Background: RNA silencing-based antiviral breeding is a promising strategy for developing VIRUS-resistant plants. Objectives: This study employed viral sense, anti-sense, and hairpin constructs to induce resistance against BEET CURLY TOP VIRUS (BCTV) and BEET CURLY TOP Iran VIRUS (BCTIV). Materials and Methods: For this purpose, a 120-bp conserved sequence of Rep- and C2-BCTV and a 222-bp conserved sequence of CP-, Reg-, and MP-BCTIV were selected for construct production.  The efficiency of constructs was investigated in transient expression in Nicotiana benthamiana and sugar BEET plants and stable expression in N. benthamiana. Results: In transient expression, all designed constructs induced effective resistance to BCTV and BCTIV; the hairpin constructs were more effective against both VIRUSes.  The stability of the achieved resistance by hairpin constructs was also confirmed in the T1 generation of transgenic plants. Conclusions: This study showed that employing conserved coding sequences of BCTVs leads to effective resistance against BCTVs infection.  The lack of protein production from transgene and degradation of its transcript due to the gene silencing mechanism makes this method safe for biosecurity.  In stable transformation, the inheritance of induced resistance against BCTVs was confirmed in the T1 generation.  These advantages make this mechanism commercially useful for the production of resistant plants to VIRUSes.

Objective BEET CURLY TOP Iran VIRUS (BCTIV) is a destructive VIRUS causing damage to agriculture industry annually. BCTIV has a circular single-stranded DNA genome with five open reading frames (ORFs): V1, V2 and V3 on genomic strand, and C1 and C2 on complementary strand. V2 acts as a suppressor of silencing interfering with this defense pathway. This study was aimed to determine the evolutionary characteristics and genetic structure of V2 among different BCTIV isolates. Materials and methods Total number of 31 BCTIV V2 nucleotide sequences were extracted from the GenBank and analyzed. The nucleotide sequence was aligned and the phylogenetic tree was drawn. Nucleotide polymorphism analysis was determined. Also, the occurrence of insertion-deletion polymorphism (InDel) was investigated. Nonsynonymous (dN) and synonymous (dS) substitution rates and dN/dS ratio were calculated in order to check the selection pressure on nucleotide sequences and V2 codons. Entropy analysis was also performed to investigate possible variations in nucleotide positions. Results Based on the host and geographical location, V2 sequences were placed in different clusters of the phylogenetic tree. 20 haplotypes and 94 polymorphic loci was detected. Haplotype and nucleotide diversity was calculated as 0.963 and 0.06517, respectively. The mean nucleotide differences was 23.463. InDel polymorphism was not observed. The rates of dN and dS were calculated as -0.60699 and 0.03020, respectively, both of which were not significant. The dN/dS ratio was also -20.10201. Total number of 26 positions among the codons showed a positive value of dN/dS ratio, while 34 positions had a negative value. Also, at least 9 recombination events were observed. Entropy analysis showed that nucleotide position 247 has the most changes with an entropy rate of 0.93. Conclusions The results suggest that the variation in the nucleotide sequence of BCTIV V2 can be effective in suppressing gene silencing and the pathogenicity of different isolates in different hosts. The variability of this part of the VIRUS genome may in the future lead to an increase in the range of host range of the VIRUS or to overcome the resistance of plant varieties.

Background and Objectives: BEET CURLY TOP Iran VIRUS (BCTIV) and BEET CURLY TOP VIRUS (BCTV) are the causal agents of BEET CURLY TOP disease in Iran. It has been reported that both VIRUSes are distributed in different geographical regions and host plants in Iran. Development of a simple, fast and low cost serological method such as ELISA for detection of these VIRUSes, is a necessary demand. To do serological tests such as ELISA, an antibody is needed. Production of polyclonal antibodies against geminiVIRUSes is a hard candidate because of low concentration and localization of their particles in the phloem tissue of their hosts. Therefore, expression and production of geminiviral recombinant proteins in bacterial system is an alternative method to obtain polyclonal antibodies. Material and Methods: Specific forward and reverse primers containing BamHI/HindIII digestion sites at the 5′ end of each primer, respectively, were designed to amplify the full-length fragment of BCTIV V1 open reading frame encoding the viral coat protein (CP). The amplified 751 bp fragment was cloned into the pTZ57R/T cloning vector. V1 gene was released from pTZ57R/T by enzymatic digestion and cloned into the expression vector pQE30. The recombinant pQE30-BCTIV V1 was transformed into Escherichia coli strain M15. The expression of protein was induced by adding 1 mM of isopropylthio-d-galactoside (IPTG) to the transformed bacterial cells and grown at 37 º C in liquid LB medium. Expression of recombinant protein was analyzed by SDS-PAGE electrophoresis and Western blot hybridization. Results: Analysis of the expressed proteins by SDS-PAGE electrophoresis, revealed a protein band with a position corresponding to molecular weight of approximately 30 kDa that was consistent with the predicted molecular weight of BCTIV CP. Time course of induction by IPTG influenced on level of the new recombinant protein expression, thus four hours induction had the highest level of expression. Western blot analysis confirmed the identity of expressed protein via using anti-His antibody to detect expressed recombinant His-BCTIV CP. Discussion: One of the most popular bacterial protein expression system is IPTG inducible system wherein IPTG regulate level of new recombinant protein expression. Our results by SDS-PAGE electrophoresis and Western blot analysis revealed a high level of expression of the fusion protein in IPTG-induced bacterial cells. However, a negligible rate of expression of recombinant protein was also observed in non-induced bacterial cells. This suggests low appearance and activity of responsible promotor even without inducing protein expression with IPTG. Expression of conserved plant viral proteins in bacterial systems following purification of the resulted recombinant proteins would facilitate production of specific polyclonal antibodies.

BEET CURLY TOP is a disease of sugar BEET which has been in the center of attention due to its unpredictable epidemics and its economic importance. It has been shown that due to short life cycle and high populations of the leafhopper vector, the disease often spreads very rapidly in a region. At least five VIRUS species of the genus CurtoVIRUS (Geminiviridae) are recognized as the cause of BEET CURLY TOP disease. Two of these species, namely, BEET severe CURLY TOP VIRUS (BSCTV) and BEET CURLY TOP Iran VIRUS (BCTIV) have been reported from Iran. Two leafhopper species, Circulifer haematoceps and C. tenellus, have been reported as the vector of BEET CURLY TOP VIRUS before recognition of different species (Fattahi et al., 2010). Therefore, it was not certain whether one or both VIRUSes are transmitted by the leafhoppers. Recently, it was shown that C. haematoceps could transmit BSCTV from sugar BEET plants infected via an infectious clone of the VIRUS. In the present study, the possibility of transmission of BCTIV by C. haematoceps was examined. Leafhoppers were collected in sugar BEET fields of northern Fars province using D-Vac. The dominant species was C. haematoceps. The leafhoppers were allowed to breed on healthy sugar BEET in the greenhouse. The newly born nymphs were placed on sugar BEET plants previously agroinoculated with an infectious clone of BCTIV (Zarghan isolate) for an acquisition access period of four days, and then transferred to healthy seedlings at 2-leaf stage (5 leafhoppers/3 seedlings). Typical symptoms of CURLY TOP appeared on all inoculated seedlings after two weeks. Presence of BCTIV in symptomatic test plants was verified by PCR using specific BCTIV primers. The experiment was repeated two more times with similar results. It is concluded that like BSCTV, BCTIV is transmitted by C. haematoceps under greenhouse condition.

In order to study the response of new sugar BEET hybrids and cultivars to CURLY TOP disease in the field conditions, an experiment was carried out in a randomized complete block design with four replications and sixteen treatments in 2007 in Fasa. Each plot included a hybrid or cultivar planted in three rows with eight meter length and 50cm between- row spacing. After weeding and first thinning, percentages of the infected plants were calculated based on the morphological characteristics of the young plants. Disease index was also measured for each treatment. At the end of growth period, the quantity (root yield and white sugar yield) and quality (sugar content) of in all treatments were measured and compared. The results showed that there was a significant difference in the percentage of infected plants and disease index among the hybrids and cultivars (a=1%). Two hybrids 28957 and 28914, had the lowest infection percentage and disease index of 3.970, 1.175 and 3.940, 1.280 percentages respectively. Cultivars H2301, BRANCO and CHINOOK had the lowest infection percentage and severity to CURLY TOP. There was also a significant difference among the hybrids and cultivars regarding root yield, white sugar yield and sugar content (a=1%). The highest root yields (78.57, 77.98 and 71.90 tha-1) and white sugar yield (11.75, 12.27 and 11.55 tha-1) were recorded for H2301, BRANCO and CHINOOK cultivars, respectively. The hybrid 28957 with 9.30 tha-1 white sugar yield was superior to the other hybrids. The maximum sugar content obtained from the cultivar CHINOOK and hybrid 28910, 18.39% and 18.23%, respectively. Therefore the hybrid 28957 are recommended as two CURLY TOP tolerant hybrids for producing resistant cultivars.

BEET CURLY TOP Iran VIRUS (BCTIV, genus BecurtoVIRUS) and BEET severe CURLY TOP VIRUS (BSCTV-IR, genus CurtoVIRUS) induce BEET CURLY TOP disease in different regions of Iran. In addition to sugar BEET, tomato and pepper are important hosts of these VIRUSes. Also tomato yellow leaf curl disease (TYLCD) is a serious disease of tomato in Iran. The Iranian isolate of Tomato yellow leaf curl VIRUS (TYLCV-[Ab]), a severe strain of TYLCV (genus BegomoVIRUS), is a major component of TYLCD in southern Iran. Until now, no information is available regarding comparative host range of these VIRUSes. To determine the natural hosts of BSCTV-IR, BCTIV and TYLCV-[Ab], samples of sugar BEET, tomato, pepper, turnip and spinach were collected from different regions in Fars province. For the experimental host range determination, a number of plants were grown in the greenhouse and their seedlings were agroinoculated with the infectious clone of each VIRUS. The results showed that BSCTV-IR has a wider natural and experimental host range compared to BCTIV. These results indicated that member of Solanaceae, Brassicaceae, Fabaceae and Amaranthaceae are most frequently infected by BSCTV-IR and BCTIV. Natural host range of TYLCV-[Ab] seemed to be very narrow as, in the present study, this VIRUS was isolated only from tomato. However, under greenhouse conditions, bean, nasturtium, petunia, redroot pigweed, datura, night shade and ground cherry were found to be infected by this VIRUS.

In order to evaluate the effect of sugar BEET genotype and method of plant inoculation on resistance to Iranian isolate of BEET severe CURLY TOP VIRUS (BSCTV-Ir), a completely randomized experimental design was performed using 18 sugar BEET cultivars. Eighteen plants of each cultivar were grown in 6 pots. Two methods of inoculation, namely, agroinoculation using the infectious clone of BSCTVIr and inoculation by viruliferous leafhopper (Circulifer haematoceps) were used. The VIRUS replication in inoculated plants was analyzed by PCR, 21 and 35 days post-inoculation (dpi) in the first experiment and 21 dpi in the second experiment. The severity of disease symptoms of inoculated plants was graded using a 0-3 scale (0, symptomless; 1, mild; 2, moderate and 3 severe symptoms). Data analysis of the first experiment indicated that the cv Brigita was the only cultivar in which all the inoculated plants were infected by the VIRUS and all of them showed severe symptoms. Consequently, this cultivar was considered as a highly sensitive control cultivar and used in comparisons. Statistical analysis of the disease symptom severity index showed highly significant differences among cultivars when compared with the control cultivar. As a result, three distinct groups were identified. The first group including the cultivars BR1, FIMMA, HM1990, 7233, and H5505, and the second group including the cultivars Rasoul, Afshari, hybrid of Balk Shiraz, P.P.8, P.P.22, Dorothea, Zarghan, Rhizofouret, IC, Flores, Hilma and Polyrow were regarded as tolerant and sensitive groups, respectively. The third group included only Brigita cultivar which was regarded as very sensitive. Comparison of the data obtained from two inoculation methods indicated that in most cultivars, the VIRUS infection rate was lower with vector inoculation than with agroinoculation.