George N. Skaracis

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 (HrpZPsph). 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 HrpZPsph 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/HrpZPsph 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/HrpZPsph 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 Gowers 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.

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.

Efficiency of Rz1-based rhizomania resistance and molecular studies on BNYVV isolates from sugar beet cultivation in Greece

A survey was carried out to investigate the current situation concerning rhizomania disease incidence in sugar beet cultivation of Greece. A systematic field evaluation over locations and years revealed a consistent disease severity pattern according to favourable agroclimatic conditions and pointed to the so far effectiveness of the Rz1 gene-based resistance, as no major disease outbreaks were observed. Molecular analyses aiming at the characterization of the type and genetic diversity of the virus further confirmed the widespread occurrence of BNYVV in the country, as evidenced by RT-PCR amplification of all five known genomic molecules and nested-PCR assays. None of the isolates contained an RNA 5, typically found in pathotype P. On the basis of RFLP patterns, all BNYVV isolates analysed were classified as pathotype A. Sequence determination of the full-length RNA 3-encoded p25 protein, responsible for symptom development, revealed amino acid motifs ACHG/VCHG in the hypervariable region aa67–70. The presence of valine in position 67 did not appear associated with increased pathogenicity and resistance breaking properties, as earlier reported.

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 HrpZPsph protein in a secreted form (SP/HrpZPsph) 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.

Cercospora Leaf Spot Disease of Sugar Beet

Cercospora leaf spot, incited by the fungus Cercospora beticola Sacc., is the most widespread and damaging sugar beet (Beta vulgaris L.) foliar disease. Under favorable for the disease agro-climatic conditions, insufficient control of epidemics leads to significant sugar yield reduction and consequent heavy economic losses. Disease control strategies basically rely on a combination of fungicide applications, growing resistant cultivars, and appropriate crop rotation. The need for a more efficient and sustainable disease management, primarily dictates the production of varieties with elevated genetic resistance and the rational use of chemical treatments to avoid the development of pathogen resistance to fungicides. Both classical and molecular breeding approaches are followed to obtain better resisting varieties, the main aims being the exploitation of additional genetic variation from natural gene pools as well as the incorporation of novel resistance traits through genetic engineering. Alternating and combining fungicides differing in mode of action, restricted use of chemicals easily provoking pathogen resistance as well as systematic risk assessment for resistance development when incorporating any novel compound, could ensure a prolonged effectiveness of the spraying programs. Designing and employing various effective integrated pest management systems is hoped to contribute towards a more effective and environmentally sound disease control.

Genetic diversity in four cabbage populations based on random amplified polymorphic DNA markers

Genetic diversity in four local Greek cabbage open-pollinated populations was investigated using RAPD (Random Amplified Polymorphic DNA) DNA markers in 18 individual plants from each population. A total of 24 random primers detected 90 polymorphic bands in the four populations studied, with an average of 3·75 bands/primer. The mean between-population differentiation was close to 40%, leaving 60% for within-population diversity. The individual plants were grouped, based on the Jaccard coefficient, by clustering (Unweighted Pair Group Method and Arithmetic Average – UPGMA) and an ordination (Principal Coordinates Analysis – PCO) methods, resulting in 7 and 6 groups, respectively. In general, there was a notable similarity in the grouping of the individuals with these two methods. In addition, Neis standard genetic distance between populations, as calculated on the basis of within-population gene frequencies, was employed to group the populations by the UPGMA method. Clustering results were in good agreement with previously reported results based on morphological descriptors applied to the same populations. It was concluded that RAPD markers could be exploited as alternative or supplementary tools to already established methods for the evaluation and classification of cabbage genetic resources.

The Sea Beet (Beta vulgaris L. ssp. maritima) of the Adriatic Coast as Source of Resistance for Sugar Beet

ConclusionsThe sea beet is considered the ancestral species from which the different types of cultivated beet originate. Its hybridation is easy because of its evolutionary proximity, unlike with other species of the GenusBeta. These traits have long made the sea beet the subject of research aimed at targeting and isolating its useful characters which could then be transferred to cultivated beet.The sea beet collected at the mouth of the Po di Levante river in the early 1900s has given more appreciable results than any of the other biotypes from different origins. Indeed, it was from this biotype that today’s most developed genetic resistance to serious diseases such as cercospora and rhizomania was isolated.

Development of a quantitative analysis protocol of heat shock protein 70 in heat stressed sugar beet (Beta vulgaris L.) plants

Quantitative real-time PCR (RT-PCR) has already been used to study the expression profile of several plant gene families (Yokoyama and Nishitani, 2001; Mladek et al., 2003; Charrier et al., 2002). This study demonstrates the potential of RT-PCR to measure the expression of heat shock protein (HSP) genes in sugar beet. Young plants underwent heat stress for 5.5 h followed by RT-PCR ofhsp70 mRNA using the housekeeping gene tubulin (tub) as a reference gene and the fluorogenic dye SYBR Green I. Expression ofhsp70 mRNA peaked at 1 h of heat stress and decreased at 5.5 h. This method proves very sensitive, quantitating the targethsp70 transcript in 6.25 ng total RNA.

Global metabolomics analysis reveals distinctive tolerance mechanisms in different plant organs of lentil (Lens culinaris) upon salinity stress

Background and AimsOmic technologies in the past years have provided a variety of data in model plants. In legumes, results οn Lotus japonicus and Medicago truncatula have highlighted the biochemistry which takes place inside cells under a variety of abiotic stresses. Here we conducted metabolomics in the forage legume lentil (Lens culinaris) upon salinity stress on acclimated and non-acclimated plants and compared results from leaf and root analyses.MethodsWe used two lentil varieties, originated from different geographical locations and studied differences in their global metabolite profile i) using gradual or initial application of salt stress, ii) between leaves and roots, and iii) between the varieties.ResultsMost important differences were noted in salinity induced diminished abundance of organic acids in both varieties’ leaves and roots, accumulation of sugars and polyols in leaves, and accumulation of other key-metabolites, such as L-asparagine, D-trehalose, allantoin and urea in the roots. We also demonstrated the driver of deleterious Cl− accumulation in leaves for potential compartmentalization in the vacuole, a defensive mechanism for withstanding salinity stress in plants. Finally, a model is suggested of how legumes upregulate a metabolic pathway, which involves purines catabolism in order to assimilate carbon and nitrogen, which are limited during salinity stress.ConclusionsFuture omics works with lentil can help understanding the regulation of the biochemical “arsenal” against abiotic stresses such as salinity and render the selection of better crops.