CRISPR-based resistance to grapevine virus A

Abstract

Introduction: Grapevine (Vitis vinifera) is an important fruit crop which contributes significantly to the agricultural sector worldwide. Grapevine viruses are widespread and cause serious diseases which impact the quality and quantity of crop yields. More than 80 viruses plague grapevine, with RNA viruses constituting the largest of these. A recent extension to the clustered regularly interspaced, short palindromic repeat (CRISPR) armory is the Cas13 effector, which exclusively targets single-strand RNA. CRISPR/Cas has been implemented as a defense mechanism in plants, against both DNA and RNA viruses, by being programmed to directly target and cleave the viral genomes. The efficacy of the CRISPR/Cas tool in plants is dependent on efficient delivery of its components into plant cells.

Methods: To this end, the aim of this study was to use the recent Cas13d variant from Ruminococcus flavefaciens (CasRx) to target the RNA virus, grapevine virus A (GVA). GVA naturally infects grapevine, but can infect the model plant Nicotiana benthamiana, making it a helpful model to study virus infection in grapevine. gRNAs were designed against the coat protein (CP) gene of GVA. N. benthamiana plants expressing CasRx were co-infiltrated with GVA, and with a tobacco rattle virus (TRV)-gRNA expression vector, harbouring a CP gRNA.

Results and discussion: Results indicated more consistent GVA reductions, specifically gRNA CP-T2, which demonstrated a significant negative correlation with GVA accumulation, as well as multiple gRNA co-infiltrations which similarly showed reduced GVA titre. By establishing a virus-targeting defense system in plants, efficient virus interference mechanisms can be established and applied to major crops, such as grapevine.

Keywords: CRISPR/Cas; Cas13; CasRx; GIGS; GVA interference; virus interference; virus resistance.

Figure 1

Schematic representing the transient expression experiment and the stable expression experiment. Agrobacterium mixtures containing the binary pCasRx construct harbouring CasRx and a desired gRNA (or without gRNA for the control), and the GVA infectious clone, were mixed in a 1:1 ratio and infiltrated into wild-type N. benthamiana (left). Agrobacterium mixture of pTRV1 and pTRV2 harbouring a desired gRNA were mixed in a 1:1 ratio and infiltrated into N. benthamiana stably-expressing CasRx (CasRx-EMPTY plants), followed by the GVA infectious clone 48 hours later. Created with BioRender.com.

Figure 2

Agrobacterium-mediated stable transformation and regeneration of wild-type N. benthamiana leaf disc organogenesis process. (A) Leaf discs on co-cultivation medium immediately after incubation with the Agrobacterium harbouring the respective constructs. (B) Callus stage with shoots beginning to form on selective medium. (C) Isolated shoots on rooting medium containing selection. (D) Fully regenerated putative transgenic plantlet on selective medium.

Figure 3

A schematic representing the GVA genome organization and the designed gRNAs, and the assembled constructs. (A) GVA genome organization, ORF1 encodes replication-related proteins; ORF2 encodes a protein with an unknown function; ORF3 encodes the movement protein (MP); ORF4 encodes the CP, and ORF5 encodes a silencing-suppressor protein. Three gRNAs were designed to target ORF4 of GVA. The seed region of the gRNA sequence is represented in red. The black horizontal lines between the gRNA and target RNA represent the nucleotides complementing single-stand RNA. (B) Schematic representation of the T-DNA region of the pCasRx-gRNA or pCasRx-EMPTY constructs, assembled using Golden Gate cloning. The neomycin phosphotransferase type II gene (NPTII) is driven by 2x35S promoters. The cauliflower mosaic virus (CaMV) 35S promoter drives the CasRx expression, while the human U6 (hU6) promoter is responsible for expressing the respective gRNA and gRNA scaffold. NLS, nuclear localization signal; LB, left border; RB, right border. Created with BioRender.com.

Figure 4

Relative quantification of GVA for transient experiment analysis. The control was infiltrated with the pCasRx-ns vector, while the test sample was infiltrated with pCasRx : CP-T1. The error bars represent the minimum and maximum values of n=3 technical replicates. Statistical analysis performed using a two-tailed unpaired Student’s t-test; with the difference being significant at p ≤ 0.05 with respect to the control group. * p=0.0163, ** p=0.0019, *** p=0.0004.

Figure 5

Gene expression analysis of stably-transformed N. benthamiana (CasRx-EMPTY plants). (A) Relative fold expression of GVA and T1, subsequent to the GVA clone and pTRV2:CP-T1 co-infiltration into CasRx-EMPTY plants. ** p<0.0027, **** p<0.0001. (B) Relative fold expression of GVA and T2, subsequent to the GVA clone and pTRV2:CP-T2 co-infiltration in CasRx-EMPTY plants. * p=0.0038, *** p<0.0005. Correlation coefficient (r) is –0.7738 (p=0.024). (C) Relative fold expression of GVA and T3, subsequent to GVA and pTRV2:CP-T3 co-infiltration into CasRx-EMPTY plants. The error bars represent the minimum and maximum values of n=3 technical replicates. Statistical analysis performed using a two-tailed unpaired Student’s t-test; with the difference being significant at p ≤ 0.05 with respect to the control group. Correlation analysis performed using the Pearson correlation coefficient (r); with the correlation being significant at p ≤ 0.05. ** p<0.0095, *** p=0.0007.

Figure 6

GVA interference by CRISPR/CasRx compared to guide-induced gene silencing. (A) Relative fold expression of GVA in CasRx-EMPTY plants, normalized to the reference sample ‘control 1’. Samples ‘control 1 to 3’ infiltrated with TRV-EMPTY, and GVA two-dpi. Samples labelled ‘sample 1 to 4’ infiltrated with the multi-gRNA TRV mixture, and GVA two-dpi. The error bars represent the minimum and maximum values of n=3 technical replicates. (B) Relative quantification of GVA expression in wild-type N. benthamiana. Data represents the mean relative gene expression, ± SEM of the biological replicates (n=3). (C) Relative quantification of GVA expression of the ‘two-gRNA’ samples in CasRx-EMPTY and wild-type plants. The control sample was infiltrated with TRV-EMPTY, and GVA two-dpi, in wild-type N. benthamiana. Data represents the mean relative gene expression, ± SEM of the n=3 samples (controls), n=3 samples (GIGS) and n=5 samples (two-gRNA). (D) Relative quantification of GVA expression of the ‘three-gRNA’ samples in CasRx-EMPTY and wild-type plants. The control sample was infiltrated with TRV-EMPTY, and GVA two-dpi, in wild-type N. benthamiana. Data represents the mean relative gene expression, ± SEM of the n=3 samples (controls), n=3 samples (GIGS) and n=5 samples (three-gRNA). Statistical analysis performed using a two-tailed unpaired Student’s t-test; with the difference being significant at p ≤ 0.05 with respect to the control.

Figure 7

Relative expression levels of the T2 gRNA from a regular T-DNA binary vector and a TRV viral vector. (A) Relative fold expression of T2 gRNA expressed under the hU6 promoter, from the T-DNA of the pCasRx : CP-T2 vector, compared to the expression of T2 gRNA under the PEBV promoter, from the pTRV2:CP-T2 vector. The error bars represent the minimum and maximum values of n=3 technical replicates. (B) Relative quantification of T2 gRNA expression of the T-DNA samples and TRV2 samples grouped biologically. Data represents the mean relative gene expression, ± SEM of the three and four biological replicates, respectively (n=3, n=4). Statistical analysis performed using a two-tailed unpaired Student’s t-test; with the difference being significant at p ≤ 0.05 with respect to the TRV2 group. *** p=0.0008.