Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize

Abstract

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

Figure 1

Schematic representation of viral constructs derived from CMV. A, Schematic representation of the full-length cDNA clones of CMV. pCMVZ1, pCMVZ2, and pCMVZ3 were constructed from CMV-ZMBJ, whereas pCMVF1, pCMVF2, and pCMVF3 were from CMV-Fny. pCMVFZ3 and pCMVZF3 were two hybrid constructs. CMV genome encodes four proteins: 1a, 2a, MP, and coat protein (CP). Each vector contains double Cauliflower mosaic virus (CaMV) 35S promoters (2 × 35S, indicated by arrow heads) and a ribozyme sequence (Rz) derived from Tobacco ring spot virus (TRSV). B, The organization of pCMVZ2_2bN81 (Wang et al., 2016) for use in Pr CMV VIGS system. The MCS was used for the insertion of target gene. C, The organization of pCMVZ2_2bN81-LIC for use in Pr CMV-LIC-based VIGS system. The LIC site was used for the insertion of target gene by LIC approach (Dong et al., 2007). Nucleotide sequences underlined represent the LIC adaptors.

Figure 2

CMV infection in maize plants. A–K, Leaf phenotypes of maize infected with CMV. Plants at 2–3-leaf stage were inoculated with saps of N. benthamiana leaves agroinfiltrated with CMV. Plant leaves were photographed at 35 dpi for each pseudorecombinant and chimeric CMV. Scale bars are 2 cm. L, Western blot analysis of CP in maize plants infected with CMV at 14 and 28 dpi. The protein expression of eukaryotic translation initiation factor 4A (eIF4A) was used as the internal control. F, viral RNA from CMV-Fny; Z, viral RNA from CMV-ZMBJ; FZ, chimeric viral RNA with 5′-UTR and MP gene from Fny-RNA3 and CP gene and 3′-UTR from ZMBJ-RNA3; ZF, chimeric viral RNA with 5′-UTR and MP gene from ZMBJ-RNA3 and CP gene and 3′-UTR from Fny-RNA3.

Figure 3

Silencing of the ZmIspH gene in maize by Pr CMV VIGS. A and B, Phenotypes of maize plants infected with Pr CMV::ZmIspH or Pr CMV::LUC control. Photographs were taken at 14 dpi for the first systemic leaf (1SL), at 21 dpi for the 2SL, at 28 dpi for the 3SL, at 35 dpi for the 4SL, at 42 dpi for the 5SL, at 49 dpi for the 6SL, at 56 dpi for the 7SL and at 105 dpi for the 13SL, respectively. Scale bars are 2 cm in (A) and 10 cm in (B). C, RT-qPCR quantification of relative mRNA levels of ZmIspH gene in maize infected with Pr CMV::ZmIspH or Pr CMV::LUC control. Relative mRNA levels of ZmIspH were normalized against those of elongation factor 1-alpha (EF1α) as the reference gene. The bars represent the mean ± SE from at least three independent experiments. In all panels, black asterisks denote significant differences with respect to the control plants infected with Pr CMV::LUC determined by Student’s t test at ***P < 0.001. D, RT-PCR analysis of CP from leaves of maize infected with Pr CMV::ZmIspH or Pr CMV::LUC control. Plant EF1α mRNA was used as the internal control. Lane M is the 2 kb plus DNA marker.

Figure 4

Genetic stability of gene insertion of ZmIspH in upper infected with Pr CMV::ZmIspH. Leaf samples were collected at 14 dpi for the first systemic leaf (1SL), at 21 dpi for the 2SL, at 28 dpi for the 3SL, at 35 dpi for the 4SL, at 42 dpi for the 5SL, at 49 dpi for the 6SL, at 56 dpi for the 7SL, and at 105 dpi for 13SL, respectively. Total RNA was isolated from the samples and subjected to RT-PCR amplification with primers flanking the insertion of ZmIspH (upper panel). The vector plasmid with the ZmIspH insert or no sequence insertion was amplified as the positive (lane PC) or negative (lane NC) control. CP was amplified and used as an internal control (lower panel). Lane M is the 2 kb plus DNA marker.

Figure 5

Silencing of the ZmIspH, ZmChlH, and ZmPDS genes in maize by Pr CMV-LIC VIGS vectors. A, Leaf phenotype of maize infected with Pr CMV-LIC::ZmIspH, Pr CMV-LIC::ZmChlH, Pr CMV-LIC::ZmPDS, or Pr CMV-LIC::LUC control. The second systemic leaf (2SL) was photographed 21 dpi for each construct. Scale bars are 2 cm. B, RT-qPCR quantification of relative mRNA levels of target genes in maize infected with Pr CMV-LIC VIGS vectors. Relative mRNA levels of target gene (ZmIspH, ZmChlH, or ZmPDS) were normalized against those of EF1α as the reference gene. The bars represent the mean ± SE from at least three independent experiments. In all panels, black asterisks denote significant differences with respect to the control plants infected with Pr CMV-LIC::LUC determined by Student’s t test at ***P < 0.001. C, RT-PCR analysis of CP (upper panel) from leaves of maize infected with Pr CMV-LIC VIGS vectors. Plant EF1α mRNA was used as the internal control. Lane M is the 2 kb plus DNA marker.