Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid.

Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in "Rutgers" tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage; (ii) early symptoms; (iii) full spectrum of symptoms and (iv) the so-called 'recovery' stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis.

Viability and genetic stability of potato spindle tuber viroid mutants with indels in specific loops of the rod-like secondary structure.

Maintenance of the rod-like structure of potato spindle tuber viroid (PSTVd), which contains over 20 loops and bulges between double-stranded helices, is important for viroid biology. To study tolerance to modifications of the stem-loop structures and PSTVd capacity for mutation repair, we have created 6 mutants carrying 3-4 nucleotides deletions or insertions at three unique restriction sites, EagI, StyI and AvaII. Differences in the infectivity of these in vitro generated PSTVd mutants can result from where the mutations map, as well as from the extent to which the secondary structure of the molecule is affected. Deletion or insertion of 4 nucleotides at the EagI and StyI sites led to loss of infectivity. However, mutants with deletion (PSTVd-Ava-del) or insertion (PSTVd-Ava-in) of 3 nucleotides (221GAC223), at the AvaII site (loop 20) were viable but not genetically stable. In all analyzed plants, reversion to the wild type PSTVd-S23 sequence was observed for the PSTVd-Ava-in mutant a few weeks after agroinfiltration. Analysis of PSTVd-Ava-del progeny allowed the identification of 10 new sequence variants carrying various modifications, some of them having retained the original three nucleotide deletion at the AvaII site. Interestingly, other variants gained three nucleotides in the deletion site but did not revert to the original wild type sequence. The genetic stability of the progeny PSTVd-Ava-del sequence variants was evaluated in tomato leaves (early infection) and in both leaves and roots (late infection), respectively.

Electrochemical label-free and reagentless genosensor based on an ion barrier switch-off system for DNA sequence-specific detection of the avian influenza virus.

This paper concerns the development of genosensors based on redox-active monolayers incorporating (dipyrromethene)2Cu(II) and (dipyrromethene)2Co(II) complexes formed step by step on a gold electrode surface. They were applied for electrochemical determination of oligonucleotide sequences related to avian influenza virus (AIV) type H5N1. A 20-mer probe (NH2-NC3) was covalently attached to the gold electrode surface via a reaction performed in the presence of ethyl(dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC/NHS) between the amine group present in the probe and carboxylic groups present on the surface of the redox-active layer. Each modification step has been controlled with Osteryoung square-wave voltammetry. The genosensor incorporating the (dipyrromethene)2Cu(II) complex was able to detect a fully complementary single-stranded DNA target with a detection limit of 1.39 pM. A linear dynamic range was observed from 1 to 10 pM. This genosensor displays good discrimination between three single-stranded DNA targets studied: fully complementary, partially complementary (with only six complementary bases), and totally noncomplementary to the probe. When the (dipyrromethene)2Co(II) complex was applied, a detection limit of 1.28 pM for the fully complementary target was obtained. However, this genosensor was not able to discriminate partially complementary and totally noncomplementary oligonucleotide sequences to the probe. Electrochemical measurements, using both types of genosensors in the presence of different supporting electrolytes, were performed in order to elaborate a new mechanism of analytical signal generation based on an ion barrier "switch-off" system.

Influence of cooking and microwave heating on microstructure and mechanical properties of transgenic potatoes.

The transgenic potato clones of cultivar Irga with improved resistance to a necrotic strain of potato virus Y (PVY(N)) were subjected to heat treatment in order to determine their technological quality. The technological quality was determined on the basis of differences between mechanical properties of unmodified potato and transgenic clones during cooking and microwave heating. The compression test was applied in order to evaluate the mechanical resistance of raw, cooked and microwave-treated potatoes. Compression resistance was expressed by fracture stress F (kPa), fracture strain D (mm/mm), and Young modulus E (kPa). The differences in microstructure of potato tubers (unmodified and modified) were investigated using scanning electron microscopy (SEM). The observed differentiation in the mechanical properties of heat-treated potatoes was less connected with genetic modification but most of all with a kind of the process used. The heat processes caused a distinct decrease in mechanical resistance in all the examined tubers. However, the process of microwave heating resulted in more significant changes in mechanical properties of tubers than cooking. Deformation of parenchyma cells during cooking was directly connected with starch, gelatinisation and gel formation. Microwave heating affected significantly cellular water evaporation which resulted in intercellular failure, collapsing of cells, and limitation of starch gelatinisation.

The ORFO product of Potato leafroll virus is indispensable for virus accumulation.

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.