Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava.

Hongqiu Zeng,Wei Hu,Guoyin Liu,Yunxie Wei,Haitao Shi,Yanwei Xie

doi:10.3390/ijms20163976

Abstract

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

Highlights

Aiming at analyzing genomic sequences and functions, functional genomics has rapidly developed as a result of sequencing projects of different species, especially of important crops
To investigate the effect of Green fluorescent protein (GFP) transient expression, the 35S::GFP vector [33] was transformed into Agrobacterium AGL-1 and GV3101, and the transformed strains were infiltrated into cassava leaves, respectively (Figure 1)
Genome sequencing of cassava has been completed for several years little progress has been made in determining the functional genomics of cassava [34]

Summary

Introduction

Aiming at analyzing genomic sequences and functions, functional genomics has rapidly developed as a result of sequencing projects of different species, especially of important crops. Agrobacterium-mediated transient overexpression is widely used in gene function studies in plants [3]. Green fluorescent protein (GFP) and β-glucuronidase (GUS) are two convenient reporter genes in biology which can be observed by fluorescent microscopy or direct staining [6,7] They can continue to express, reaching their peak at 3 days postinfiltration (dpi), and exert obvious expression for at least one week [4,8]. They can be efficiently expressed in a wide range of plant species.

Methods

Results

Conclusion