Serological Characterization Of Citrus Tristeza Virus And The Identification Of Differentially Expressed Genes Induced By CTV In Citrus Aurantifolia

Citrus play an importantlly economical role in the fruit industry in the world. Citrus tristeza, caused by Citrus tristeza virus (CTV) is one of the most devastating and economically important viral diseases of citrus production worldwide. In many contries, measures to control losses caused by CTV include using of virus-free seedlings, utilizing mild strain cross protection (MSCP) and plant quarantine systems to prevent CTV spread. However, CTV detection is an important step in these measures. The serological method is widely used for virus detection in fruit trees, due to its high sensitivity, speediness, economy, and adapting for detection of a large of samples. It has been proved that CTV has several different isolates that produce significantly different symptoms, however, the characteristics of CTV-host interactions are poorly understood.In this paper, polyclonal and and monoclonal antibodies against Chinese CTV isolates were prepared by the modified protocol for purifying CTV, and their efficiencyfor detecting virus was analyzed. In addition, the effect of CTV infection on the growth, morphogenesis and gene expression of in-vitro cultured citrus were analyzed. The aim of this paper was to provide effective reagents for establish high-through and fast CTV detection techniques and establish the foundation for further studying the properties of Chinese CTV strains, screening the mild strains in our country, researching into the virus-host interactions and identifying the resistant-related genes. The main results obtained are listed below:1. CTV was purified from sweet orange samples (Citrus sinensis) L5 and S4 that induced stem pitting on branches by a modified protocol, the yield of purified virions was about 1 mg from 100 g citrus tissues. Polyclonal antibody (PAb-L5) was prepared by immunizing rabbits with the purified CTV preparation from L5 samples, and the titer was 1:25 600 in indirect ELISA test. Eighteen hybridoma-cell lines secreting monoclonal antibodies (MAbs-S4) against CTV were screened atter the fusion of mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the virus preparation from S4 samples. The titers of 18 ascetic fluids against these hybridoma cell lines ranged from 1: 12 800 to 1: 204 800 in indirect ELISA. Four hybridoma-cell lines were selected randomly for later analysis. The results indicated that the epitopes for four MAbs were different, and their isotypes and subclasses were IgG2a for 2G and 3H and IgG2b for 1E and 4H. These four Mabs were used to detect CTV in citrus samples in different sources. Results showed that TAS-ELISA with polyclonal antibody as trapping antibody and monoclonal antibody as testing antibody had a higher specificity and sensitivity than PAS-ELISA. Four Mabs showed different intensities of serological reaction with different CTV isolates. However, much work remains for realizing the characteristics and the serological relationships among these isolates.2. A CTV isolate HB1 (CTV-HB1) showing unusual serological characteristic was identified when three antibodies from different sources were used to detect CTV in fieldZ samples. CTV-HB 1 could not be recognized by PAb-Ⅰraised against an Italy isolate 0002 and the cocktail of ten MAbs-S4, but it showed a positive reaction with PAb-L5 in both ELISA and Western blot analysis. To understand the relationship between the anomalous serological property and the biological characteristics, and to reveal the molecular mechanisms underlying such unusual serological property, the biological activities of CTV-HB 1 were identified on Mexican lime (C. aurantifolia) and Pineapple sweet orange (C. sinensis), and the RFLP and the sequences of capsid protein (CP) genes were determined for these three isolates. Results indicated that three CTV isolates all induced typical stem pitting on two indicators. The CP/Hinf I RFLP pattern of CTV-HB1 was single, and was the same with that of CTV-L5, but was different from that of CTV-S4. Comparison of the CP gene sequence from CTV-HB1 with those of other isolates published in GenBank revealed that CTV-HB1 was closely related to CTV-L5, SY568, and NUagA at nucleotide and deduced amino acid levels, the similarities were higher than 97%. Moreover, three amino acid residues [84 (S→L), 98 (G→D) and 190 (G→C)] in the CP gene from CTV-HB 1 were different from that of CTV-L5, and these three sites were conserved in all other sequenced isolates. By investigating the prediction of Antigenic Indexes and Surface Probability in four CTV isolates (HB1, S4, L5 and 0002) with the Peptidestructure and Plotstructure sol, wares, it was found that the CTV-HB1 profile showed two small specific peaks at aa 84 and 190, however, CTV-HB1 and CTV-L5 showed a similar small peak at the 5' end which did not occur in the other two CTV isolates. Such a relationship between serological reactions and molecular characteristics suggested that the specific structure of 5' end might result in failure in recognition of CTV-HB I by PAb-IⅠand MAbs-S4. This study sheds a new light on the epitopes for some different CTV isolates and looking for new epitopes that are strain specific.3. The citrus tissue culture technique was established for Mexican lime, Pineapple sweet orange and Arizona Etrog citron (C. medica) from young citrus, and Fisher navel from adult citrus. In addition, the optimum initiated medium was identified for stem segments culture of Gillete navel, Frost navel and Atwood navel from adult citrus. In this study, citrus nodal stem segments from CTV infected and un-infected citrus were cultured in vitro. Regeneration frequency, growth vigor and rooting capacity of regenerated shoots were reduced as a result of CTV infection. In three species of young citrus, the effect was very severe on explants from Etrog citron, and moderate on Mexican lime and sweet orange. The vigor of all cultures from Etrog citron was strongly reduced and those from sweet orange and Mexican lime showed the reduction of growth vigor after 18 sub-culturing cycles. In four cultivars of adult citrus, Frost navel was the most sensitive to CTV infection.Virus titers and graft infectivity of CTV in in-vitro cultures of three young citrus over periodical sub-culturings were investigated by TAS-ELISA, IC-RT-PCR and shoot-tip graining, respectively. The result indicated that virus titers and gtaft infectivity showed a correlated reduction tendency with the effect on cultures during subcuituring. The titer and graft infectivity of CTV in in-vitro cultures did not changed significantly before the first three cycles, but they greatly decreased after the fifteenth sub-cultruing for Mexican lime and Sweet orange, and after the sixth sub-culturing for Etrog citron. These results revealed that differences in sensitivity to CTV presented in hosts and suggested that the expression of some genes related to the development and resistance of hosts might be suppressed or induced by the virus.4. Two differentially expressed genes libraries-a library of up-regulated genes and a library of down-regulated genes in CTV-infected Mexican lime (C aurantifolia) of in-vitro cultures were prepared by suppression subtractive hybridization (SSH). Inserts of 600 randomly selected colonies from the forward-subtractive library and of 300 colonies from the reverse-subtractive library were identified with the reverse Northern dot-blot screening method. Sequencing results showed that 203 ESTs represented 180 different gene fragments, among which 80 genes are up-regulated expression and 100 genes are down-regulated expression by CTV infection.Among the 180 ESTs isolated and identified in Mexican lime, only 17 ESTs had been reported on citrus in GenBank database, the others are mostly from Arabidopsis, tobacco and tomato. The function of 93 (about 52%) gene fragments was unknown, while the rest 48% had putative function clarifying as disease defense, transcription, signal transduction, energy, metabolism and protein synthesis. The results indicated that the process of CTV-host interactions was complex and many genes could participate in the process. In this paper, we first reported the differentially expressed genes of Mexican lime induced by CTV infection, which established the foundation for understanding the molecular mechanisms of the virus-host interactions.