The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1.

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.

Major latex protein-like encoding genes contribute to Rhizoctonia solani defense responses in sugar beet.

Sugar beets are attacked by several pathogens that cause root damages. Rhizoctonia (Greek for "root killer") is one of them. Rhizoctonia root rot has become an increasing problem for sugar beet production and to decrease yield losses agronomical measures are adopted. Here, two partially resistant and two susceptible sugar beet genotypes were used for transcriptome analysis to discover new defense genes to this fungal disease, information to be implemented in molecular resistance breeding. Among 217 transcripts with increased expression at 2 days post-infection (dpi), three resistance-like genes were found. These genes were not significantly elevated at 5 dpi, a time point when increased expression of three Bet v I/Major latex protein (MLP) homologous genes BvMLP1, BvMLP2 and BvML3 was observed in the partially resistant genotypes. Quantitative RT-PCR analysis on diseased sugar beet seedlings validated the activity of BvMLP1 and BvMLP3 observed in the transcriptome during challenge by R. solani. The three BvMLP genes were cloned and overexpressed in Arabidopsis thaliana to further dissect their individual contribution. Transgenic plants were also compared to T-DNA mutants of orthologous MLP genes. Plants overexpressing BvMLP1 and BvMLP3 showed significantly less infection whereas additive effects were seen on Atmlp1/Atmlp3 double mutants. The data suggest that BvMLP1 and BvMLP3 may contribute to the reduction of the Rhizoctonia root rot disease in sugar beet. Impact on the defense reaction from other differential expressed genes observed in the study is discussed.

The RsRlpA Effector Is a Protease Inhibitor Promoting Rhizoctonia solani Virulence through Suppression of the Hypersensitive Response.

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.

[Creation of transgenic sugar beet lines expressing insect pest resistance genes cry1C and cry2A].

Impact of insect pests makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into plant genome is one of the promising strategies to ensure plant resistance. The aim of this work was to obtain sugar beet lines (based on the MM 1/2 line) transformed with cry2A and cry1Cgenes. We have optimized transformation protocol and direct plant let regeneration protocol from leaf explants using 1 mg/l benzylaminopurine as well as 0,25 mg/l benzylaminopurine and 0,1 mg/l indole-butyric acid. Consequently, transgenic sugar beet lines transformed with vector constructs pRD400-cry1C and pRD400-cry2A have been obtained. PCR analysis revealed integration of cry2A and cry1C into genome of transgenic lines and expression of these genes in leaf tissues was shown by reverse transcription PCR.

USE OF GREEN MANURE CROPS AND SUGAR BEET VARIETIES TO CONTROL HETERODERA BETAE.

Although it is less studied than the white beet cyst nematode (Heterodera schachtii), the yellow beet cyst nematode (H. betae) has been found in many countries in Europe. For example in The Netherlands, France and Spain. H. betae causes yield losses on sandy soils. A high infestation can result in loss of complete plants. In The Netherlands, this nematode is especially found in the south eastern and north eastern part, where it occurs on 18% and 5% of the fields, respectively. From a project of the Dutch Sugar beet Research Institute IRS (SUSY) on factors explaining differences in sugar yield, this nematode was one of the most important factors reducing sugar yields on sandy soils. Until 2008, the only way to control H. betae was by reducing the number of host crops in the crop rotation. Host crops are crops belonging to the families of Cruciferae, Chenopodiaceae, Polygonaceae, Caryophyllaceae and Leguminosea. In order to find more control measures, research was done to investigate the host status of different green manure crops and the resistance and tolerance of different sugar beet varieties to H. betae. White mustard (Sinapis alba) and oil seed radish (Raphanus sativus spp. oleiferus) varieties resistant to H. schachtii were investigated for their resistance against H. betae. A climate room trial and a field trial with white mustard and oil seed radish were conducted in 2007 and 2008, respectively. Results show that H. betae could multiply on susceptible white mustard and susceptible oil seed radish, but not on the H. schachtii resistant varieties. In climate room trials in 2009, 2010 and 2011 and field trials in 2010, 2011 and 2012, the effect of different sugar beet varieties on the multiplication of H. betae and the effect of H. betae on yield at different infestation levels was investigated. Sugar beet varieties with resistance genes to H. schachtii (from Beta procumbens or B. maritima) were selected. Varieties with resistance genes from these sources were not totally resistant to H. betae, but limited the multiplication of this nematode in comparison with susceptible varieties considerably. Only the varieties with resistance genes from B. maritima gave higher yields in comparison with susceptible varieties. Growing these varieties was already profitable from very light infestation levels (75 eggs and larvae/100 ml soil) of H. betae. Therefore, resistant cruciferous green manure crops and resistant and tolerant sugar beet varieties are good tools for growers to control H. betae.

Systemic resistance induced by Bacillus lipopeptides in Beta vulgaris reduces infection by the rhizomania disease vector Polymyxa betae.

The control of rhizomania, one of the most important diseases of sugar beet caused by the Beet necrotic yellow vein virus, remains limited to varietal resistance. In this study, we investigated the putative action of Bacillus amylolequifaciens lipopeptides in achieving rhizomania biocontrol through the control of the virus vector Polymyxa betae. Some lipopeptides that are produced by bacteria, especially by plant growth-promoting rhizobacteria, have been found to induce systemic resistance in plants. We tested the impact of the elicitation of systemic resistance in sugar beet through lipopeptides on infection by P. betae. Lipopeptides were shown to effectively induce systemic resistance in both the roots and leaves of sugar beet, resulting in a significant reduction in P. betae infection. This article provides the first evidence that induced systemic resistance can reduce infection of sugar beet by P. betae.

[Activity of phenylalanine-ammonia-lyase in callus cultures of sugar beat infected by Acholeplasma].

The effect of Acholeplasma laidlawii var. granulum 118 on activity of phenylalanine-ammonia-lyase (PAL) in callus cultures of sugar beat was researched. The optimal conditions of enzyme reaction were: using the L-phenilalanine as a substrate, pH 8.4-8.8, the temperature optimum 38-40 degrees C. It was established that at the infecting of sugar beat callus culture by phytopathogenic mollicute the PAL activity was temporarily increased and reached its maximum after 2 h of infecting. Then it gradually decreased and in 24 h reached its initial level. An increase of PAL activity of plant is considered as protective reaction in response to the action of pathogen.

Inducing effect of PGRs on small regulatory si/miRNA in resistance to sugar beet cyst nematode.

Sugar beet cyst nematode Heterodera schachtii Schmidt is an economically important plant parasite of sugar beet in Ukraine. The pest control options are limited. Sugar beet cyst nematode resistant varieties are not available on the market. Carbamate and organophosphate pesticides have been banned due to the high toxicity. The problem is aggravated by continuously increasing of oilseed rape (which is suitable host for H. schachtii) growing area due to biofuel demands. Several studies' results indicate that PGRs have role in management of plant parasitic nematodes but for sugar beet it is not studied well. We had an objective- studying of the role of four compositional PGRs created based of avermectin in suppression of sugar beet cyst nematode population on sugar beet and oilseed rape caused by enhancing of endogenous si/miRNA complementary to H. schachtii mRNA. Laboratory study was conducted in 2011 with using method DOT-blot hybridization si/miRNA with mRNA and by testing inhibitory activity in cell free system protein biosynthesis. That was shown that application of the PGRs enhances sugar beet and oilseeds rape plant immune-protective properties and resistance against plant-parasitic nematode Heterodera schochtii through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the parasitic organisms. As a result, translation of mRNA of the nematode is blocked and causes the mortality of plant parasite juveniles.

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.

Ambivalent effects of defective DNA in beet curly top virus-infected transgenic sugarbeet plants.

Beet curly top virus (BCTV) limits sugarbeet production considerably. Previous studies have shown that infections are associated with the generation of defective DNAs (D-DNA) which may attenuate symptoms. Transgenic sugarbeet lines were established carrying a partial direct repeat construct of D-DNA in order to examine whether they are useful as a means of generating tolerance against BCTV. Thirty four independent transgenic lines were challenged. Viral full-length and D-DNAs were monitored by polymerase chain reaction (PCR) or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP). The differential accumulation of both DNA species was compared with symptom severity during the course of infection. RCA/RFLP allowed the discrimination of two D-DNA classes which were either derived from the transgenic construct (D(0)) or had been generated de novo (D(n)). The statistical analysis of the results showed that the presence of D(0)-DNA correlated with increased symptom severity, whereas D(n)-DNAs correlated with attenuated symptoms.

Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil.

The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.

A survey on basal resistance and riboflavin-induced defense responses of sugar beet against Rhizoctonia solani.

We examined basal defense responses and cytomolecular aspects of riboflavin-induced resistance (IR) in sugar beet-Rhizoctonia solani pathsystem by investigating H(2)O(2) burst, phenolics accumulation and analyzing the expression of phenylalanine ammonia-lyase (PAL) and peroxidase (cprx1) genes. Riboflavin was capable of priming plant defense responses via timely induction of H(2)O(2) production and phenolics accumulation. A correlation was found between induction of resistance by riboflavin and upregulation of PAL and cprx1 which are involved in phenylpropanoid signaling and phenolics metabolism. Application of peroxidase and PAL inhibitors suppressed not only basal resistance, but also riboflavin-IR of sugar beet to the pathogen. Treatment of the leaves with each inhibitor alone or together with riboflavin reduced phenolics accumulation which was correlated with higher level of disease progress. Together, these results demonstrate the indispensability of rapid H(2)O(2) accumulation, phenylpropanoid pathway and phenolics metabolism in basal defense and riboflavin-IR of sugar beet against R. solani.

Characterization of resistance mechanisms to powdery mildew (Erysiphe betae) in beet (Beta vulgaris).

Beet powdery mildew incited by Erysiphe betae is a serious foliar fungal disease of worldwide distribution causing losses of up to 30%. In the present work, we searched for resistance in a germplasm collection of 184 genotypes of Beta vulgaris including fodder (51 genotypes), garden (60 genotypes), leaf (51 genotypes), and sugar (22 genotypes) beet types. Resistant genotypes were identified in the four beet types under study. In addition, mechanisms underlying resistance were dissected through histological studies. These revealed different resistance mechanisms acting at different fungal developmental stages, i.e., penetration resistance, early and late cell death, or posthaustorial resistance. Most genotypes were able to hamper fungal development at several stages. The later are interesting for breeding aiming to resistance durability. Furthermore, characterization of defense mechanisms will be useful for further cellular and molecular studies to unravel the bases of resistance in this species.

Identification of amino acids of the beet necrotic yellow vein virus p25 protein required for induction of the resistance response in leaves of Beta vulgaris plants.

The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.

Plant-derived EpCAM antigen induces protective anti-cancer response.

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.

Cross-reactivity between Parietaria pollen and beet.

Allergy to beet is very rare. Until now, only a few reports about asthma induced by inhaling the vapor of cooked beet have been published. We describe two patients with allergic rhinitis and positive skin prick tests to Parietaria and beet only. To investigate possible cross-reactivity between Parietaria pollen and beet, we performed laboratory assays that showed beet-specific IgE in the sera of both patients and possible cross-reactivity between Parietaria and beet in one patient.

Superoxide dismutase transgenes in sugarbeets confer resistance to oxidative agents and the fungus C. beticola.

Sugarbeets carrying superoxide dismutase transgenes were developed in order to investigate the possibility of enhancing their resistance to oxidative stress. Binary T-DNA vectors carrying the chloroplastic and cytosolic superoxide dismutase genes from tomato, were used for Agrobacterium-mediated transformation of sugarbeet petioles. The transgenic plants were subjected to treatments known to cause oxidative stress, such as the herbicide methyl viologen and a natural photosensitizer toxin produced by the fungus Cercospora beticola, namely cercosporin. The transgenic plants exhibited increased tolerance to methyl viologen, to pure cercosporin, as well as to leaf infection with the fungus C. beticola.

A monoclonal antibody to feruloylated-(1-->4)-beta-D-galactan.

We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(1-->4)-beta-D-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno-assays and immunofluorescence microscopy indicated that LM9 binding is specific to samples and cell walls obtained from species belonging to this family. In a series of competitive-inhibition enzyme-linked immunosorbent assays with potential oligosaccharide haptens, the most effective inhibitor was O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose (Gal2F). LM9 is therefore a useful antibody probe for the analysis of phenolic substitution of cell wall pectic polymers and of cell wall structure in the Amaranthaceae including sugar beet (Beta vulgaris L.) and spinach (Spinacia oleracea L.).