| Citrus tristeza caused by Citrus tristeza virus (CTV) is one of the most devastating and economically important viral diseases of citrus worldwide. Symptoms induced by CTV depend on the viral strains and citrus species. Some severe CTV strains can induce the stem pitting on citrus or the decline and even death of citrus propagated on sour orange(Citrus aurantium L.) rootstock. Mild strains usually induce very mild leaf chlorosis or symptomless on host plants. Many studies have indicated that symptoms of host plants with virus infection were associated with the gene expression changes induced by virus. The CTV mild isolate can replicate and move in host plants as a severe isolate does, although it does not usually induce visible symptoms. Therefore, it could be expected that there were interactions between the mild virus and its host. In order to better understand the complex interaction implicated in a mild CTV strain and citrus, the differential expressed genes of citrus plants induced by CTV-N4infection was identified, and the temporal expression of some candidate genes in lime plants were comparatively analyzed in this study. Meanwhile, the biological characteristics of the mild strain and a severe strain and the effects of their mixed infections on host growth and their replication were evaluated. The main results obtained are listed below:1. Two libraries of genes induced and repressed in lime plants with the infection of CTV-N4were constructed by suppression subtractive hybridization (SSH). A total of589expressed sequence tags (ESTs) were screened from two SSH libraries by dot blot analysis. Sequence alignment and homology searches indicated that589ESTs represented202genes. Among these genes,111genes showed up-regulated expression and91genes were down-regulated. The differential expression of13selected genes was confirmed by quantitative real-time RT-PCR (qRT-PCR). Results revealed that relative expression levels of eight genes selected from the forward library were up-regulated about1.1-47folds after CTV-N4infection and the expression levels of five genes selected from the reverse library was slightly down-regulated (about1.2-1.8folds) in CTV-N4infected Mexican lime. The largest set of genes up-regulated by CTV-N4infection was assigned to the stress and disease defense responses (27.9%), while genes involved in signal transduction constituted the smallest group (5.4%). Genes implicated in cell structure and transport were about15.3%of the selected cDNA collection. Genes involved in transcription, metabolism, energy, and protein synthesis were12.7%,11.7%,9.0%and6.3%, respectively. In the reverse library, down-regulated genes were mostly involved in stress response and disease defense (17.6%), energy (13.2%), and cell structure and transport (13.1%). Genes involved in signal transduction constituted the smallest group (4.4%). ESTs with unknown functions accounted for11.7%and22.0%of up-and down-regulated genes.2. The transcriptional levels of three genes, encoding acidic class â…¡ chitinase, caffeic acid3-O-methyltransferase and lipid-transfer protein, in lime plants with the infection of a mild isolate N4and a severe isolate N21were comparatively analyzed by qRT-PCR. Results showed that the infection of both isolates N4and N21could induce the expression of the three genes. Meanwhile, the transcriptional changes of three selected genes were more greatly induced by CTV-N21than those induced by CTV-N4. Acidic class â…¡ chitinase showed the highest expression levels at60days post inoculation (dpi), and was up-regulated about31-fold and14-fold by CTV-N21and N4, respectively. Later, their expression levels dramatically decreased, but was still significantly up-regulated about6.4-fold and1.9-fold by CTV-N21and CTV-N4until120dpi. Caffeic acid3-O-methyltransferase also showed the highest expression levels at60dpi with infection of both isolates (up-regulated5.7-fold for CTV-N21and2.3-fold for CTV-N4), and then its expression level dramatically decreased in CTV infected plants. The expression levels of lipid-transfer protein were relatively less up-regulated, ranging from1.6-to2.5-fold and1.1-to1.4-fold in plants infected by CTV-N21and N4, respectively. Moreover, it was found that the previous infection of a viral isolate could decrease the relative expression levels of those genes induced by a followed CTV isolate.3. The biological tests showed that the severe CTV strain N21induced chlorosis, yellowing and dwarf symptoms on Mexican lime, sweet orange, and HB pomelo seedlings. The immuno-inoculation with the mild CTV strains N3and N4could only decrease the symptoms on sweet orange induced by the severe CTV strain N21.4. The relative titers of different CTV strains in citrus plants previously inoculated with mild the strain N3or N4and followed by the infection of a severe CTV strain N21were evaluated by RT-PCR with strain specific primers. Results showed that the positive rate of CTV-N3and CTV-N4in three host plants was gradually decreased at90days post N21inoculation (dpi), and the rates of plants positive to CTV-N3and CTV-N4dropped from100%at30dpi to50%and45%at150dpi, respectively. In contrast, the titers of CTV-N21in citrus plants pre-inoculated with N3or N4was greatly increased as infection period prolonged. The rates of plants positive to CTV-N21in RT-PCR tests were over95%at120dpi. The titers of different CTV strains in citrus plants were also related to citrus species. Both the decreasing level of titers of N3and N4and the increasing level of N21titer were relatively lower in sweet orange than that in other two host plants. Furthermore, semi-nested RT-PCR was employed to detect the N3and N4in plants which showed negative reaction in previous RT-PCR tests at210dpi. The mild CTV strain specific fragment was amplified from all test plants. Furthermore, our research showed that the replication level of N21was over that of N4or N21infection might inhibit the replication of N4in lime plants.5. RT-PCR products of CTV CP gene from lime plants with the mixed infections of CTV mild and severe strains were cloned and sequenced at18months post CTV inoculation. Results showed that the copies of CP gene of the mild strain in lime plants were much lower than those of the severe CTV strain. Moreover, the titer of CTV mild strain was affected by the time course of both N4and N21. CP copies of N4in plants with pre-inoculation of N21followed by N4inoculation (N21+N4) was much lower than those in plants with pre-inoculation of N4followed by N21inoculation (N4+N21), which was in accordance with RT-PCR results. Sequence alignments showed that the pre-inoculated mild CTV strain had somewhat affacted the population structure of severe CTV strain N21, ten mutation sites of amino acids in two CP clones were identifed.
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