Marta Budziszewska

The nucleotide sequence of a Polish isolate of Tomato torrado virus

A new virus was isolated from greenhouse tomato plants showing symptoms of leaf and apex necrosis in Wielkopolska province in Poland in 2003. The observed symptoms and the virus morphology resembled viruses previously reported in Spain called Tomato torrado virus (ToTV) and that in Mexico called Tomato marchitez virus (ToMarV). The complete genome of a Polish isolate Wal’03 was determined using RT-PCR amplification using oligonucleotide primers developed against the ToTV sequences deposited in Genbank, followed by cloning, sequencing, and comparison with the sequence of the type isolate. Phylogenetic analyses, performed on the basis of fragments of polyproteins sequences, established the relationship of Polish isolate Wal’03 with Spanish ToTV and Mexican ToMarV, as well as with other viruses from Sequivirus, Sadwavirus, and Cheravirus genera, reported to be the most similar to the new tomato viruses. Wal’03 genome strands has the same organization and very high homology with the ToTV type isolate, showing only some nucleotide and deduced amino acid changes, in contrast to ToMarV, which was significantly different. The phylogenetic tree clustered aforementioned viruses to the same group, indicating that they have a common origin.

How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by Peanut stunt virus in the presence or absence of its satellite RNA

Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains associated with a satellite RNA (satRNA) modify the symptoms of infected plants and interfere with plant metabolism. We used PSV‐P genomic transcripts (GTs) with and without PSV‐P satRNA and a comparative proteomic 2D‐DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV‐P genomic transcripts‐infected (no satRNA present) and mock‐inoculated plants were compared 29 differentially regulated proteins were found. When comparisons were made for plants infected with PSV‐P‐GT in the presence or absence of satRNA, and for mock‐infected plants and those infected with the satRNA‐associated PSV‐P‐GT, 40 and 60 such proteins, respectively, were found. The presence of satRNA mostly decreased the amounts of the affected host proteins. Proteins involved in photosynthesis and carbohydrate metabolism, for example ferredoxin‐NADP‐reductase and malate dehydrogenase, are among the identified affected proteins in all comparisons. Proteins involved in protein synthesis and degradation were also affected. Such proteins include chaperonin 60β—whose abundance of the proteins changed for all comparisons—and aminopeptidase that is a satRNA‐ or PSV‐P‐GT/satRNA‐responsive protein. Additionally, the levels of the stress‐related proteins superoxide dismutase and acidic endochitinase Q increased in the PSV‐P‐GT‐ and PSV‐P‐GT/satRNA‐infected plants. This study appears to be the first report on plant proteome changes in response to a satRNA presence during viral infection and, as such, may provide a reference for future studies concerning the influence of satRNAs during viral infections.

Construction of infectious clones of tomato torrado virus and their delivery by agroinfiltration

The first biologically active infectious clones of tomato torrado virus (ToTV) were generated and delivered into Nicotiana benthamiana and Solanum lycopersicum plants viaAgrobacterium tumefaciens. The engineered constructs consisted of PCR-amplified complementary DNAs derived from the ToTV RNA1 and RNA2 components, individually inserted into an engineered pGreen binary vector between the CaMV 35S promoter and nopaline synthase terminator. These constructs were introduced into the plant hosts by means of A. tumefaciens-mediated infiltration. In the presence of the progeny virus, typical symptoms of ToTV infection developed in N. benthamiana and S. lycopersicum. Moreover, the virus was sap-transmissible when isolated from agroinfiltrated plants and induced symptoms similar to those caused by the wild-type virus. The presence of viral particles and viral genetic material was confirmed by electron microscopy and re-inoculation to S. lycopersicum and N. benthamiana, as well as by reverse transcription polymerase chain reaction and high-resolution melt analysis.

One-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of tomato torrado virus

Abstract‘Torrado’ disease caused by tomato torrado virus (ToTV) is responsible for considerable losses in tomato production. Therefore, a one-step reverse transcription loop-mediated isothermal amplification protocol for early and fast detection of ToTV isolates has been developed. The RNA extracted from ToTV-infected plants was tested using this protocol with a set of six primers specific for the Vp35 coat protein gene sequence. The amplified products were analyzed using amplification curves, electrophoresis, and direct staining of DNA. The sensitivity of the protocol was tenfold higher than that of conventional RT-PCR. This new protocol is inexpensive, rapid, simple, and very sensitive.

Analysis of two strains of Peanut stunt virus : satRNA-associated and satRNA free

Peanut stunt virus (PSV) is a pathogen of legumes, vegetables, trees, and weeds occurring worldwide. The species is characterized by significant genetic variability. PSV strains are classified into four subgroups on the basis of their nucleotide sequence homology. Here, we are presenting two further, fully sequenced PSV strains—PSV-Ag and PSV-G, that could be considered as I subgroup representatives. However, their sequence homology with other typical I subgroups members, similarly as another strain—PSV-P, characterized by our group previously, is lower than 90%. This lead us to propose further subdivision of the I subgroup into IA, IB, and IC units, and to classify PSV-Ag and PSV-G strains to the last one. In this article, we are showing that identity level of PSV-Ag and PSV-G is very high and apart from the presence of satRNA in the first one, they differ only by a few nucleotides in their genomic RNAs. Nevertheless, symptoms they cause on host plants might differ significantly, just as the levels in infected plants. Effect of single amino acid changes between strains on the three-dimensional structure of viral proteins was analyzed. Differences occur mainly on the protein surfaces which can possibly affect protein–protein interaction in infected cells, which is discussed.

Changes in the expression of mitochondrial cytochrome oxidase subunits due to pyrethroid intoxication in pyrethroid-resistant pollen beetles, Meligethes aeneus (Coleoptera: Nitidulidae)

Pollen beetle (Meligethes aeneus, Coleoptera: Nitidulidae) is the most important pest of oilseed rape in Europe, causing great yield losses. Due to the heavy use of pyrethroid insecticides in controlling Meligethes, a widespread build-up of resistance to pyrethroid active substances has arisen, reported in many countries where the pest occurs. Mutations in the voltage-sensitive sodium channels (VSSC), which constitute the main targets for the pharmacological action, as well as increased oxidative metabolism of pyrethroid active substances in resistant populations are considered, as they are the main molecular mechanisms of the development of pyrethroid resistance.In this study we have analyzed the level of expression of mitochondrial cytochrome oxidase subunit genes (mtCOI, mtCOII) in esfenvalerate-treated populations of M. aeneus collected in 2011 by using the real-time PCR approach. Our results indicate that the esfenvalerate-treated beetles have a significantly higher mtCOI gene expression compared with the untreated ones and that the mtCOII transcript level is also slightly induced. This enhanced expression might, in part, be responsible for the increased oxidative metabolism in pyrethroid-challenged pollen beetle populations.

High Stability of a Mitochondrial Genetic Marker mtCOII in Polish Colorado Potato Beetle Populations

Colorado potato beetle (CPB) (Leptinotarsa decemlineata (Say in Journal of the Academy of Natural Sciences of Philadelphia 3: 298–331, 1824)) (Coleoptera: Chrysomelidae) is one of the most serious potato pests. It has been reported worldwide, from North America to Europe and Asia. In this study we analyzed the genetic diversity of a mitochondrial DNA marker – a second subunit of cytochrome oxidase (mtCOII) in Polish CPB populations to assess the possible changes of this gene sequence over time and over the country, influencing the intra-specific variability of CPB. During a three-year survey in Polish potato fields the beetles were collected from 20 evenly spaced locations of varying climatic and geographic conditions, and the nucleotide sequence of this marker was analyzed. Our research revealed that in spite of three years of sampling the mitochondrial haplotype in all individuals was fixed, and no single nucleotide change was found in any individual, indicating a high stability of this maternally inherited marker in L. decemlineata. This finding about the level of biodiversity is of importance for plant protection strategies.ResumenEl escarabajo de la papa de Colorado (CPB) (Leptinotarsa decemlineata (Say in Journal of the Academy of Natural Sciences of Philadelphia 3: 298–331, 1824)) (Coleoptera: Chrysomelidae) es una de las plagas más serias en papa. Se ha reportado en todo el mundo, desde Norteamérica a Europa y Asia. En este estudio analizamos la diversidad genética de un marcador de ADN mitocondrial – una segunda subunidad de citocromo oxidasa (mtCOII) en poblaciones polacas de CPB para ver los posibles cambios en la secuencia de este gene en el tiempo y en el país, que estuvieran influenciando la variabilidad intraespecífica del CPB. Durante los tres años del estudio en los campos de papa polacos se colectaron los escarabajos de veinte localidades espaciadas uniformemente de condiciones climáticas y geográficas variables, analizando la secuencia de nucleótidos de este marcador. Nuestra investigación reveló que a pesar de los muestreos por tres años, el haplotipo mitocondrial era fijo en todos los individuos, y no se encontró un solo cambio de nucleótido en ningún individuo, indicando una gran estabilidad de este marcador heredado maternalmente en L. decemlineata.