Bioinformatic-based approach for mutagenesis of plant immune Tm-22 receptor to confer resistance against tomato brown rugose fruit virus (ToBRFV).

Nucleotide-binding leucine-rich repeat (NLR) plant immune receptors mediate the recognition and activation of defense signaling pathways in response to intra- and extracellular pathogens. Several NLR such as Tm-2 and Tm-22 have been introgressed into commercial solanaceous varieties to confer protection against different tobamoviruses. Particularly, Tm-22 was used during recent decades to confer resistance against tobacco mosaic virus, tomato mottle mosaic virus and tomato mosaic virus, which recognizes the viral movement protein (MP). However, tomato brown rugose fruit virus(ToBRFV), a novel tobamovirus, can avoid the protection conferred by Tm-22 due to the presence of key substitutions in the MP. The aim of this work was to identify the key amino acid residues involved in the interaction between Tm-22 and ToBRFV MP through bioinformatic analyses, and to identify potential Tm-22 mutations that could generate greater binding affinity. In silico 3D structure prediction, molecular docking, and computational affinity methods were performed. We predicted that R350, H384 and K385 Tm-22 residues are relevant for the interaction with MP, and two mutations (H384W and K385L) were identified as putative sites to increase the affinity of Tm-22 to the MP with the potential elicitation of resistance against ToBRFV.

Characterization of a ToMV isolate overcoming Tm-22 resistance gene in tomato.

Tomato mosaic virus (ToMV) is easily transmitted in soil and by contact. By these reasons, it is relatively difficult to control ToMV disease in tomato. Incorporation of the Tm-22 gene has been widely used as a control method for ToMV, but ToMV isolates that overcome this resistance gene have been reported worldwide in recent years. In this study, we determined the entire nucleotide sequences of ToMV isolate [named ToMV-KMT (LC650928)], which was isolated from tomato plants showing symptoms of systemic necrosis in Kumamoto prefecture, Japan. We also analyzed the viral gene of ToMV-KMT that overcome the Tm-22 gene by constructing its infectious cDNA clone and by generating chimeric viruses with a non-breaking strain. According to previous research, Tm-22 recognizes the viral movement protein (MP) and exerts resistance by inducing hypersensitive reaction or hypersensitive cell death. We discovered that a mutation in the 240th amino acid (aspartic acid to tyrosine) of the MP of ToMV-KMT, which may stabilize the protein's structure, is responsible for the ability of this isolate to overcome the resistance of Tm-22.

Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harbouring the Tm-22 resistance gene.

Tomato cultivars containing the Tm-22 resistance gene have been widely known to resist tobacco mosaic virus (TMV) and tomato mosaic virus. Tomato brown rugose fruit virus (ToBRFV), a new emerging tobamovirus, can infect tomato plants carrying the Tm-22 gene. However, the virulence determinant of ToBRFV that overcomes the resistance conferred by the Tm-22 gene remains unclear. In this study, we substituted the movement protein (MP) encoding sequences between ToBRFV and TMV infectious clones and conducted infectivity assays. The results showed that MP was the virulence determinant for ToBRFV to infect Tm-22 transgenic Nicotiana benthamiana plants and Tm-22 -carrying tomato plants. A TMV MP chimera with amino acid residues 60-186 of ToBRFV MP failed to induce hypersensitive cell death in the leaves of Tm-22 transgenic N. benthamiana plants. Chimeric TMV containing residues 60-186 of ToBRFV MP could, but chimeric ToBRFV containing 61-187 residues of TMV MP failed to infect Tm-22 transgenic N. benthamiana plants, indicating that 60-186 residues of MP were important for ToBRFV to overcome Tm-22 gene-mediated resistance. Further analysis showed that six amino acid residues, H67 , N125 , K129 , A134 , I147 , and I168 of ToBRFV MP, were critical in overcoming Tm-22 -mediated resistance in transgenic N. benthamiana plants and tomato plants. These results increase our understanding of the mechanism by which ToBRFV overcomes Tm-22 -mediated resistance.

The Tomato Brown Rugose Fruit Virus Movement Protein Overcomes Tm-22 Resistance in Tomato While Attenuating Viral Transport.

Tomato brown rugose fruit virus is a new virus species in the Tobamovirus genus, causing substantial damage to tomato crops. Reports of recent tomato brown rugose fruit virus (ToBRFV) outbreaks from around the world indicate an emerging global epidemic. ToBRFV overcomes all tobamovirus resistances in tomato, including the durable Tm-22 resistance gene, which had been effective against multiple tobamoviruses. Here, we show that the ToBRFV movement protein (MPToBRFV) enables the virus to evade Tm-22 resistance. Transient expression of MPToBRFV failed to activate the Tm-22 resistance response. Replacement of the original MP sequence of tomato mosaic virus (ToMV) with MPToBRFV enabled this recombinant virus to infect Tm-22-resistant plants. Using hybrid protein analysis, we show that the elements required to evade Tm-22 are located between MPToBRFV amino acids 1 and 216 and not the C terminus, as previously assumed. Analysis of ToBRFV systemic infection in tomato revealed that ToBRFV spreads more slowly compared with ToMV. Interestingly, replacement of tobacco mosaic virus (TMV) and ToMV MPs with MPToBRFV caused an attenuation of systemic infection of both viruses. Cell-to-cell movement analysis showed that MPToBRFV moves less effectively compared with the TMV MP (MPTMV). These findings suggest that overcoming Tm-22 is associated with attenuated MP function. This may explain the high durability of Tm-22 resistance, which had remained unbroken for over 60 years.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Hsp90 Interacts With Tm-22 and Is Essential for Tm-22-Mediated Resistance to Tobacco mosaic virus.

The tomato resistance gene Tm-22 encodes a coiled coil-nucleotide binding site-leucine rich repeat type resistance protein and confers effective immune response against tobamoviruses by detecting the presence of viral movement proteins (MPs). In this study, we show that the Nicotiana benthamiana Heat shock protein 90-kD (Hsp90) interacts with Tm-22. Silencing of Hsp90 reduced Tm-22-mediated resistance to Tobacco mosaic virus (TMV) and the steady-state levels of Tm-22 protein. Further, Hsp90 associates with SGT1 in yeast and in plant cells. These results suggest that Hsp90-SGT1 complex takes part in Tm-22-mediated TMV resistance by functioning as chaperone to regulate Tm-22 stability.