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Developmental- and tissue-specific expression of NbCMT3-2 encoding a chromomethylase in Nicotiana benthamiana Yu-Ting 1 Lin (林郁婷) , Huei-Mei 1 Wei , Syue-Yu 1 Lyu , Yung-I 2 Lee , and Shih-Feng 1* Fu (傅士峰) 1Department of Biology, National Chunghua University of Education (彰化師範大學生物系) 2Botany Department, National Museum of Natural Science (自然科學博物館植物園) 2. Expression of NbCMT3 and NbCMT3-2 in tissues of N. benthamiana Abstract DNA methylation is a heritable epigenetic process controlling gene expression and developmental programs in various organisms. The chromomethylase (CMT) protein family A is unique to plants and controls non-CpG methylation. Here, we investigated the B developmental expression of CMT3-2 in Nicotiana benthamiana (NbCMT3-2) and its significance by analyzing plants with silencing of NbCMT3-2 and analyzing leaf tissues transiently expressing the N-terminal polypeptide. Alignment of the NbCMT3-2 amino acid sequence with other plant CMT3s showed a specific N-terminal extension required for nuclear localization. Transient expression of the N-terminal polypeptide in N. benthamiana resulted in the formation of chlorotic lesions, which indicates its vital role in leaf development. NbCMT3-2 was expressed mainly in proliferating tissues such as the shoot apex and developing leaves. We generated transgenic N. benthamiana harboring a fusion reporter construct linking the NbCMT3-2 promoter region and -glucuronidase (GUS) reporter (pNbCMT3-2::GUS) to analyze the tissue-specific expression of NbCMT3-2. NbCMT3-2 was expressed in shoot and root apical meristem and leaf primordia in young seedlings and highly Fig. 2 Expression of NbCMT3 and NbCMT3-2 in different tissues. (A) expressed in developing leaves and ovary as well as lateral buds in mature plants. Virus- Diagrams of gene-specific primers corresponding to NbCMT3 and NbCMT3-2 induced gene silencing used to knock down NbCMT3-2 expression led to partial loss of cDNA sequences. Apical shoots (A), young leaves (Y), mature leaves (M), flowers (F) and roots (R).The tissue culture (T) of mature leaf explants during genomic CHG DNA methylation. Silencing NbCMT3-2 interfered with leaf development and shoot regeneration for 2 weeks were collected for gene expression analysis. the expression of genes involved in jasmonate homeostasis. The differential roles between NbEF1 was a reference gene. NbCMT3 and NbCMT3-2 were investigated and compared. We reveal the expression patterns of NbCMT3-2 in proliferating tissues. NbCMT3-2 may play an essential role in leaf 3. Shoot apex- and root tip-specific expression of development by modulating jasmonate pathways. NbCMT3 and NbCMT3-2 promoter Results 1. Nuclear localization of NbCMT3-2 protein C F ig . 1 Su b cellu lar lo caliz atio n o f NbCMT3-2 proteins in N. benthamiana. (A)Schematic diagram of the constructs used for transient expression of proteins in leaf epidermal cells. The unique N-terminal extension (N) is in yellow, the Bromoadjacent homology (BAH) domain is in green, and the chromo domain (CD) is in red. The blue boxes indicate the conserved methyltransferase catalytic motifs I, IV, VI, VIII, IX and X. (B) Agrobacterium carrying the fusion constructs under the control of Cauliflower mosaic virus 35S promoter was infiltrated into N. benthamiana epidermal cells. At 4 days after agroinfiltration, fluorescence of NbCMT3-derived proteins was observed by confocal laser scanning microscopy (C) Western blot analysis of NbCMT3-derived protein fusions expressed from pSITEII4C1 vectors in agroinfiltrated leaves Fig. 3 Histochemical localization of GUS activity in transgenic N. benthamiana plants expressing GUS gene driven by the NbCMT3 or NbCMT3-2 promoter region. Cotyledons (Cy), Center zone (CZ), Peripheral zone (PZ), RZ (Ribzone), PM (Primary meristem), AM (Apical meristem), RC (Root cap), Co (Cortex), E (Epidermis). 4. Expression of NbCMT3 or NbCMT3-2 promoter in stigma 6. Suppression of endogenous NbCMT3 or NbCMT3-2 expression by VIGS Fig. 6 Molecular Fig. 4 Distribution of pNbCMT3-2::GUS activity in various organs of mature transgenic N. benthamiana plants. (A) lateral bud, (B) developing leaves, (C) ovary and (D) stigma. 5. Accumulation of NbCMT3– or NbCMT3-2–GFP-GUS fusion protein during organogenesis Fig. 5 Promoter NbCMT3- or NbCMT3-2-driven GFP and GUS expression during callus formation and organogenesis in vitro. Callusinducing medium (CIM). Shoot inducing medium (SIM) (A-F). (G and H ) We s t e r n b l o t analysis of GFP expressed from t r a n s g e n i c p Nb CM T 3 : : G FP GUS or pNbCMT32::GFP-GUS leaf explants. Wild-type N. benthamiana plants were a negative control. The star (*) denotes the non-specific cross-reaction of a n t i b o d i e s . characterization o f NbCMT3-, NbCMT3-2or NbCMT3/3-2silenced N. benthamiana plants .(A) NbCMT3 or NbCMT32 cDNA structures and the VIGS construct containing the cDNA fragments. (B) Suppression of NbCMT3 or NbCMT32 transcripts by virusinduced gene suppression (VIGS). (C) Quantification of g l o b a l D N A methylation in NbCMT3-silenced p l a n t s . 7. Regulation of leaf development by NbCMT3 and NbCMT3-2 Fig. 7 Alternation in N. benthamiana N b C M T 3 - 2 expression led to abnormality in plant g row th an d development. (A) P h e n o t y p i c characterization of N b C M T 3 - , NbCMT3-2- and NbCMT3/3-2s i l e n c e d N.benthamiana p l a n t s . (B)Evaluation of g row th an d development using transient gene expression from a PVX-based vector. Conclusions and future work NbCMT3-2 and its promoter activity were expressed in proliferating tissues such as shoot apex and root tips. The unique Nterminal extension of NbCMT3-2 has a role in nuclear localization and leaf development. Further investigation into the roles of the unique N-terminal extension will help elucidate the molecular actions of NbCMT3-2 in plant epigenetic regulation.