The host specificity of Fusarium oxysporum (Fox) formae speciales has been reported to be linked to effector proteins known as Secreted in Xylem (SIX). These genes are associated with the non-autonomous mobile element miniature impala (mimp), normally distributed on the accessory chromosomes. The pattern of mimp associated with effector genes has been used to predict candidate effector profiles which characterize Fox formae speciales. In this study, we demonstrate the pathogenicity of strains Fusarium oxysporum f.sp. radicis-lycopersici (Forl) ZUM2407 and Fusarium oxysporum f.sp. radicis-cucumerinum (Forc) V03-2g in a common host plant (cucumber) and compare their genomes. The Forl ZUM2407 genome lacks SIX genes and their homologs, in contrast to Forc V03-2g. We predicted the total number of mimp elements in the genome of Forl ZUM2407 to be three-fold less than that of Forc V03-2g (10 and 36 copies, respectively). The mimp distribution pattern in Forl ZUM2407 was completely different from that present in Forc V03-2g. Candidate effector profile analysis did not predict that Forl ZUM2407 was able to infect cucumber plants like Forc V03-2g. Therefore, we assume that Forl ZUM2407 has a different type of genome organization associated with pathogenicity, whose effector profile cannot be described using the mimp-based approach.

Transcription factors (TFs) play a central role in the gene regulation associated with a plant's development and its response to the environmental factors. The work of TFs is well regulated at each stage of their activities. TFs usually consist of three protein domains required for DNA binding, dimerization, and transcriptional regulation. Alternative splicing (AS) produces multiple proteins with varying composition of domains. Recent studies have shown that AS of some TF genes form small proteins (small interfering peptide/small interfering protein, siPEP/siPRoT), which lack one or more domains and negatively regulate target TFs by the mechanism of protein interference (peptide interference/protein interference, PEPi/PROTi). The presence of an alternative form for the transcription factor CCA1 of Arabidopsis thaliana, has been shown to be involved in the regulation of the response to cold stress. For the PtFLC protein, one of the isoforms was found, which is formed as a result of alternative splicing and acts as a negative repressor, binding to the full-length TF PtFLC and therefore regulating the development of the Poncirus trifoliata. For A. thaliana, a FLM gene was found forming the FLM-б isoform, which acts as a dominant negative regulator and stimulates the development of the flower formation process due to the formation of a heterodimer with SVP TF. Small interfering peptides and proteins can actively participate in the regulation of gene expression, for example, in situations of stress or at different stages of plant development. Moreover, small interfering peptides and proteins can be used as a tool for fundamental research on the function of genes as well as for applied research for permanent or temporary knockout of genes. In this review, we have demonstrated recent studies related to siPEP/siPROT and their involvement in the response to various stresses, as well as possible ways to obtain small proteins.

Here we present the method for the target-specific elimination of certain intracellular proteins in plants using the ubiquitin-proteasome system. We modified the E3 ubiquitin ligase Chip of A. thaliana to obtain two variants carrying the deletions at the N-terminus and the GFP recognition domain. The interaction of the GFP protein and the chimeric ubiquitin ligase was confirmed via yeast two-hybrid assay. Fluorescence microscopy and fluorimetry showed that, when an infiltration of the gfp-expressing N. benthamiana plants was performed with agrobacteria carrying the hybrid E3 gene of the ubiquitin ligase Chip with the GFP recognition domain, a significant decrease in the fluorescence was observed for both variants: carrying the N-terminal deletion of 100 amino acids or the deletion of 140 amino acids.

Experimental data obtained in this study had shown the involvement of A. thaliana immunophilin genes At2g16600, At4g33060, and At5g48570 in plant defense responses to the Xanthomonas campestris invasion. We had found not only that the expression levels of these genes changed upon bacterial infection, but also that the plant’s resistance to the pathogen was increased if the expression levels of the immunophilin genes were elevated in the host cells.

DOI of original article: http://dx.doi.org/10.1016/j.gene.2014.01.029. ⁎ Corresponding author at: 219 Bessey Hall, Department of Plant Pathology and Microbiology, Iowa State University, Ames 50014, IA, USA. Tel.: +1 515 294 3120. E-mail addresses: gennady@iastate.edu, gpogorelko@yandex.ru (G.V. Pogorelko), marja-2007@yandex.ru (M. Mokryakova), oksfursova@yandex.ru (O.V. Fursova), insaz@yandex.ru (I. Abdeeva), eleopiru@vigg.ru (E.S. Piruzian), sergey.bruskin@gmail.com (S.A. Bruskin).

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.

Gene-targeted and site-directed mutagenesis. Reverse genetics methods (loss of function)Using transgenic plant with insertion/deletion or site-specific mutations.Host gene is replaced with mutant allele.The most conventional approach to the analysis of gene function is loss-of-function mutagenesis by chemicals or fast neutrons that introduce random mutations or deletions in the genome (Ostergaard and Yanofsky 2004).www.intechopen.com

Одним из главных инструментов патогенеза грамотрицательных бактерий являются белки, секретируемые при помощи транспортной системы третьего типа (T3TS) внутрь растительной клетки. Класс белков, доставляемых в растительную клетку через T3TS, имеет общее название белков-эффекторов. На основе анализа геномных последовательностей восьми полностью секвенированных геномов ксантомонад нами был определен состав потенциальных генов белков-эффекторов, куда вошли 19 генов как с известными, так и с неизвестными функциями. Методом ПЦР был определен состав генов белков-эффекторов, характерный для каждого штамма ксантомонад в исследуемой популяции. Был показан высокий уровень генетической изменчивости состава генов-эффекторов в пределах одного вида и его патовариантов. Впервые, у ряда штаммов Xanthomonas campestris pv. campestris были обнаружены гены-эффекторы, несвойственные типовым штаммам этого вида, и выдвинуто предположение о связи ряда генов с симптомами вызываемых поражений на растении-хозяине, а также показана связь числа генов-эффекторов с основными расами патогена.

A new strategy for creating experimental models for functional genomics has been proposed. It is based on the expression in transgenic plants of genes from thermophilic bacteria encoding functional analogues of plant proteins with high specific activity and thermal stability. We have validated this strategy by comparing physiological, biochemical and molecular properties of control tobacco plants and transgenic plants expressing genes of beta-glucanases with different substrate specificity. We demonstrate that the expression of bacterial beta-1,3-1,4-glucanase gene exerts no significant influence on tobacco plant metabolism, while the expression of bacterial beta-1,3-glucanase affects plant metabolism only at early stages of growth and development. By contrast, the expression of bacterial beta-1,4-glucanase has a significant effect on transgenic tobacco plant metabolism, namely, it affects plant morphology, the thickness of the primary cell wall, phytohormonal status, and the relative sugar content. We propose a hypothesis of beta-glucanase action as an important factor of genetic regulation of metabolic processes in plants.

Enzymes grouped into a superfamily of peptidyl-prolyl-cis/trans-isomerases (PPIases) possess chaperone activity and fold proteins into active configuration by catalyzing slow cis/trans isomerization on proline-peptide bonds. PPIases are conservative and abundant among Pro- and Eukaryotes. Many proteins involved in disease processes require modification; thus, PPIases can play an important role in pathogenesis. Analysis of the experimental data published so far allows us to classify the involvement of PPIases in pathologic process as follows: (1) Modification of outer membrane porines and channels, components of secretion systems, and secreted proteins by FkpA-like and SurA-like PPIases. Such modification helps to avoid or suppress host cell immune response; (2) Use of host PPIases (like ROC1 of Arabidopsis thaliana) for modification of effector proteins necessary for pathogenesis. Bacterial protein surA participates in suppression of host immune reaction and can be recognized as elicitor of immune response in other host. SurA is essential for formation of bacterial flagella and pile. We suggest that SurA protein can be involved in activation of some effector proteins, while other effectors, for their activation in host cell, may need PPIases of ROC1-type. Viruses and some other obligate parasites use host PPIases for their own protein modification as an essential step for the pathogen multiplication. In this case modified host PPIases are capable to suppress the pathogenesis. That makes the PPIases potential targets for gene therapy.

Experimental models of primary potato transgenic plants that express the cry3aM-licBM2 hybrid gene were created. The molecular analysis and biotests of the experimental models allow a new system of cry genes expression in plants to be proposed. This system is based on the expression of hybrid genes containing the reporter lichenase gene sequence and the use of a light-induced promoter ensuring preferential expression of the regulated genes only in green plant tissues (leaves), the target tissues for pests, as a regulatory element. In is shown that the presence of lichenase in hybrid proteins facilitates selection and analysis of the level of expression of hybrid proteins in transgenic plants. Judging by the properties of the reporter protein lichenase in hybrid proteins, it seems possible to use this reporter system for transgene monitoring in agrocenosis, because this system is fairly simple and precise and does not need considerable material and time expenses.

Transgenic potato plants have been created that express recombinant proteins that are analogues of spidroin 1, the protein of the cobweb dragline. Expression of spidroin 1 hybrid genes possessing some repeated sequences is retained on going from model test tube-grown plants to crops. The expression level of the synthetic spidroin 1 genes and the level of accumulation of their products in plants depend on the type of promoter, number of repeats, organ specificity, and plant species, but not on the duration of plant material storage. The results show that the strategy based on construction and expression of hybrid proteins including a reporter protein makes it easier to select and analyze the expression of hybrid proteins in transgenic organisms.

Transgenic popato plants have been created which express recombinant proteins, analogues of spidroin 1, the protein of the cobweb skeleton thread. Expression of the hybrid spidroin 1 genes possessing some repeated sequences retains both in the model test-tube-growing plants and in the crops. Expression level of the synthetic spidroin 1 genes and the level of accumulation of their products in plants depend on the type of promoter, number of repeats, organ specificity and plant species but not on the duration of plant material storage. The results show that the strategy based on constuction and expression of hybrid proteins which include the reporter protein makes it easier to select and analyse expression of hybrid proteins in transgenic organisms.

The functional analysis of plant genes employs various comprehensive approaches that include transcriptome analysis using microchips, gene knockout, RNA interference, and various experimental models. We have proposed a novel experimental model approach for plant functional genomics. It is based on creating and studying transgenic plants that express bacterial genes which are functionally similar to plant genes. The validity of this approach owes to the similarity of basic pro- and eukaryotic metabolic pathways and gene networks controlling the functioning of these organisms under normal conditions and in response to abiotic and biotic stresses.Our studies using molecular biology, physiology and biochemistry methods have demonstrated adequateness of the proposed strategy. It allows the modeling of processes taking place in the plant cell and differential assessment of contribution of individual enzymes. Our results indicate that the proposed approach is highly effective for functional genomics, namely, for determining the function of a gene product in vivo. The experimental data can be further used as a basis for elaboration of gene networks controlling particular physiological processes and stress responses of plants to biotic and abiotic environmental factors.

An original yeast system with two reporter genes oriented oppositely to each other was developed to study the regulatory functions of noncoding genomic sequences. A fragment of HERV-K LTR from region 22-19 of human chromosome 7p22 was tested for promoter activity. The LTR 22-19 fragment was shown to initiate transcription of a reporter regardless of its orientation, and to control both reporters simultaneously. The promoter activity was compared for the LTR 22-19 fragment and two potent promoters in Saccharomyces cerevisiae cells. The activity of the LTR 22-19 fragment accounted for about 0.34% of the activity of the inducible GAL1 promoter and for 0.25% of the activity of the constitutive TDH promoter.