TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation

Abstract

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A-TOM1-TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.

Keywords: ToMV; evolution; genome editing; host factor; resistance; tobacco mosaic virus.

FIGURE 1

Phylogeny of TOM1 family members in Nicotiana benthamiana and multiplex mutation of members does not affect plant growth. (a) Phylogenetic analysis of TOM1 homologues in N. benthamiana. Predicted full‐length TOM1 protein sequences from N. benthamiana and Arabidopsis thaliana were used for phylogenetic tree construction. Arabidopsis TOM1 homologues are indicated in red. Bootstrap values are indicated with red numbers. (b) Schematic representation of CRISPR/Cas9 construct used for TOM1s engineering. The construct contains seven tandemly arrayed gRNAs that target the nine TOM1 homologues in N. benthamiana. tRNAs are indicated with rounded rectangles of different colours; gRNA are indicated with rhombus of different colours; gRNA scaffolds are indicated with green rectangles; AtU6‐26p, Arabidopsis U6‐26 promoter; U6‐26t, Arabidopsis U6‐26 terminator; 35Sp, 35S promoter; NPTII, neomycin phosphotransferase II. (c) CRISPR/Cas9‐induced mutations in NbTOM1 homologues. gRNA and protospacer adjacent motif (PAM) sequences are indicated with red and green letters, respectively; gaps are indicated with dash; dots indicate nucleotides that are not shown. Sequence mutations in each TOM1 homologues were indicated at the end of each sequence. Three other TOM1 homologues (NbTOM1‐L3, L4 and L5) that are not successfully engineered are not shown. (d) Phenotype of wild‐type and sextuple mutant (n64) plants without virus inoculation. Photographs were taken 52 days after germination. Scale bar, 4 cm.

FIGURE 2

Knockout of the NbTOM1 family enhanced TMV and ToMV resistance. (a) Phenotype of wild‐type (WT) and n64 mutant leaves co‐infiltrated with TMV‐GFP + empty vector (EV) or TMV‐GFP + NtTOM1 at 4 days postinfiltration (dpi). GFP fluorescence was viewed under a portable UV light source at 4 dpi. Infiltration was performed on the third and fourth leaves of 30‐day‐old plants. At least three biological replicates were performed. Photographs were taken at 4 dpi. Representative photographs are shown. (b) Immunoblot analysis of TMV coat protein (CP) accumulation from infiltrated Nicotiana benthamiana leaves at 4 dpi. TMV levels accumulated in the infiltration sites were analysed using immunoblotting with anti‐TMV coat protein (CP) antibody as previously described (Hu et al., 2021). An immunoblot using anti‐Actin antibody was used as loading control. (c) Phenotype of wild‐type and n64 mutant leaves co‐infiltrated with TMV‐GFP + EV or TMV‐GFP + NtTOM1 at 7 dpi. The yellow arrows indicate the inoculated leaves; the red arrows indicate the upper noninoculated leaves. TMV‐GFP + EV and TMV‐GFP + NtTOM1 are infiltrated on the left and right sides of inoculated leaves, respectively. (d) Phenotype of wild‐type and n64 plants inoculated with ToMV sap at 4 dpi and 7 dpi. Yellow and white arrowheads indicate the inoculated and systemic leaves, respectively. At least three biological replicates were performed for each experiment. Representative photographs are shown. (e) ToMV viral RNA accumulation in wild‐type and n64 systemic leaves at 4 dpi.

FIGURE 3

Influence of NbTOM1 homologues on TMV accumulation. (a) Schematic illustration of the infiltration sites on n64 mutant leaves. NC, infiltration site where TMV‐GFP and empty vector (EV) were co‐infiltrated, which served as negative control. PC, infiltration sites where TMV‐GFP and NtTOM1 from Nicotiana tabacum were co‐infiltrated, which served as positive control; TOM1, infiltration site where TMV‐GFP and TOM1 homologues were co‐infiltrated. (b–j) Phenotypes of n64 leaves co‐infiltrated with TMV‐GFP and TOM1 homologues from Nicotiana benthamiana. Organization of the infiltration sites is indicated in Figure 3a. (k) Immunoblot analysis of TMV CP from infiltrated n64 leaves shown in Figure 3b–j. TMV accumulation levels in the infiltration sites were analysed as in Figure 2b. The n64 leaves inoculated with TMV‐GFP + EV or TMV‐GFP + NtTOM1 were used as negative and positive control, respectively. Protein analysed in lane b through lane j was derived from the “TOM1” sites (where TMV‐GFP + TOM1 homologues were co‐infiltrated) of leaves shown in Figure 3b through Figure 3j. (l) Relative GFP fluorescence levels in n64 mutant leaves overexpressing NbTOM1 homologues. GFP fluorescence was quantified as previously described (Ye et al., 2022). The relative GFP fluorescence level was calculated as the ratio between GFP fluorescence obtained by overexpressing TOM1 homologues and NtTOM1. Different letters above bars indicate a statistically significant difference (p < 0.05) based on four different biological replicates. Statistical analysis was performed using the Tukey HSD test implemented in SPSS software. Error bars indicate standard deviation (SD) based on four biological replicates. It should be noted that the relative GFP fluorescence in the positive control (TMV‐GFP + NtTOM1) was set to 1 in all biological replicates and thus the error bar for the positive control group was too small (SD = 0) to be seen above the boxplot.

FIGURE 4

Influence of TOM1 orthologues on the multiplication of TMV. (a–g) Phenotypes of n64 mutant leaves co‐infiltrated with TMV‐GFP and TOM1 orthologues from Coccomyxa subellipsoidea (a), Physcomitrella patens (b), Selaginella moellendorffii (c), Picea abies (d), Amborella trichopoda (e), Oryza sativa (f), and Solanum lycopersicum (g). Plant growth, experimental conditions, and figure presentation are the same as in Figure 3. (h) TMV accumulation levels in infiltrated leaves. The method for the detection of TMV levels in the infiltration sites is described in Figure 2. The n64 mutant leaves co‐infiltrated with TMV‐GFP + empty vector (EV) or TMV‐GFP + NtTOM1 were used as negative and positive control, respectively. Protein analysed in lane a to lane g was derived from the infiltration site “TOM1” (where TMV‐GFP + TOM1 orthologues were co‐infiltrated) of leaves shown in Figure 4a to Figure 4g. (i) Relative GFP fluorescence levels in n64 mutant leaves overexpressing TOM1 orthologues from seven plant species. Data were analysed and are presented in the same way as in Figure 3l.

FIGURE 5

Phylogenetic analysis of TOM1 homologues from eight plant species. Amino acid sequences from the conserved DUF1084 TOM1 domains were used for phylogenetic tree construction. The phylogenetic tree was constructed using the neighbour‐joining method with 1000 bootstrap replicates using MEGA v. 7. TOM1 homologues were named “AbcTOM1 or “AbcTOM1‐L#,” where “A” is the first letter of the genus name, “bc” is the first two letters of the species name, and “#” represents the serial number. Arabidopsis TOM1 homologues are indicated in red. The relevant species names and protein accession numbers are shown in parentheses. Gene names for the tomato TOM1 homologues used by Kravchik et al. (2022) and Ishikawa et al. (2022) are indicated to the right of the parentheses. The functionally characterized genes are indicated with green circles (promote TMV multiplication), blue triangles (partially promote TMV multiplication), or red triangles (could not promote TMV multiplication). The numbers associated with tree branches are bootstrap values.

FIGURE 6

Interaction between TOM1 and TOM2A or TOM1 and TMV replication protein. (a) Schematic representation of bait and prey constructs. The TOM1 orthologue was fused at its C‐terminus with Cub‐PLV and served as bait construct. TOM2A or TMV Hel was fused at its N‐terminus with a mutant form of the ubiquitin N‐terminal fragment (NubG) and served as prey construct. CYC1pro, cytochrome C‐1 (CYC1) promoter; CYC1ter, CYC1 terminator; SUC, cleavable signal sequence from the yeast SUC2 gene; Cub, C‐terminal fragment of ubiquitin; PLV, a protein A‐LexA‐VP16 reporter polypeptide; NubG, mutant form of the ubiquitin N‐terminal fragment. (b, c) Split‐ubiquitin membrane yeast two‐hybrid (MbY2H) assay showing the interactions between TOM1 and TOM2A (b) and between TOM1 and TMV Hel (c). The bait construct (TOM1‐Cub‐PLV) was transformed together with prey constructs (NubG‐TOM2A or NubG‐TMV Hel) using a standard lithium transformation method. Co‐transformation of NtTOM1‐Cub‐PLV with either pPR3‐N or pAI‐AIg5 served as negative and positive control, respectively. Co‐transformation of pBT3‐N and NubG‐SlyTOM2A or TMV Hel served as another negative control. After transformation, yeast was grown on SD/−Trp−Leu and protein interaction was analysed on SD/−Trp−Leu−His−Ade. Photographs were taken 4 days after plating.

FIGURE 7

The N‐terminal fragment of TOM1 is crucial for TMV multiplication. (a) Schematic representation of the structure of PpaTOM1‐L1 and its variants. TM, transmembrane domain; blue and green boxes represent PpaTOM1‐L1 and SlyTOM1‐L1 fragments, respectively. (b) Contribution of Sl‐PpaTOM1 chimeric variants to TMV accumulation. Thirty‐day‐old n64 mutant leaves were infiltrated with Agrobacterium containing appropriate constructs and TMV‐GFP. Photographs were taken at 4 days postinfiltration. Each leaf was infiltrated on four different sites. EV, TMV‐GFP + empty vector; NC, TMV‐GFP + PpaTOM1‐L1; PC, TMV‐GFP + SlyTOM1‐L1; Var, TMV‐GFP + Sl‐PpaTOM1 chimeric variant. (c) Relative GFP fluorescence levels in Sl‐PpaTOM1 chimeric variants shown in Figure 7b. Data were analysed and are presented in the same way as in Figure 3l. (d) Schematic representation of Sl‐PpaTOM1 chimeric variants with their N‐termini replaced by the corresponding fragments from SlyTOM1‐L1. (e) Contribution of Sl‐PpaTOM1 chimeric variants to TMV accumulation. Representation of the infiltration sites and scoring of the contribution of each variant to TMV accumulation were performed in the same way as in Figure 7b. Four biological replicates were performed for each experiment. (f) Relative GFP fluorescence levels in Sl‐PpaTOM1 chimeric variants shown in Figure 7e. Data were analysed and are presented in the same way as in Figure 3l.