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.

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.

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.

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.