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. 2023 Mar 30:14:1164921.
doi: 10.3389/fpls.2023.1164921. eCollection 2023.

Artificial microRNA-mediated resistance against Oman strain of tomato yellow leaf curl virus

Maha R Al-Roshdi  1 , Ume Ammara  1 , Jamal Khan  1 , Abdullah M Al-Sadi  1 , Muhammad Shafiq Shahid  1
Affiliations

Artificial microRNA-mediated resistance against Oman strain of tomato yellow leaf curl virus

Maha R Al-Roshdi et al. Front Plant Sci. .

Abstract

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman.

Keywords: RNA interference; agrobacterium-infiltration; artificial microRNA; gene silencing; southern blotting.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of Rep-amiRNA1 (A) and Rep-amiRNA2 (B) cloned at the EcoRI/HindIII site of the pCAMBIA2300 plant expression vector, comprising the 35S2 promoter, Nos terminator, and NptII as selectable markers (C).
Figure 2
Figure 2
A fragment of 8.7 kb of expression vector pCAMBIA2300 Rep-amiRNA1 (Lane 1) and Rep-amiRNA2 (Lane 2) with a size of 300 bp. M= DNA marker (A). Agrobacterium transformed with pCAMBIA2300 harboring Rep-amiRNA1 (Lane 1-4) and Rep-amiRNA2 (Lane 5-8). A fragment of 8.7 kb and 300 bp was yielded. M=DNA marker (B).
Figure 3
Figure 3
Transient assay with Rep-amiRNA1 and Rep-amiRNA2 constructs in N. benthamiana plants agroinfiltrated with Rep-amiRNA1 (B) and Rep-amiRNA2(C). (A) is a healthy plant, shown here in comparison with a symptomatic plant (D).
Figure 4
Figure 4
Southern hybridization blotting investigation of N. benthamiana plants inoculated with Rep-amiRNA1 (Lane 3-7) and Rep-amiRNA2 (Lane 8-11) and challenged with TYLCV. DNA from symptomatic plant inoculated with TYLCV only (Lane 2). Lane 1 is plasmid DNA. The position of single-stranded (ss), supercoiled (sc), and linear (lin) viral DNA forms is indicated with arrow. N= Negative control (A). Southern blot analysis of F1-transformed tomato plants inoculated with Rep-amiRNA1 (Lane 4-8) and Rep-amiRNA2(Lane 3) and challenged with TYLCV-OM. DNA from symptomatic plant inoculated with TYLCV-OM as control (Lane 2). Lane 1 is plasmid DNA. N= Negative control. The position of single-stranded (ss), supercoiled (sc), and linear (lin) viral DNA forms is indicated with arrow (B).
Figure 5
Figure 5
Tomato transformation. (A) Seedlings of tomato germinated on MS medium. (B) Cotyledon explants on pre-culture medium. (C) Cotyledon explants on selection medium after co-cultivation with Agrobacterium. (D, E) Callus with regeneration shoots on selection medium. (F) Shoots regenerating from a single callus growing on selection medium. (G) Cotyledon explant was discarded. (H) Young plantlet on shoot elongation medium. (I) Plantlet with well-developed roots on rooting medium. (J) Fully regenerated plant hardened in soil. (K) Two months after hardening. (L) Plant at flowering stage. (M, N) Mature transgenic tomato plants with fruits.
Figure 6
Figure 6
PCR results for Rep-amiRNA1 construct amplified from transformed tomato plants. Amplicon of 300bp was observed in 11 plants transformed with Rep-amiRNA1 construct. P1, P2= Positive control, N= Negative control, M= 1kb marker DNA (A). PCR products were analyzed for Rep-amiRNA1 and Rep-amiRNA2 constructs amplified from transformed tomato plants. Amplicon of 300bp was observed in 10 plants transformed with Rep-amiRNA1 (Lane 1-9, 11) and three plants transformed with Rep-amiRNA2 (Lane 12, 13, 15). P1, P2= Positive control, N= Negative control, M= 1kb marker DNA (B). PCR products were analyzed for Rep-amiRNA1 and Rep-amiRNA2 constructs amplified from transformed tomato plants. Amplicon of 300 bp was observed in three plants transformed with Rep-amiRNA1 (Lane 1, 3, 9) and two plants transformed with Rep-amiRNA2 (Lane 5, 6). P1, P2= Positive control, N= Negative control, M= 1kb marker DNA (C).
Figure 7
Figure 7
Electron micrograph of a phloem (A) and parenchymal (B) cells from agroinoculated, symptomless transgenic plant. Nucleolus is obvious in the nucleus “N”, and a small clump “C” is also visible.
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