Differences in the 3' intergenic region and the V2 protein of two sequence variants of tomato curly stunt virus play an important role in disease pathology in Nicotiana benthamiana

Abstract

Tomato production in South Africa is threatened by the emergence of tomato curly stunt virus (ToCSV), a monopartite Begomovirus transmitted by the whitefly vector Bemisia tabaci (Genn.). We investigated the role of sequence differences present in the 3' intergenic region (IR) and the V2 coding region on the differing infectivity of ToCSV sequence variant isolates V30 and V22 in the model host Nicotiana benthamiana. Using virus mutant chimeras, we determined that the development of the upward leaf roll symptom phenotype is mediated by sequence differences present in the 3' IR containing the TATA-associated composite element. Sequence differences present in the V2 coding region are responsible for modulating disease severity and symptom recovery in V22-infected plants. Serine substitution of V22 V2 Val27 resulted in a significant increase in disease severity with reduced recovery, the first study to demonstrate the importance of this V2 residue in disease development. Two putative ORFs, C5 and C6, were identified using in silico analysis and detection of an RNA transcript spanning their coding region suggests that these ORFs may be transcribed during infection. Additional virus-derived RNA transcripts spanning multiple ORFs and crossing the boundaries of recognised polycistronic transcripts, as well as the origin of replication within the IR, were detected in ToCSV-infected plants providing evidence of bidirectional readthrough transcription. From our results, we conclude that the diverse responses of the model host to ToCSV infection is influenced by select sequence differences and our findings provide several avenues for further investigation into the mechanisms behind these responses to infection.

Fig 1. Comparison of ToCSV sequence variant isolates V30 and V22.

(A) Genome organisation of V30 (left) and V22 (right). Accepted virion-sense and complementary-sense ORFs indicated with bold labels, putative C5 and C6 ORFs indicated with italicised labels. Intergenic region (IR) highlighted in grey with stem-loop element containing origin of replication (Ori) shown. Recombinant fragment present in V22 spanning part of the 3’ IR and the first half of the V2 ORF boxed in yellow. (B) Pairwise partial 3’ IR nt sequence alignment for V30 and V22 done using MUSCLE alignment program with manual adjustments. Conserved region containing TATA box in green. TATA-associated composite element (TACE) in blue. V2 start codon in magenta. (C) Pairwise V2 protein aa sequence alignment generated using PRALINE with colour key indicating aa conservation. Hydrophobic domains (H1 and H2), putative protein kinase C (PKC) phosphorylation motif, and CxC motif boxed in black. Hyphens indicate alignment-generated gaps. Asterisks indicate sequence conservation.

Fig 2. Single infection of Nicotiana benthamiana with V30 intergenic region (IR)-swap mutant infectious clones.

(A) Symptoms in plants inoculated with V30, V30ΔIR-s, and V30ΔIR-sr at 20 dpi (top row) and 36 dpi (middle and bottom rows). Bar = 2 cm. Mock inoculated with Agrobacterium tumefaciens C58C1 carrying empty pCAMBIA2300. (B) Corresponding upward leaf roll (ULR) and swollen vein (SV) severity scores at four-day intervals from 12 to 36 dpi. (C) Log viral fold change at 20 and 28 dpi. Bars represent mean ± SD. Student’s t-test levels of significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3. Single infection of Nicotiana benthamiana with V22 intergenic region (IR)-swap mutant infectious clones.

(A) Symptoms in plants inoculated with V22, V22ΔIR-s, and V22ΔIR-sr at 20 dpi (top and middle rows) and 36 dpi (bottom row). Bar = 2 cm. Mock inoculated with Agrobacterium tumefaciens C58C1 carrying empty pCAMBIA2300. (B) Corresponding upward leaf roll (ULR) and swollen vein (SV) severity scores at four-day intervals from 12 to 36 dpi. (C) Log viral fold change at 20 and 28 dpi. Bars represent mean ± SD. Student’s t-test levels of significance: *, p < 0.05; **, p < 0.01.

Fig 4. Single infection of Nicotiana benthamiana with V30 V2-swap mutant infectious clone.

(A) Symptoms in plants inoculated with V30, and V30ΔV2-s at 20 dpi (top row) and 36 dpi (middle and bottom rows). Bar = 2 cm. Mock inoculated with Agrobacterium tumefaciens C58C1 carrying empty pCAMBIA2300. (B) Corresponding upward leaf roll (ULR) and swollen vein (SV) severity scores at four-day intervals from 12 to 36 dpi. (C) Log viral fold change at 20 and 36 dpi. Bars represent mean ± SD. Student’s t-test levels of significance: *, p < 0.05; **, p < 0.01.

Fig 5. Single infection of Nicotiana benthamiana with V22 V2-swap mutant and V2 site-directed mutant infectious clones.

(A) Symptoms in plants inoculated with V22, V22ΔV2-s, V22ΔV2-V27S, and V22ΔV2-T58S at 20 dpi (top row) and 36 dpi (middle and bottom rows). Bar = 2 cm. Mock inoculated with Agrobacterium tumefaciens C58C1 carrying empty pCAMBIA2300. (B) Corresponding upward leaf roll (ULR) and swollen vein (SV) severity scores at four-day intervals from 12 to 36 dpi. (C) Log viral fold change at 20 and 36 dpi. Bars represent mean ± SD. Student’s t-test levels of significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 6. Identification of virus-derived RNA transcripts in V30-infected Nicotiana benthamiana using strand-specific RT-PCR.

V30 dsDNA on the left with virion-sense (vs) ORFs in blue, complementary-sense (cs) ORFs in red, and IR in grey. Corresponding positions of virus-derived RNAs as RT-PCR products (R1-R8) depicted relative to V30 ssDNA vs and cs strands with 5′-3′ direction indicated. Agarose gel images of respective RT-PCR products shown on the right. M, DNA molecular weight marker; tDNA, PCR of total DNA extracted from plant inoculated with V30 (positive control); cDNA, RT-PCR of total RNA extracted from plants inoculated with V30 or mock; RT-, negative reverse transcriptase enzyme control; NTC, PCR no template control. Mock is RT-PCR of total RNA extracted from plant inoculated with Agrobacterium tumefaciens C58C1 carrying empty pCAMBIA2300.