Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots

Abstract

Aim: We report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots.

Background: VIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function.

Results: We developed an African cassava mosaic virus-based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots.

Conclusions: The African cassava mosaic virus-based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.

Keywords: ACMV; Agrobacterium tumefaciens; Agroinoculation; Cassava; Geminivirus; Gene silencing; VIGS.

Fig. 1

Construction of a VIGS vector based on the African Cassava Mosaic Virus (ACMV) infectious clone ACMV-[NOg]. a The genome of this bipartite geminivirus was previously cloned into two pCambia 1300 plasmids containing either the DNA-A or DNA-B ACMV-[Nog] sub-genomes CRA: Common Region of DNA-A; AV1: coat protein gene; AV2: Protein V2 gene; AC3: Replication enhancer protein gene; AC2: Transcriptional activator protein gene; AC1: Replication associated protein gene; AC4: RNA silencing suppressor gene [24]. b The cassava VIGS vector was constructed by modifying the DNA-A sub-genome by replacing part of the AV1 gene with a 30-bp multiple cloning site (MCS) for insertion of gene targeting DNA fragments

Fig. 2

Overview of the VIGS assay in cassava. Six week-old cassava plants were injected with a 29G syringe filled with a suspension of mixed Agrobacteria containing either the DNA-A (VIGS vector) or DNA-B plasmids in at least three sites close to axillary meristems. Additional superficial cuts in the lower part of the stem were made using the same syringe to induce the T-DNA release from the Agrobacteria

Fig. 3

VIGS assays using leaves from infected transgenic 35S::uidA cassava plants. a Mock treatment, b infection with DNA-A + DNA-B, c infection with VIGS-uidA + DNA-B, d infection with VIGS-35S + DNA-B. GUS staining of representative leaves from VIGS-infected plants 2 months post-inoculation. One of the three biological replicates per treatment is shown. Infection rates are presented in Additional file 1: Table S1 A, E and F

Fig. 4

VIGS assays using roots of infected transgenic 35S::uidA cassava plants. a Mock treatment, b infection with DNA-A + DNA-B, c infection with VIGS-uidA + DNA-B, d infection with VIGS-35S + DNA-B. Adventitious roots from sterile stem cuttings of VIGS-infected cassava were grown in tissue culture and tested for GUS activity after 4 weeks. Intermediate and storage roots of VIGS-infected cassava plants grown in the greenhouse were taken from the soil and tested for GUS activity 6 months post-inoculation. One of the three biological replicates per treatment is shown. Infection rates are presented in Additional file 1: Table S1 A, E and F

Fig. 5

Summary of the VIGS assay for silencing of the Chl1 gene in wild-type cassava. Representative images of control and infected leaves showing silencing of the Chl1 gene encoding the Mg²⁺-chelatase enzyme (Manes.17G053100). Images were recorded 2 months post-inoculation. Infection rates are presented in Additional file 1: Table S1 D

Fig. 6

Evaluation of silencing in leaves, fibrous roots, root tips and storage roots for a leaf- and root-expressed gene in wild-type cassava. . Plants infected with VIGS constructs targeting different endogenous genes were evaluated by RT-qPCR 6 months p.i. a Chl1 gene, coding for the Mg²⁺-chelatase (Manes.17G053100). b Lysin motif (LysM) receptor kinase gene (Manes.12G071800). Control plants were infected with the VIGS-uidA construct, targeting the uidA transgene. Bars represent means and standard deviations of expression levels calculated according to the Pfaffl method (2001) from three biological replicates displaying CMD symptoms (asterisks indicate significant differences in two-tailed t tests, p <0.05). The reference gene used for normalization is PP2A (Manes.09G039900). Fold change in gene expression levels as compared to the VIGS-uidA control samples is indicated above the bar for the biological samples displaying a significant reduction in gene expression. Infection rates are presented in Additional file 1: Table S1 D, E and G