Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity.

The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for protein⁻protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; АТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens.

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall.

BACKGROUND: Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants. METHODS: Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests. KEY RESULTS: An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3-4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass. CONCLUSIONS: This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.

Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast.

The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana ß-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, ß-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and ß-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

A zebrafish model of granulin deficiency reveals essential roles in myeloid cell differentiation.

Granulin is a pleiotropic protein involved in inflammation, wound healing, neurodegenerative disease, and tumorigenesis. These roles in human health have prompted research efforts to use granulin to treat rheumatoid arthritis and frontotemporal dementia and to enhance wound healing. But how granulin contributes to each of these diverse biological functions remains largely unknown. Here, we have uncovered a new role for granulin during myeloid cell differentiation. We have taken advantage of the tissue-specific segregation of the zebrafish granulin paralogues to assess the functional role of granulin in hematopoiesis without perturbing other tissues. By using our zebrafish model of granulin deficiency, we revealed that during normal and emergency myelopoiesis, myeloid progenitors are unable to terminally differentiate into neutrophils and macrophages in the absence of granulin a (grna), failing to express the myeloid-specific genes cebpa, rgs2, lyz, mpx, mpeg1, mfap4, and apoeb. Functionally, macrophages fail to recruit to the wound, resulting in abnormal healing. Our CUT&RUN experiments identify Pu.1, which together with Irf8, positively regulates grna expression. In vivo imaging and RNA sequencing experiments show that grna inhibits the expression of gata1, leading to the repression of the erythroid program. Importantly, we demonstrated functional conservation between the mammalian granulin and the zebrafish ortholog grna. Our findings uncover a previously unrecognized role for granulin during myeloid cell differentiation, which opens a new field of study that can potentially have an impact on different aspects of human health and expand the therapeutic options for treating myeloid disorders such as neutropenia or myeloid leukemia.

A new technique for activation tagging in Arabidopsis.

We have created and applied to Arabidopsis thaliana a new system of two vectors. The first vector (pEnLox) is intended for activation tagging and contains a multimerized transcriptional enhancer from the cauliflower mosaic virus (CaMV) 35S gene in T-DNA flanked by two loxP-sites and the second vector (pCre) contains the cre gene. Using pEnLox we have generated more than a hundred mutants resistant to the herbicide ammonium glufosinate, and about ten helper-lines resistant to the antibiotic hygromycin obtained with the use of pCre vector and also more than ten double mutants resistant to both selective markers. In at least 3 cases among 40 mutant lines that have been analyzed we observed constitutive ectopic expression of the genes adjacent to the T-DNA insertion that causes development of the mutant phenotype. Also, reversion of the mutants to the wild-type phenotype after removing the CaMV enhancer has been demonstrated. The system presented here provides a new and easier way to analyze A. thaliana gain-of-function mutants.

A highly efficient miPCR method for isolating FSTs from transgenic Arabidopsis thaliana plants.

The exact localization of an insertion in the genome of transgenic plants obtained by Agrobacterium-mediated transformation is an integral part of most experiments aimed at studying these types of mutants. There are several methods for isolating unknown nucleotide sequences of genomic DNA which flank the borders of T-DNA integrated in the genome of plants. However, all the methods based on PCR have limitations which in some cases do not permit the desired objective to be achieved. We have developed a new technique for isolating flanking sequence tags (FSTs) via modified inverse PCR. This method is highly efficient and simple, but also retains the advantages of previously well-documented approaches.

Bacterial community in ancient permafrost alluvium at the Mammoth Mountain (Eastern Siberia).

Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised >5% of the OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top families (Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae) held >90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction of reads. The bacterial community in the studied ancient alluvium differs from other permafrost sediments, mainly by predominance of Bacteroidetes (>52%). The diversity of this preserved bacterial community has the potential to cause effects unknown if prompted to thaw and spread with changing climate. Therefore, this study elicits further reason to study how reintroduction of these ancient bacteria could affect the surrounding ecosystem, including current bacterial species.

Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defense response against Pseudomonas syringae.

Plant immunophilins are a broadly conserved family of proteins, which carry out a variety of cellular functions. In this study, we investigated three immunophilin genes involved in the Arabidopsis thaliana response to Pseudomonas syringae infection: a cytoplasmic localized AtCYP19, a cytoplasmic and nuclear localized AtCYP57, and one nucleus directed FKBP known as AtFKBP65. Arabidopsis knock-out mutations in these immunophilins result in an increased susceptibility to P. syringae, whereas overexpression of these genes alters the transcription profile of pathogen-related defense genes and led to enhanced resistance. Histochemical analysis revealed local gene expression of AtCYP19, AtCYP57, and AtFKBP65 in response to pathogen infection. AtCYP19 was shown to be involved in reactive oxygen species production, and both AtCYP57 and AtFKBP65 provided callose accumulation in plant cell wall. Identification of the involvement of these genes in biotic stress response brings a new set of data that will advance plant immune system research and can be widely used for further investigation in this area.

Identification of ICE2, a gene involved in cold acclimation which determines freezing tolerance in Arabidopsis thaliana.

Several transcription factors are presently known to regulate the response to cold stress. Here we describe a new positive regulator, ICE2, which is a transcription factor of the bHLH family that participates in the response to deep freezing through the cold acclimation-dependent pathway in Arabidopsis thaliana plants. An overexpression of ICE2 (as we named the At1g12860 locus) in transgenic Arabidopsis plants results in increased tolerance to deep freezing stress after cold acclimation. The seeds of transgenic lines that overexpressed ICE2 were characterized by decreased levels of carbohydrate and increased levels of lipids. The analysis of expression of CBF1 gene (also known as DREB1B), which have been shown to be required for the complete development of cold acclimation response in Arabidopsis indicated a difference between expression of the CBF1 gene in transgenic plants and the wild-type control plants, Col-0. These results suggested that the CBF1 transcription factor, known as one of the regulators of the cold stress response, has a dominant role in providing freezing tolerance in transgenic plants characterized by overexpression of ICE2.

IDentification and Analysis of the Arabidopsis Thaliana Atfas4 Gene Whose Overexpression Results in the Development of A Fasciated Stem.

Using a new pEnLox vector employed to generate gain-of-function mutants in Arabidopsis thaliana, the AtFAS4 mutant has been obtained and analyzed. The mutant is characterized by super-expression of the At1g33390 gene, which leads to the occurrence of a mutant phenotype - stem fasciation. The level of expression of the AtFAS4 gene in normally developing A.thaliana plants is extremely low thus accounting for almost complete absence of information on EST's of this gene. The generated AtFAS4 mutant has permitted full-length cDNA of the At1g33390 gene to be obtained and analyzed for the first time.