BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach.

Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species.

High level resistance against rhizomania disease by simultaneously integrating two distinct defense mechanisms.

With the aim of achieving durable resistance against rhizomania disease of sugar beet, the employment of different sources of resistance to Beet necrotic yellow vein virus was pursued. To this purpose, Nicotiana benthamiana transgenic plants that simultaneously produce dsRNA originating from a conserved region of the BNYVV replicase gene and the HrpZ(Psph) protein in a secreted form (SP/HrpZ(Psph)) were produced. The integration and expression of both transgenes as well as proper production of the harpin protein were verified in all primary transformants and selfed progeny (T1, T2). Transgenic resistance was assessed by BNYVV-challenge inoculation on T2 progeny by scoring disease symptoms and DAS-ELISA at 20 and 30 dpi. Transgenic lines possessing single transformation events for both transgenes as well as wild type plants were included in inoculation experiments. Transgenic plants were highly resistant to virus infection, whereas in some cases immunity was achieved. In all cases, the resistant phenotype of transgenic plants carrying both transgenes was superior in comparison with the ones carrying a single transgene. Collectively, our findings demonstrate, for a first time, that the combination of two entirely different resistance mechanisms provide high level resistance or even immunity against the virus. Such a novel approach is anticipated to prevent a rapid virus adaptation that could potentially lead to the emergence of isolates with resistance breaking properties.

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Description and analysis of genetic diversity among squash accessions

In this work, the part of the squash core collection, maintained in the Greek Gene Bank, was assessed using the morphological and molecular data. Sixteen incompletely classified accessions of the squash were characterized along with an evaluation of their resistance against two isolates of Fusarium oxysporum. A molecular analysis using Random Amplified Polymorphic DNA (RAPD) markers was also performed, revealing high level of polymorphism. To study the genetic diversity among the squash accessions, a clustering procedure using Unweighed Pair Group Method and Arithmetic Average (UPGMA) algorithm was also adopted. Two independent dendrograms, one for the morphophysiological and one for molecular data were obtained, classifying the accessions into two and three main clusters, respectively. Despite the different number of the clusters there were many similarities between these two dendrograms, and a third dendrogram resulting from their combination was also produced, based on Gower's distance and UPGMA clustering algorithm. In order to determine the optimal number of clusters, the upper tail approach was applied. The more reliable clustering of the accessions was accomplished using RAPD markers as well as the combination of the two different data sets, classifying the accessions into three significantly different groups. These groups corresponded to the three different cultivated species of C. maxima Duch., C. moschata Duch., and C. pepo L. The same results were also obtained using Principal Component Analysis.

A abobrinha de inverno compõe um cultivo agrícola com valor econômico determinado exercendo, no entanto, um papel importante em zonas caracterizadas por um cultivo menos intensivo. Na Grécia, o cultivo da abobrinha se baseia, principalmente, em variedades locais conservadas a muitos anos por agricultores locais. Uma parte do cultivo nuclear da abobrinha, que é conservada pelo Banco Grego de Genes, foi melhorada utilizando-se dados morfológicos e moleculares, especialmente dezesseis cultivos de abobrinha classificados incompletamente, que foram diferenciados apenas com base em características morfológicas, em relação a uma avaliação à resistência contra o Fusarium Oxysporum, em dois isolamentos. Foi realizada uma análise molecular utilizando DNA Polimórficos Casual Amplificados índices (RAPDs), revelando um alto nível de polimorfismo. Para estudar a diversidade genética entre a coleção de abobrinhas, um procedimento de agrupamento foi realizado usando-se o algoritmo U.P.G.M.A. Dois dendrogramas independentes, um morfofisiológico e outro para dados moleculares, foram coletados, classificando as coleções em dois e três grupos básicos, respectivamente. Apesar do número diferente dos grupos, foram introduzidas muitas semelhanças entre os dois dendrogramas e um terceiro dendrograma foi produzido como resultado da combinação dos dois primeiros, baseado na distância de Gower e no algoritmo de agrupamento U.P.G.M.A. Para determinar o número ótimo dos grupos, a aproximação "upper tail" foi aplicada. O grupo mais aceitável das coleções foi conseguido usando-se índices RAPD, assim como a combinação dos dois grupos de dados diferentes, classificando as coleções em três grupos consideravelmente diferentes. Os grupos que correspondem às três espécies cultivadas diferentemente, que correspondem às três espécies cultivadas diferentemente por C.máxima Duch., C.moschata Duch. e C. pepo L. além disso, os mesmos resultados foram conseguidos usando-se a "Principal...