A multiSite gateway™ vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Recombinatorial cloning using the Gateway^TM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway^TM compatible vectors. The MultiSite Gateway^TM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast <it>Saccharomyces cerevisiae</it>, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.</p> <p>Results</p> <p>Here, we present a set of three-fragment MultiSite Gateway^TM destination vectors that have been developed for gene expression in <it>S. cerevisiae</it> and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.</p> <p>Conclusion</p> <p>Our vectors make MultiSite Gateway^TM cloning accessible in <it>S. cerevisiae</it> and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.</p

A dual sgRNA approach for functional genomics in Arabidopsis thaliana

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase theta (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes

Glutaredoxin GRXS17 associates with the cytosolic iron-sulfur cluster assembly pathway

Cytosolic monothiol glutaredoxins (GRXs) are required in iron-sulfur (Fe-S) cluster delivery and iron sensing in yeast and mammals. In plants, it is unclear whether they have similar functions. Arabidopsis (Arabidopsis thaliana) has a sole class II cytosolic monothiol GRX encoded by GRXS17. Here, we used tandem affinity purification to establish that Arabidopsis GRXS17 associates with most known cytosolic Fe-S assembly (CIA) components. Similar to mutant plants with defective CIA components, grxs17 loss-of-function mutants showed some degree of hypersensitivity to DNA damage and elevated expression of DNA damage marker genes. We also found that several putative Fe-S client proteins directly bind to GRXS17, such as XANTHINE DEHYDROGENASE1 (XDH1), involved in the purine salvage pathway, and CYTOSOLIC THIOURIDYLASE SUBUNIT1 and CYTOSOLIC THIOURIDYLASE SUBUNIT2, both essential for the 2-thiolation step of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNAs. Correspondingly, profiling of the grxs17-1 mutant pointed to a perturbed flux through the purine degradation pathway and revealed that it phenocopied mutants in the elongator subunit ELO3, essential for the mcm5 tRNA modification step, although we did not find XDH1 activity or tRNA thiolation to be markedly reduced in the grxs17-1 mutant. Taken together, our data suggest that plant cytosolic monothiol GRXs associate with the CIA complex, as in other eukaryotes, and contribute to, but are not essential for, the correct functioning of client Fe-S proteins in unchallenged conditions

Із зали засідань Президії НАН України

20 червня 2012 року відбулося виїзне засідання Президії Національної академії наук України на запрошення президента — генерального конструктора Державного підприємства «АНТОНОВ» академіка НАН України Д.С. Ківи

FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation

The RING E3 ligase KEEP ON GOING modulates JASMONATE ZIM-DOMAIN12 stability

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCFCOI1) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCFCOI1 components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability

Subfunctionalization of Paralog Transcription Factors Contributes to Regulation of Alkaloid Pathway Branch Choice in Catharanthus roseus

Catharanthus roseus produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available C. roseus transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the C. roseus flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.</jats:p