The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.     

Strategies for the Detection of Potential Beet necrotic yellow vein virus Genome Recombinations which might Arise as a Result of Growing A type Coat Protein Gene-expressing Sugarbeets in Soil Containing B Type Virus

We have searched for beet necrotic yellow vein virus (BNYVV) populations with a recombined genome which could possibly arise when transgenic sugarbeets expressing the coat protein gene of A type BNYVV are grown in soil containing Polymyxa betae carrying B type BNYVV, in soil samples from previous field release experiments and in a greenhouse model experiment. In order to accelerate the potential evolution of virus populations with recombined genomes in the model experiment, eight successive crops of sugarbeet plantlets were grown in the same soil samples over a period of 3 years. For the sensitive detection of recombined BNYVV genomes, we used nested PCRs with sense primers that are preferentially extended on the A type BNYVV sequence in the region of the coat protein gene and antisense primers which are preferentially extended on the B type BNYVV sequence in a region downstream of the coat protein gene which is not present in the transgene. Controls with mixtures of sap from plants which were singly infected with A or with B type BNYVV only revealed that, unless proper precautions are taken, PCR-mediated recombination artifacts may readily be produced. A method was developed that is able to detect A type/B type recombinant RNA molecules up to dilutions of one to a million in pure B type RNA molecules. Inspite of this high sensitivity we failed to detect any BNYVV with a recombined genome in the transgenic plants of the model experiment or at the sites of the previous field release experiments.     

Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

甜菜坏死黄脉病毒内蒙分离物自然缺失突变体缺失区域的定位分析

以甜菜坏死黄脉病毒内蒙分离物（ＢＮＹＶＶ ＮＭ）总ＲＮＡ为模板,经ＲＴ－ＰＣＲ扩增,分别获得ＲＮＡ２、ＲＮＡ３和ＲＮＡ４自然缺失突变体ｃＤＮＡ克隆。序列分析结果表明,ＲＮＡ２自然缺失突变体在７５ｋＤ通读蛋白编码区Ｃ端缺失３４８个核苷酸（缺失位置ｎｔ１４８８￣ｎｔ１８３５）。ＲＮＡ３在其２５ｋＤ蛋白编码区内缺失３６０个核苷酸（缺失位置ｎｔ７２９￣ｎｔ１０８８）。ＲＮＡ４的自然缺失区域位于３１ｋＤ蛋白     

Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.     

Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon

Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the boundary mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV.     

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.     

Two-component signal transduction systems and regulation of virulence factors in <Emphasis Type=Italic>Xanthomonas</Emphasis>: a perspective

Two-component signal transduction systems (TCSTSs), consisting of a histidine kinase and a response regulator, play a critical role in regulating virulence gene expression in Gram-negative phytopathogenic bacteria Xanthomonas spp.. To date, 12 TCSTS genes have been identified, accounting for approximately 10% of the TCSTS genes in each genome that have been experimentally identified to be related to pathogenesis. These TCSTSs modulate the expression of a number of virulence factors through diverse molecular mechanisms such as interacting with DNA, protein-binding and involvement in second messenger metabolism, which generates a high level of regulatory versatility. Here we summarize the current knowledge in this field and discuss the emerging themes and remaining questions that are important in deciphering the signaling network of TCSTSs in Xanthomonas.     

Erwinia amylovora catalases KatA and KatG are virulence factors and delay the starvation‐induced viable but non‐culturable (VBNC) response

The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H₂O₂) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H₂O₂‐neutralizing enzymes, such as catalases, during plant–pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non‐host environments was determined using catalase gene mutants and expression, as well as catalase activity analyses. The participation of E. amylovora exopolysaccharides (EPSs) in oxidative stress protection was also investigated. Our study revealed the following: (i) a different growth phase regulation of each catalase, with an induction by H₂O₂ and host tissues; (ii) the significant role of E. amylovora catalases as virulence and survival factors during plant–pathogen interactions; (iii) the induction of EPSs by H₂O₂ despite the fact that apparently they do not contribute to protection against this compound; and (iv) the participation of both catalases in the detoxification of the starvation‐induced intracellular oxidative stress, favouring the maintenance of culturability, and hence delaying the development of the viable but non‐culturable (VBNC) response.     

Improved myocardial perfusion in chronic diabetic mice by the up‐regulation of pLKB1 and AMPK signaling

Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up‐regulating pAMPK‐pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild‐type and streptozotocin (STZ)‐treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO‐1, 3‐nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time‐dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L‐NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK‐3 expression. Cobalt protoporphyrin treatment to induce HO‐1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up‐regulation of HO‐1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK‐3, and peNOS levels and a decrease in myocardial superoxide and 3‐nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes. J. Cell. Biochem. 109: 1033–1044, 2010. © 2010 Wiley‐Liss, Inc.     

INPP1 up‐regulation by miR‐27a contributes to the growth,migration and invasion of human cervical cancer

Inositol polyphosphate‐1‐phosphatase (INPP1) is an enzyme that is responsible for glycolysis and lipid metabolism. Here, we discovered that INPP1 expression was up‐regulated in CC tissues compared to that in adjacent normal tissues by RT‐qPCR. Inositol polyphosphate‐1‐phosphatase overexpression promoted and INPP1 knockdown suppressed cell viability, cellular migration/invasion and EMT in CC cells. To explore the mechanism of dysregulation, INPP1 was predicted to be a target of miR‐27a, and a pmiRGLO dual‐luciferase reporter assay showed that miR‐27a bound to the 3′ UTR of INPP1. RT‐qPCR revealed that miR‐27a was also up‐regulated and had a positive correlation with INPP1 expression in CC tissues. Furthermore, shR‐INPP1 could favour the malignant phenotype reversion induced by miR‐27a, suggesting that miR‐27a up‐regulates INPP1 to promote tumorigenic activities. Altogether, our findings show that the up‐regulation of INPP1 by miR‐27a contributes to tumorigenic activities and may provide a potential biomarker for CC.