The Study On The Effects And Mechanism Of Petunia Transcription Factors PhOBF1 And PhERF2 On Virus-induced Gene Silencing Efficiency

Virus-induced gene silencing(VIGS) is a commonly used technique for assaying gene function in plant molecular biology. The inoculation with virus vector carrying a fragment of candidate gene results in the decreased transcript levels of homologous m RNA in plants. So VIGS belongs to the post-transcriptional gene silencing. PDS and CHS are frequently used reporter genes for identifying the function of related genes in the leaves and floral tissues, respectively, through VIGS method. Their silencing-triggered visual phenotypes could be used to indicate VIGS silencing position or efficiency. Virus-induced RNA silencing participates in plant antiviral response, and requires RNA-dependent RNA polymerases(RDRs), Dicer-like RNase III enzymes(DCLs) and Argonaute proteins(AGOs). However, the transcriptional regulation of these important components is largely unknown. In this study, one b ZIP transcription factor, designated PhOBF1, and one ERF transcription factor, designated PhERF2, were isolated through transcriptomic sequencing during various developmental stages of flower in petunia. The petunia(Petunia × hybrid) purple-corollas cultivar ‘Primetime Blue' and white-corollas cultivar ‘Mitchell Diploid' were chosen as plant materials. Expression profiles of PhOBF1 or PhERF2 in petunia leaves following TRV inoculation, and treatments with abiotic stresses and stress-associated hormones were studied. Transcriptional regulation of RNA silencing-related genes and influence on Tobacco rattle virus(TRV)-induced gene silencing efficiency by PhOBF1 or PhERF2 were analyzed. Furthermore, the resistance variations to different viruses in PhOBF1 or PhERF2 transgenic petunia plants were examined. The results are shown as follows:1. Among the differentially-expressed genes in the c DNA library constructed using transcriptomic analysis during flower development in petunia, PhOBF1 and PhERF2 fragments were obtained. BLAST search in the NCBI revealed the m RNA sequences containing the complete open reading frame(ORF) of PhOBF1 and PhERF2. The PhOBF1 m RNA sequence was 915 bp in length. Its ORF region was 489 bp in length, encoding a protein of 162 amino acids. The 5' un-translated region of PhOBF1 harbors a sucrose-controlled u ORF region, whose length was 147 bp encoding a protein of 48 amino acids. Gen Bank accession no. for PhOBF1: FN001301. The length of PhERF2 m RNA sequence was 1308 bp, and of its ORF region was 1137 bp, encoding a protein of 378 amino acids. Gen Bank accession no. for PhERF2: HQ259596. The amino acids of PhOBF1 and PhERF2 contain a conserved b ZIP domain and AP2/ERF domain, respectively. Phylogenetic analysis suggested that PhOBF1 is a member of subfamily S of b ZIP transcription factors, and is highly homologous to Arabidopsis Atb ZIP11. PhERF2 belongs to subfamily VII of ERF transcription factors, and is highly homologous to At RAP2.12 and At RAP2.2 in Arabidopsis.2. After inoculation with TRV, the inoculated and uppermost systemically-infected leaves of petunia plants showed elevated transcription levels of PhOBF1 or PhERF2. The elevated trend of PhOBF1 or PhERF2 transcripts was consistent with the accumulation levels of TRV RNA1(or TRV1) and RNA2(or TRV2). Apart from this biotic factor, various abiotic factors and hormones were used to treat petunia leaves for examining the expression of PhOBF1 or PhERF2. The results indicated that the treatments with low temperature, dehydration, abscisic acid(ABA), ethylene, gibberellin(GA3), and salicylic acid(SA) induced the expression of PhOBF1, while the treatments with low temperature, high salinity, dehydration, abscisic acid(ABA), ethylene, salicylic acid(SA), and methyl jasmonate(Me JA) induced the expression of PhERF2. Under abiotic stresses, low temperature led to the maximum induction of PhOBF1 or PhERF2 transcripts, followed by drought. Of all hormones tested, SA treatment results showed the maximum induction of PhOBF1 or PhERF2 expression. These data indicated that the two transcription factors may play important roles in petunia plant tolerance to abiotic stresses. In addition, ethylene treatment significantly increased the transcript abundances of PhOBF1 or PhERF2. As petunia plant is sensitive to ethylene, which is the primary signal controlling the process from flower anthesis to completed wilting, predictably PhOBF1 or PhERF2 is probably involved in the regulation of petunia flower senescence.3. In VIGS assay, TRV was used as virus vector and PDS or CHS was used as reporter gene. The PhOBF1 or PhERF2 fragment was inserted into TRV-Ph PDS/CHS, TRV-Ph PDS or TRV-Ph CHS constructs. The Agrobacterium bearing all kinds of TRV constructs was used to inoculate the leaves of wild-type petunia seedling using injection method, and the inoculation with non-transformed Agrobacterium was referred to as mock control. At different stages post-inoculation, the PDS-silenced leaf photobleaching or CHS-silenced white-corollas phenotype was not observed in uppermost systemically-infected leaves or flowers. Semi-quantitative RT-PCR results of virus accumulation levels showed that TRV RNA1(or TRV1) and RNA2(or TRV2) carrying PhOBF1 or PhERF2 insert could infect the topmost systemic leaves. These findings indicated that the inserted PhOBF1 or PhERF2 fragment did not inhibit the replication and movement of TRV vector, allowing us to exclude the possibility that the suppressed TRV proliferation resulted in the failure of reporter gene silencing. Quantitative real-time PCR results suggested that PDS transcript levels in TRV-Ph PDS/OBF1- or TRV-Ph PDS/ERF2-infected leaves were reduced by 30-40% compared to mock control. PDS transcripts in TRV-Ph PDS-infected leaves showing photobleaching phenotype were reduced by approximately 90%. Down-regulation of PhOBF1 or PhERF2 resulted in decreased expression levels of RNA silencing-related genes, including RDRs, DCLs and AGOs, and nearly undetectable si RNAs accumulation levels. These data demonstrated that PhOBF1 or PhERF2 affected TRV-induced gene silencing efficiency probably through transcriptional regulation of key genes in RNA silencing pathway.4. Agrobacterium-mediated transformation was employed to generate PhOBF1-RNAi silencing(#2, #6 and #8) and-overexpressing(#B, #D and #H), or PhERF2-RNAi(#1 and #4) and-overexpressing(#C, #D and #I) transgenic petunia plants. The observation of plant growth phenotypes showed that PhOBF1 affected the plant height, diameter of stem and shoot, leaf thickness, and seed size. PhERF2-RNAi silencing caused a reduced plant growth rate, while its overexpression led to an enhanced growth rate. PhOBF1 silencing and overexpression significantly suppress and promote the expression levels of RDR1, RDR2, DCL1, DCL2, DCL4 and AGO2, respectively. PhERF2 silencing and overexpression significantly decreased and increased the transcript levels of RDR2, RDR6, DCL2 and AGO2, respectively. PhOBF1- or PhERF2-silenced and-overexpressing transgenic petunia plantlets were inoculated with TRV-Ph PDS, and the wild-type plants inoculated with TRV-Ph PDS were used as control. The observation of leaf phenotypes post-inoculation showed that PhOBF1- or PhERF2-RNAi silencing substantially reduced the efficiency of PDS silencing, as shown by no obvious PDS-silenced leaf photobleaching phenotype in the uppermost systemic leaves. But PhOBF1 or PhERF2 overexpression restored and even enhanced PDS-silenced leaf photobleaching. Quantitative real-time PCR analysis indicated that PDS transcript levels in systemic leaves of PhOBF1- or PhERF2-silenced transgenic plants inoculated with TRV-Ph PDS were reduced by 30-40%, whereas PDS transcript levels in systemic leaves of wild-type plants infected with TRV-Ph PDS were reduced by about 90%. PhOBF1 or PhERF2 overexpression also resulted in a 90% reduction in PDS transcript abundances in TRV-Ph PDS-infected systemic leaves. There was an obvious discrepancy in accumulation levels of TRV RNA1(or TRV1) and RNA2(or TRV2) between wild-type and PhOBF1 or PhERF2 transgenic plants inoculated with TRV-Ph PDS, suggesting that PhOBF1 or PhERF2 are involved in not only regulation of TRV-induced gene silencing but also plant defense response against TRV infection. Moreover, PhOBF1 silencing and overexpression decreased and increased the expression of core genes, including DAHPS, SK1, EPSPS, CS, CM1, ADT1, PAL1 and PAL2, in the shikimate and phenylpropanoid pathway, and contents of endogenous SA, respectively. Exogenous SA treatment remarkably induced the expression of RDR1, RDR2, DCL1, DCL2, DCL4 and AGO2. These findings suggested that SA may be an important intermediate signal linking PhOBF1 and downstream RNA silencing pathway.5. The inoculation with TRV empty vector and TRV-GFP construct was carried out using injection method. The resistance response to TRV infection in PhOBF1- or PhERF2-RNAi-silenced and-overexpressing transgenic plants was investigated. The mechanical method was performed for inoculation with Tobacco mosaic virus(TMV) and Cucumber mosaic virus(CMV). We tested the resistance to TMV in PhOBF1 transgenic plants and to CMV in PhERF2 transgenic plants, respectively. After inoculation with TRV-GFP, the inoculated leaves of PhOBF1-silenced transgenic plants showed bigger relative fluorescent area than those of wild-type plants, while the inoculated leaves of PhOBF1-overexpressing plants showed smaller one. The systemic leaves of PhOBF1- or PhERF2-silenced plants inoculated with TRV empty vector showed severer symptoms, such as mosaic, mottling and chlorosis than TRV empty vector-infected wild-type plants. Quantitative real-time PCR analysis indicated that accumulation levels of TRV RNA1(or TRV1) and RNA2(or TRV2) in PhOBF1- or PhERF2-silenced plants were much higher than those in wild-type plants. The systemic leaves of PhOBF1-overexpressing transgenic plants inoculated with TRV empty vector showed no obvious mosaic, mottling and chlorosis symptoms. Quantitative real-time PCR analysis indicated that PhOBF1 overexpression led to reduced accumulation levels of TRV RNA1(or TRV1) and RNA2(or TRV2), compared to wild-type plants. Furthermore, PhOBF1 silencing and overexpression increased and decreased transcript levels of TMV-CP, encoding a coat protein of TMV, and PhERF2 silencing and overexpression increased and decreased transcript abundances of CMV-CP, encoding a coat protein of CMV, respectively. These results suggested that PhOBF1 or PhERF2 is possibly involved in extensive resistance responses to a broad range of viruses.