Modification of vectors for functional genomic analysis in plants

Li, J T; Yu, Gang; Chen, Jia; Du, Qian; Li, Q Y; Pan, Hongyu

doi:10.4238/2014.september.26.20

Cited by 15 publications

(7 citation statements)

References 30 publications

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“…The 35S:ccdB cassette-LUC-NOS was then inserted into pCAMBIA1300 via Pst I and EcoR I and designated as pGX301 for cloning 5′ leader sequences through replacement of the Apa I-flanked ccdB cassette 24 . Similarly, the 35S:RLUC-HA-rbs terminator construct was made through fusion of PCR fragments of 35S from pMDC140 23 , RLUC from pmirGLO (Promega, E1330) and rbs terminator from pCRG3301 25 . The 35S:RLUC-HA-rbs fragment flanked with EcoR I was inserted into pTZ-57rt (Thermo fisher, K1213) via TA cloning to generate pGX125.…”

Section: Methodsmentioning

confidence: 99%

Global translational reprogramming is a fundamental layer of immune regulation in plants

Greene

Yoo

et al. 2017

Nature

197

212

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In the absence of specialized immune cells, the need for plants to reprogram transcription to transition from growth-related activities to defence is well understood1, 2. However, little is known about translational changes that occur during immune induction. Using ribosome footprinting (RF), we performed global translatome profiling on Arabidopsis exposed to the microbe-associated molecular pattern (MAMP) elf18. We found that during this pattern-triggered immunity (PTI), translation was tightly regulated and poorly correlated with transcription. Identification of genes with altered translational efficiency (TE) led to the discovery of novel regulators of this immune response. Further investigation of these genes showed that mRNA sequence features are major determinants of the observed TE changes. In the 5′ leader sequences of transcripts with increased TE, we found a highly enriched mRNA consensus sequence, R-motif, consisting of mostly purines. We showed that R-motif regulates translation in response to PTI induction through interaction with poly(A)-binding proteins. Therefore, this study provides not only strong evidence, but also a molecular mechanism for global translational reprogramming during PTI in plants.

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Section: Methodsmentioning

confidence: 99%

Global translational reprogramming is a fundamental layer of immune regulation in plants

Greene

Yoo

et al. 2017

Nature

197

212

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“…Full-length coding regions of Ssnsd1 and Ssfdh1 were cloned into pCG-1301-GFP and pCHF-3301-3xFLAG vectors (54) to generate pCG- Ssnsd1 -GFP and pCHF- Ssfdh1 - 3×FLAG constructs. Agrobacterium carrying different plasmids was coinfiltrated into N.…”

Section: Methodsmentioning

confidence: 99%

The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum

Zhu

Zhang

et al. 2019

mSystems

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GATA transcription factors (TFs) are common eukaryotic regulators, and glutathione-dependent formaldehyde dehydrogenases (GD-FDH) are ubiquitous enzymes with formaldehyde detoxification activity. In this study, the formaldehyde dehydrogenase Sclerotinia sclerotiorum Fdh1 (SsFdh1) was first characterized as an interacting partner of a GATA TF, SsNsd1, in S. sclerotiorum. Genetic analysis reveals that SsFdh1 functions in formaldehyde detoxification, nitrogen metabolism, sclerotium development, and pathogenicity. Both SsNsd1 and SsFdh1 harbor typical zinc finger motifs with conserved cysteine residues. SsNsd1 regulates SsFdh1 in two distinct manners. SsNsd1 directly binds to GATA-box DNA in the promoter region of Ssfdh1; SsNsd1 associates with SsFdh1 through disulfide bonds formed by conserved Cys residues. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Site-directed point mutation of these Cys residues influences the SsNsd1-SsFdh1 interaction and SsNsd1 DNA binding capacity. SsFdh1 is regulated by and functions jointly with the SsNsd1 factor, providing new insights into the complex transcriptional regulatory mechanisms of GATA factors. IMPORTANCE S. sclerotiorum is a pathogenic fungus with sclerotium and infection cushion development, making S. sclerotiorum one of the most challenging agricultural pathogens with no effective control method. We identified important sclerotium and compound appressorium formation determinants, SsNsd1 and SsFdh1, and investigated their regulatory mechanism at the molecular level. SsNsd1 and SsFdh1 are zinc finger motif-containing proteins and associate with each other in the nucleus. On other hand, SsNsd1, as a GATA transcription factor, directly binds to GATA-box DNA in the promoter region of Ssfdh1. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Our results provide insights into the role of the GATA transcription factor and its regulation of formaldehyde dehydrogenase in stress resistance, fungal sclerotium and compound appressorium development, and pathogenicity.

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“…The obtained GmFATB1A, GmFATB1B, GmFATB2A and GmFATB2B were fused to the N-terminal of green fluorescent proteins (GFP) in the pCAMBIA3301 vector. Further, plasmids for the above four genes were constructed using the pCHF3300 vector that contains the CaMV35S promoter (Li et al 2014) and transformed into wild-type (WT) A. thaliana (cv. Columbia) using the Agrobacterium-mediated floral dip method for overexpression.…”

Section: Rna Extraction Gene Cloning and Reverse Transcription Quanti...mentioning

confidence: 99%

Glycine max acyl–acyl carrier protein thioesterase B gene overexpression alters lipid content and fatty acid profile of Arabidopsis seeds

Zhao,

Yan,

Liu

et al. 2024

Funct. Plant Biol.

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The fatty acyl–acyl carrier protein thioesterase B (FATB) gene, involved in the synthesis of saturated fatty acids, plays an important role in the content of fatty acid and composition of seed storage lipids. However, the role of FATB in soybeans (Glycine max) has been poorly characterised. This paper presents a preliminary bioinformatics and molecular biological investigation of 10 hypothetical FATB members. The results revealed that GmFATB1B, GmFATB2A and GmFATB2B contain many response elements involved in defense and stress responses and meristem tissue expression. Moreover, the coding sequences of GmFATB1A and GmFATB1B were significantly longer than those of the other genes. Their expression varied in different organs of soybean plants during growth, with GmFATB2A and GmFATB2B showing higher relative expression. In addition, subcellular localisation analysis revealed that they were mainly present in chloroplasts. Overexpression of GmFATB1A, GmFATB1B, GmFATB2A and GmFATB2B in transgenic Arabidopsis thaliana plants increased the seed oil content by 10.3%, 12.5%, 7.5% and 8.4%, respectively, compared to that in the wild-type and led to significant increases in palmitic and stearic acid content. Thus, this research has increased our understanding of the FATB family in soybeans and provides a theoretical basis for subsequent improvements in soybean quality.

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Modification of vectors for functional genomic analysis in plants

Cited by 15 publications

References 30 publications

Global translational reprogramming is a fundamental layer of immune regulation in plants

Global translational reprogramming is a fundamental layer of immune regulation in plants

The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum

Glycine max acyl–acyl carrier protein thioesterase B gene overexpression alters lipid content and fatty acid profile of Arabidopsis seeds

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