Interaction of EXA1 and eIF4E Family Members Facilitates Potexvirus Infection in Arabidopsis thaliana.

Plant viruses depend on a number of host factors for successful infection. Deficiency of critical host factors confers recessively inherited viral resistance in plants. For example, loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. However, the molecular mechanism of how EXA1 assists potexvirus infection remains largely unknown. Previous studies reported that the salicylic acid (SA) pathway is upregulated in exa1 mutants, and EXA1 modulates hypersensitive response-related cell death during EDS1-dependent effector-triggered immunity. Here, we show that exa1-mediated viral resistance is mostly independent of SA and EDS1 pathways. We demonstrate that Arabidopsis EXA1 interacts with three members of the eukaryotic translation initiation factor 4E (eIF4E) family, eIF4E1, eIFiso4E, and novel cap-binding protein (nCBP), through the eIF4E-binding motif (4EBM). Expression of EXA1 in exa1 mutants restored infection by the potexvirus Plantago asiatica mosaic virus (PlAMV), but EXA1 with mutations in 4EBM only partially restored infection. In virus inoculation experiments using Arabidopsis knockout mutants, EXA1 promoted PlAMV infection in concert with nCBP, but the functions of eIFiso4E and nCBP in promoting PlAMV infection were redundant. By contrast, the promotion of PlAMV infection by eIF4E1 was, at least partially, EXA1 independent. Taken together, our results imply that the interaction of EXA1-eIF4E family members is essential for efficient PlAMV multiplication, although specific roles of three eIF4E family members in PlAMV infection differ. IMPORTANCE The genus Potexvirus comprises a group of plant RNA viruses, including viruses that cause serious damage to agricultural crops. We previously showed that loss of Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana confers resistance to potexviruses. EXA1 may thus play a critical role in the success of potexvirus infection; hence, elucidation of its mechanism of action is crucial for understanding the infection process of potexviruses and for effective viral control. Previous studies reported that loss of EXA1 enhances plant immune responses, but our results indicate that this is not the primary mechanism of exa1-mediated viral resistance. Here, we show that Arabidopsis EXA1 assists infection by the potexvirus Plantago asiatica mosaic virus (PlAMV) by interacting with the eukaryotic translation initiation factor 4E family. Our results imply that EXA1 contributes to PlAMV multiplication by regulating translation.

Target degradation specificity of phytoplasma effector phyllogen is regulated by the recruitment of host proteasome shuttle protein.

Phytoplasmas infect a wide variety of plants and can cause distinctive symptoms including the conversion of floral organs into leaf-like organs, known as phyllody. Phyllody is induced by an effector protein family called phyllogens, which interact with floral MADS-box transcription factors (MTFs) responsible for determining the identity of floral organs. The MTF/phyllogen complex then interacts with the proteasomal shuttle protein RADIATION SENSITIVE23 (RAD23), which facilitates delivery of the MTF/phyllogen complex to the host proteasome for MTF degradation. Previous studies have indicated that the MTF degradation specificity of phyllogens is determined by their ability to bind to MTFs. However, in the present study, we discovered a novel mechanism determining the degradation specificity through detailed functional analyses of a phyllogen homologue of rice yellow dwarf phytoplasma (PHYLRYD ). PHYLRYD degraded a narrower range of floral MTFs than other phyllody-inducing phyllogens, resulting in compromised phyllody phenotypes in plants. Interestingly, PHYLRYD was able to bind to some floral MTFs that PHYLRYD was unable to efficiently degrade. However, the complex of PHYLRYD and the non-degradable MTF could not interact with RAD23. These results indicate that the MTF degradation specificity of PHYLRYD is correlated with the ability to form the MTF/PHYLRYD /RAD23 ternary complex, rather than the ability to bind to MTF. This study elucidated that phyllogen target specificity is regulated by both the MTF-binding ability and RAD23 recruitment ability of the MTF/phyllogen complex.

Random mutagenesis-based screening of the interface of phyllogen, a bacterial phyllody-inducing effector, for interaction with plant MADS-box proteins.

To understand protein function deeply, it is important to identify how it interacts physically with its target. Phyllogen is a phyllody-inducing effector that interacts with the K domain of plant MADS-box transcription factors (MTFs), which is followed by proteasome-mediated degradation of the MTF. Although several amino acid residues of phyllogen have been identified as being responsible for the interaction, the exact interface of the interaction has not been elucidated. In this study, we comprehensively explored interface residues based on random mutagenesis using error-prone PCR. Two novel residues, at which mutations enhanced the affinity of phyllogen to MTF, were identified. These residues, and all other known interaction-involved residues, are clustered together at the surface of the protein structure of phyllogen, indicating that they constitute the interface of the interaction. Moreover, in silico structural prediction of the protein complex using ColabFold suggested that phyllogen interacts with the K domain of MTF via the putative interface. Our study facilitates an understanding of the interaction mechanisms between phyllogen and MTF.