1. Characterization of the Nicotiana benthamiana chromomethyltransferase genes, NbCMT3s, in leaf development by virus-induced gene silencing Abstract Results DNA methylation is essential for normal developmental processes and genome stability. DNA methyltransferases are key enzymes catalyzing DNA methylation. Chromomethylase (CMT) genes are specific to the plant kingdom and encode chromodomain-containing methyltransferases. However, the function of CMT genes in plants remains elusive. In this study, we isolated and characterized CMT genes from Nicotiana benthamiana, designated NbCMT3 and NbCMT3-2. Alignment of the NbCMT3s amino acid sequence with other plant CMT3s showed conservation of bromo-adjacent-homology and methyltransferase catalytic domains. NbCMT3-2 has unique N-terminal extension when compared to NbCMT3. We investigated the expression patterns of NbCMT3s and its function in developmental programs. NbCMT3 was expressed predominately in proliferating tissues such as apical shoots and young leaves. NbCMT3 and NbCMT3-2 protein showed a nuclear location, which could be related to its putative cellular functions. Knocking down NbCMT3 and NbCMT3-2 expression by virus-induced gene silencing revealed its vital role(s) in leaf morphogenesis. Transcriptomic analysis of VIGS-NbCMT3 plants revealed that endonuclease might be a potential target of CMT3. The formation of palisade cells was defective in NbCMT3-silenced plants as compared with controls. In summary, NbCMT3 has a role in leaf developmental programs. 4. Comparison of NbCMT3 and NbCMT3-2-silenced plants 1. Tissue-specific expression and subcellular localization of NbCMT3 Fig. 1 Organ-specific expression of NbCMT3 (A) and subcellular localization of NbCMT3 (B). Analysis of NbCMT3 gene expression in different N. benthamiana organs. Total RNA was purified from different organs of 1-month- old N. benthamiana, including apical shoots (AS), young leaves (YL), mature leaves (ML), old leaves (OL), flowers (F) and roots (R). 5. Bimolecular fluorescence complementation (BiFC) analysis of proteins that interacted with NbCMT3 2. Suppression of endogenous transcripts of NbCMT3 by VIGS Fig. 2 Suppression of NbCMT3 gene expression in N. benthamiana plants by VIGS. (A) Schematic of the NbCMT3 cDNA structure and the VIGS construct containing the cDNA fragments. The box indicates the ORF region of NbCMT3. The two primer sets corresponding to the 5’ of the NbCMT3 coding region were used in RT-PCR to assess the knockdown efficiency of the host gene by VIGS. (B) Characterization of the NbCMT3- silenced leaf tissues in the N. benthamiana plants. The cDNA fragments were amplified by RT-PCR with the gene-specific primers. (C) Analysis of the relative expression of NbCMT3 gene by real-time PCR. Fig. 3 The typical phenotypes of NbCMT3-silenced N. benthamiana plants. Growth arrest phenotypes of the NbCMT3-slienced N. benthamiana. The morphology of shoot apexes was shown at 6 weeks post inoculation. The shoot length at the similar developmental stage was compared between the control and NbCMT3-slienced plants (n=8). The experimental were repeated three times, and a representative result is shown. Fig. 5 BiFC assays showing interaction and subcellular localization of NbCMT3 in N. benthamiana cells by Agro-infiltration using a 1-ml needleless syringe. Four days after infiltration, the abaxial epidermis of infiltrated tobacco leaves was assayed for fluorescence by confocal laser-scanning microscopy. (A) N- terminal NbCMT3 containing the BAH and CD domain and C-terminal NbCMT3 fragment with the DNA methyltransferase domain was fused to N-terminal YFP and C-terminal YFP, respectively. (A) Full-length NbCMT3 and NbAGO1 (or NbTFL) was fused to N-terminal YFP and C-terminal YFP, respectively. B right field, DAPI and epifluorescence images are merged to show the nuclear localization of interacted proteins (Merged). 6. Transcriptomic analysis ofNbCMT3-silenced plants by RNA-seq Conclusions and future work 1.Subcellular localization studies revealed that the NbCMT3 protein was mainly located in the cytoplasm and nucleus. 2.The NbCMT3 gene was predominantly expressed in actively proliferating tissues such as apical shoots. 3.NbCMT3 and NbCMT3-2 may play various roles in plant growth and development. 4.Future work will focus on the identification of the downstream targets of NbCMT3s by DNA methylome analysis. 許桂婷 1 、林郁婷 1 、李勇毅 2 、傅士峰 1 1 Department of Biology, National Chunghua University of Education 1 彰化師範大學生物系 2 Botany Department, National Museum of Natural Science 2 自然科學博物館植物園 3. Aberrant development of NbCMT3-silenced plants (A) (B) (C) Fig. 4 Comparison of leaf development between NbCMT3- and NbCMT3-2-silenced N. benthamiana plants. The experimental were repeated three times with similar results. Table 2 Up-regulation (TRV2-NbCMT3/Ve) Table 1 Down-regulation (TRV2-NbCMT3/Ve) NbCMT3 NbCMT3-2 NbPR1a NbPR1b NbRDR1 NbRDR6 EF1 rRNA TRV2-Ve TRV2-NbCMT3 TRV2-CMT3-2