Activation of Tm-22 resistance is mediated by a conserved cysteine essential for tobacco mosaic virus movement

Abstract

The tomato Tm-2² gene was considered to be one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). However, an emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has overcome Tm-2² , damaging tomato production worldwide. Tm-2² encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MP^ToBRFV ) enabled the virus to overcome Tm-2² -mediated resistance. Yet, it was unknown how Tm-2² remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that a conserved cysteine (C68) in the MP of TMV (MP^TMV ) plays a dual role in Tm-2² activation and viral movement. Substitution of MP^ToBRFV amino acid H67 with the corresponding amino acid in MP^TMV (C68) activated Tm-2² -mediated resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae-infecting tobamoviruses except ToBRFV and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell and subcellular movement analysis showed that C68 is required for the movement of TMV by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-2² immune activation could explain how TMV was unable to overcome this resistance for such a long period.

Keywords: Tm-2 2; Tobamovirus; TMV; ToBRFV; movement protein; plasmodesmata.

FIGURE 1

Tm‐2 ² resistance is activated in the presence of movement protein amino acid C68. (a) Images of Nicotiana benthamiana leaves transiently expressing MP^TMV, MP^ToBRFV, MP^{ToBRFV H67C}, MP^{ToBRFV H67S} or MP^{TMV C68H} co‐expressed with an empty vector (−Tm‐2 ² ) or with Tm‐2 ² (+Tm‐2 ² ). Photographs were taken 48 h after infiltration. (b) Electrolyte leakage assay of N. benthamiana leaves expressing each construct along with an empty vector (−Tm‐2 ² ) or Tm‐2 ² (+Tm‐2 ² ). Different letters indicate significance in Tukey's HSD test (p < 0.05, n ≥ 5).

FIGURE 2

Conservation of C68 in sequence and structure. (a) Protein sequence alignment of Solanaceae‐infecting tobamovirus movement proteins. C68 is highlighted by a black arrow and ToBRFV H67 is highlighted by a black square. (b–d) AlphaFold models of MP^TMV (b), MP^ToBRFV (c), and four distant members of the 30K movement protein superfamily (MP^CaMV, MP^CMV, MP^TBSV, and MP^TSWV) (d). Purple marks the conserved β‐barrel fold. The white arrows in (b) show the hydrophobic endoplasmic reticulum association regions previously described by Peiro et al. (2014). The red arrow marks C68 in MP^TMV (b) and the yellow arrow marks H67 in MP^ToBRFV (c).

FIGURE 3

Substitution of movement protein (MP) amino acid C68 results in loss of ToMV and TMV infectivity in tomato and Nicotiana benthamiana. (a) Tomato plants (cv. Moneymaker) homozygous to the tm‐2 or Tm‐2 ² allele infected with ToMV, ToMV whose original movement protein was replaced by MP^ToBRFV, and ToMV with the C68H mutation in the MP open reading frame. (b) Western blot analysis of ToMV CP in young leaves of tm‐2 and Tm‐2 ² plants infected with ToMV expressing the various MPs. Images and leaf samples were taken at 13 days postinoculation (dpi). (c) White light (top) and UV light (bottom) images of N. benthamiana plants infected with TMV‐GFP infectious clones harbouring MP^TMV (left), MP^ToBRFV (middle), or MP^{TMV C68H} (right). (d) In vivo imaging‐based quantitative analysis of green fluorescent protein (GFP) fluorescence in systemic leaves (fourth leaf from the apex) of plants infected with TMV‐GFP harbouring the different movement proteins (MP^TMV, MP^ToBRFV, or MP^{TMV C68H}). Images were taken at 10 dpi. Different letters indicate significance in Tukey's HSD test (p < 0.05, n ≥ 4).

FIGURE 4

Substitution of MP^TMV amino acid C68 results in loss of viral cell‐to‐cell movement. (a–c) Cell‐to‐cell movement of the TMV‐GFP infectious clones harbouring MP^TMV (a), MP^ToBRFV (b), or MP^{TMV C68H} (c) at 5 days postinoculation. Magnification of the segmented section in the upper panel showing the failure of TMV‐GFP MP^{TMV C68H} to move out of the infected cell is shown in (c'). (d) Quantification of infection foci area. Different letters above bars indicate significance in Tukey's HSD test (p < 0.05, n ≥ 9). Scale: 100 μm.

FIGURE 5

C68 is essential for MP^TMV intercellular movement. (a–c) Z‐stack maximal projection confocal images of a Nicotiana benthamiana leaf epidermal cell expressing MP^TMV‐YFP (a), the nonmobile endoplasmic reticulum (ER)‐mCherry marker (b), and their overlay (c). (d–f) Z‐stack maximal projection confocal images of epidermal cells expressing MP^ToBRFV‐YFP (d), ER‐mCherry (e), and their overlay (f). (g–i) Z‐stack maximal projection confocal images of epidermal cells expressing MP^{TMV C68H}‐YFP (g), ER‐mCherry (h), and their overlay (i). (j) Quantitative analysis of the ratio between the area of MP^TMV‐YFP, MP^ToBRFV‐YFP, and MP^{TMV C68H}‐YFP signal and the area of ER‐mCherry signal. (k) Quantitative analysis of the number of cells per MP^TMV‐YFP or MP^ToBRFV‐YFP and MP^{TMV C68H}‐YFP cluster. Different letters indicate significance in Tukey's HSD test (p < 0.05, n ≥ 11). Scale: 100 μm.

FIGURE 6

C68 is essential for MP^TMV plasmodesmatal localization. Localization of MP^TMV‐YFP (a), MP^ToBRFV‐YFP (b), and MP^{TMV C68H}‐YFP (c) proteins (green) to plasmodesmata (PD) (purple). Plasmodesmata were labelled using aniline blue callose staining. Note that the MP^{TMV C68H}‐YFP (c) fails to specifically be targeted to PD. (c) Quantitative analysis of the accumulation of the various MP‐YFP proteins in PD. The calculation was based on the ratio between MP‐YFP signal in PD and the average fluorescent signal in the plasma membrane (PM). Different letters indicate significance in Tukey's HSD test (p < 0.05, n ≥ 25). Scale: 10 μm.

FIGURE 7

C68 is required for MP^TMV interaction with the endoplasmic reticulum (ER) and intracellular movement. Surface imaging of upper Nicotiana benthamiana leaf epidermal cell planes. (a–c) Expression of MP^TMV‐YFP (a), ER‐mCherry (b), and an overlay of both proteins (c). (d–f) expression of MP^{TMV C68H}‐YFP (d), ER‐mCherry (e), and an overlay of both proteins (f). (g) Quantitative analysis of MP^TMV‐YFP and MP^{TMV C68H}‐YFP colocalization with ER‐mCherry. (h) Dynamics of MP^TMV‐YFP (top) and MP^{TMV C68H}‐YFP (bottom) on the ER network. Note that while MP^TMV‐YFP are mobile on the ER, MP^{TMV C68H}‐YFP are immobile (yellow arrow). (i) Quantification of MP^TMV‐YFP and MP^{TMV C68H}‐YFP vesicle velocity. Scale = 5 μm (**p < 0.01, ****p < 0.0001 in Student's t test, n ≥ 24).