screening-identification-rna-silencing-suppressors-from-secreted

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Guizhou Institute of Tobacco Science

Tobacco is one of the most important economic and agricultural crops worldwide. miRNAs have been increasingly acknowledged for their important roles in different biological processes of tobacco. However, few miRNAs have been identified so far in tobacco impeding the development of new tobacco strains with better properties. In this study, high-throughput sequencing technology was employed to identify novel tobacco miRNAs. A total of 84 potential miRNAs were obtained in tobacco, including 33 conserved and 51 novel miRNAs. Tissue-specific and topping-related miRNAs were identified. A tobacco miRNA microarray was also constructed to investigate miRNA expression patterns in different tissues, and their expression patterns were further validated by qRT-PCR and Northern Blot. Finally, the potential targets of these miRNAs were predicted based on a sequence homology search. Thus, in the current study, we have performed the comprehensive analysis of tobacco miRNAs, including their identification, expression pattern and target prediction. Our study opens a new avenue for further elucidation for their roles underlying the regulation of diversity of physiological processes.

Identification and characterization of miRNAome in tobacco (Nicotiana tabacum) by deep sequencing combined with microarray.

Transcription activator-like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses. 

Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses.

Polyphenols are important secondary metabolites and bioactive compounds in plants. Light is a vital abiotic factor that greatly impacts the content of polyphenols in plants. In spite of their importance the mechanism of polyphenol regulation still remains unknown in tobacco. A phytochrome B homolog, NtPHYB1, was isolated from Nicotiana tabacum cv. K326 to investigate the role of light receptors in the regulation of polyphenol metabolism in tobacco leaves. Furthermore, role of NtPHYB1 in polyphenol metabolism was analyzed by over-expression and RNAi-silencing approaches. Consistent and complemented results indicated involvement of NtPHYB1 in the regulation of polyphenol metabolism in tobacco leaves. Moreover, high levels of NtPHYB1 transcripts favor the accumulation of chlorogenic acid and its isomers, the key polyphenol component in tobacco leaves. Transcriptome analysis was also carried out for exploring the regulation mechanism of NtPHYB1 in the polyphenol metabolism. Compared with WT, 1665 and 1421 differentially-expressed genes were found in NtPHYB1-GFP and NtPHYB1-RNAi transgenic lines, respectively. Among these, about 30 genes were related to phenylpropanoid pathway, which is predominantly involved in synthesis of polyphenols. Further evidences from quantitative RT-PCR confirmed that NtPHYB1 may control phenylpropanoid pathway through regulating the transcription of PAL4 (phenylalanine ammonialyase 4), 4CL1 (4-coumarate:coenzyme A ligase 1) and COMT (caffeic acid 3-O-methyltransferase) genes.

NtPHYB1, a homologous gene of Arabidopsis PHYB, positively regulates the content of phenolic compounds in tobacco.

Potato virus Y (PVY) is a globally and economically important pathogen of potato, tobacco, tomato and other staple crops and caused significant yield losses and reductions in quality.To explore the molecular PVY-host interactions, we analysed changes in the miRNA and mRNA profiles of tobacco in response to PVY infection. A total of 81 differentially expressed miRNAs belonging to 29 families and 8133 mRNAs were identified. The Gene Ontology (GO) enrichment analyses showed that genes encoding the DNA/RNA binding, catalytic activity and signalling molecules were all significantly enriched. Moreover, 88 miRNA-mRNA interaction pairs were identified through a combined analysis of the two datasets. We also found evidence showing that the virus-derived siRNAs (vsiRNAs) from the PVY genome target tobacco translationally controlled tumor protein (NtTCTP) mRNA and mediate plant resistance to PVY. Together, our findings revealed that both miRNA and mRNA expression patterns can be changed in response to PVY infection and novel vsiRNA-plant interactions that may regulate plant resistance to PVY. Both provide fresh insights into the virus-plant interactions.

Integrated analysis of tobacco miRNA and mRNA expression profiles under PVY infection provids insight into tobacco-PVY interactions.

Cas9-PF, an early flowering and visual selection marker system, enhances the frequency of editing event occurrence and expedites the isolation of genome-edited and transgene-free plants.

Article	Year
Identification and characterization of miRNAome in tobacco (Nicotiana tabacum) by deep sequencing combined with microarray. Gene\| PubMed ID: 22575711	2012
Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses. Viruses\| PubMed ID: 26308041	2015
NtPHYB1, a homologous gene of Arabidopsis PHYB, positively regulates the content of phenolic compounds in tobacco. Plant physiology and biochemistry : PPB\| PubMed ID: 27636822	2016
Integrated analysis of tobacco miRNA and mRNA expression profiles under PVY infection provids insight into tobacco-PVY interactions. Scientific reports\| PubMed ID: 28687775	2017
Cas9-PF, an early flowering and visual selection marker system, enhances the frequency of editing event occurrence and expedites the isolation of genome-edited and transgene-free plants. Plant biotechnology journal\| PubMed ID: 30963647	2019

Yushuang Guo

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