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter ( vs-1 ) specifically activated both the −82 CaMV 35S and the −76/ grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro . To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro . This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal −82 CaMV 35S promoter in vivo . Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the −76/ grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis , belong to a new family of plant bZIP proteins.     

Arabidopsis LIP5, a Positive Regulator of Multivesicular Body Biogenesis,Is a Critical Target of Pathogen-Responsive MAPK Cascade in Plant Basal Defense

Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-INTERACTING PROTEIN 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2^DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1–43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.     

The trypanosome VSG expression site encodes adenylate cyclase and a leucine-rich putative regulatory gene.

African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.     

Microtubule-associated protein 1S, a short and ubiquitously expressed member of the microtubule-associated protein 1 family

The related high molecular mass microtubule-associated proteins (MAPs) MAP1A and MAP1B are predominantly expressed in the nervous system and are involved in axon guidance and synaptic function. MAP1B is implicated in fragile X mental retardation, giant axonal neuropathy, and ataxia type 1. We report the functional characterization of a novel member of the microtubule-associated protein 1 family, which we termed MAP1S (corresponding to sequence data bank entries for VCY2IP1 and C19ORF5). MAP1S contains the three hallmark domains of the microtubule-associated protein 1 family but hardly any additional sequences. It decorates neuronal microtubules and copurifies with tubulin from brain. MAP1S is synthesized as a precursor protein that is partially cleaved into heavy and light chains in a tissue-specific manner. Heavy and light chains interact to form the MAP1S complex. The light chain binds, bundles, and stabilizes microtubules and binds to actin. The heavy chain appears to regulate light chain activity. In contrast to MAP1A and MAP1B, MAP1S is expressed in a wide range of tissues in addition to neurons and represents the non-neuronal counterpart of this cytolinker family.     

TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG.

Regulatory elements containing the sequence ACGT are found in several plant promoters and are recognized by various basic/leucine zipper (bZIP) proteins. The Arabidopsis G-box binding factor 1 (GBF1), initially identified by its ability to bind to the palindromic G-box (CCACGTGG), also interacts with the TGACGT motif if this hexamer sequence is followed by either the dinucleotide GG--as found in the Hex motif of the wheat histone 3 promoter--or GT. Here we describe the isolation of an Arabidopsis bZIP protein, denoted TGA1, that also recognizes ACGT-containing sequences. However, TGA1 differs from members of the GBF family in the spectrum of base pair permutations flanking the ACGT sequence that are required for DNA binding. TGA1 primarily requires a TGACG motif and preferentially binds to those pentamers that are followed by a T residue. We show that although both TGA1 and GBF1 bind to the Hex motif (TGACGTGG), this binding can be distinguished on the basis of their specific DNA-protein contacts. Furthermore, TGA1 also differs from members of the GBF family in that it apparently does not form heterodimers with any member of this family.