Functional characterization of the Nicotiana benthamiana chromomethyltransferase gene, NbCMT3, in developmental programs 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. Chromomethyltransferase (CMT) genes are specific to plant kingdoms and encode methyltransferase containing chromodomain. However, the functions of the CMT genes in plants remain to be elusive. In this study, we isolated and characterized a CMT gene from Nicotiana benthamiana and designated as NbCMT3. Alignment of NbCMT3 amino acids with other cloned CMT3 genes showed the structural conservation. The expression patterns of NbCMT3 and its functions were investigated with respect to developmental programs. NbCMT3 was expressed predominately in proliferating tissues such as apical shoot and young leaves. Subcellular localization of NbCMT3 protein was performed to correct with its putative cellular functions. Knocking down the expression of NbCMT3 gene by virus-induced gene silencing revealed its vital roles in leaf morphogenesis. The formation of palisade cells was defected in NbCMT3-silencing plants as compared to that of empty-vector controls. In summary, the study suggests for a role of NbCMT3 in developmental programs 1.Isolation of a full-length cDNA encoding CMT3 from N. benthamiana 4. Aberrant development of NbCMT3-silenced plants Fig. 1 Alignment of the amino acid sequences of NbCMT3 with representative plant CMT3 members. 2. Tissue-specific expression and subcellular localization of NbCMT3 Fig. 2 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. Silencing of the NbCMT3 gene damaged the mesophyll cell structure 3. Suppression of endogenous transcripts of NbCMT3 by VIGS Fig. 3 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. Fig. 4 The typical phenotypes of NbCMT3-silenced N. benthamiana plants. (A) VIGS phenotypes of the whole plants, leaves and flowers in TRV empty-vector control, NbPDS- and NbCMT3-silenced plants at 3 weeks post inoculation. The photos of upper leaves were collected from the top of the individual plant. (B) Growth arrest phenotypes of the NbCMT3-slienced N. benthamiana plants. 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 Microscopic analysis of leaf transverse sections in N. benthamiana plants. 6. Roles of NbCMT3 during organogenesis in vitro Fig. 6 In vitro organogenesis of leaf discs and evaluation of the regeneration capacity in the VIGS- treated N. benthamiana plants. Leaf discs of the control (TRV2-Ve), NbPDS-(TRV2-PDS) and NbCMT3- silenced (TRV2-CMT3) plants were cultured for 4 w on the shoot induction medium containing NAA and BA. 7. NbCMT3-silenced plants showed stress phenotypes Fig. 7 The leaves of the control and NbCMT3-silenced plants were stained with DAB and NBT for the detection of superoxide H 2 O 2 and O 2 ● −, respectively. The expression level of defense-related genes was examined in the NbCMT3-silencing plants. NbPR1a, NbPR1b, NbPR2, and NbWRKY4 gene expression were induced in the NbCMT3-silencing plants as compared to the controls. The transcript levels of the developmental-related genes such as NbCDC6 and NbNTH3 was downregulated. 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.The aberrant development of NbCMT3-silenced plants is in correlation with the imbalance of cellular ROS level. 4.Future work will focus on the identification of the downstream targets of NbCMT3. 侯品全 1 、許桂婷 1 、吳偉誌 1 、李勇毅 2 、傅士峰 1* 1 Department of Biology, National Changhua University of Education, Changhua, Taiwan 彰化師範大學生物學系 2 Botany Department, National Museum of Natural Science 自然科學博物館植物園