Both Conserved Aromatic Nature and Critical RNA Motif of Residue 67 in the ToBRFV Movement Protein May Be Essential for Evading Tm-22-Mediated Resistance.

The 68th amino acid residue (cysteine, C68) in tobacco mosaic virus (TMV) movement protein (MP) and the corresponding position in some tobamoviruses are required to trigger Tm-22-mediated resistance. In contrast, the 67th amino acid residue (histidine, H67) in tomato brown rugose fruit virus (ToBRFV) MP enables evasion, although its key property remains unclear. To assess the key property of this site, a saturated mutant pool was generated at ToBRFV MP H67 and TMV MP C68, along with two single mutants, H67C and C68H. Infection assays were conducted using two tomato cultivars, Jingpeng No. 1 (JP, Tm-22/tm-2) and Rutgers (RG, tm-2/tm-2), as well as wild-type (WT) and Tm-22 transgenic Nicotiana benthamiana. Progeny sequencing revealed species- and cultivar-specific outcomes. The mutants TAC and TAT (encoding the aromatic amino acid tyrosine, Y) and CAC (encoding the WT histidine, H), were the only ones detected in JP tomato and Tm-22 N. benthamiana. In single-infection assays, ToBRFV carrying TTC (encoding phenylalanine, F) at this position infected JP tomato, TAC (Y) enabled infection of Tm-22 N. benthamiana. TGG (encoding tryptophan, W) infected both JP tomato and Tm-22 N. benthamiana. Moreover, CAT (H) but not CAC (H), and TTC (F) but not TTT (F) infected JP plants, highlighting the role of the critical RNA motif. This suggests that the aromatic moiety and critical RNA motif at this position are essential for evading Tm-22-mediated resistance, as the imidazole ring of the WT histidine also exhibits aromatic character. Therefore, Tm-22 may be engineered to recognize aromatic amino acids at this position to confer resistance to ToBRFV.