A phytoplasma effector acts as a ubiquitin-like mediator between floral MADS-box proteins and proteasome shuttle proteins.

Plant pathogenic bacteria have developed effectors to manipulate host cell functions to facilitate infection. A certain number of effectors use the conserved ubiquitin-proteasome system in eukaryotic to proteolyze targets. The proteasome utilization mechanism is mainly mediated by ubiquitin interaction with target proteins destined for degradation. Phyllogens are a family of protein effectors produced by pathogenic phytoplasmas that transform flowers into leaves in diverse plants. Here, we present a noncanonical mechanism for phyllogen action that involves the proteasome and is ubiquitin-independent. Phyllogens induce proteasomal degradation of floral MADS-box transcription factors (MTFs) in the presence of RADIATION-SENSITIVE23 (RAD23) shuttle proteins, which recruit ubiquitinated proteins to the proteasome. Intracellular localization analysis revealed that phyllogen induced colocalization of MTF with RAD23. The MTF/phyllogen/RAD23 ternary protein complex was detected not only in planta but also in vitro in the absence of ubiquitin, showing that phyllogen directly mediates interaction between MTF and RAD23. A Lys-less nonubiquitinated phyllogen mutant induced degradation of MTF or a Lys-less mutant of MTF. Furthermore, the method of sequential formation of the MTF/phyllogen/RAD23 protein complex was elucidated, first by MTF/phyllogen interaction and then RAD23 recruitment. Phyllogen recognized both the evolutionarily conserved tetramerization region of MTF and the ubiquitin-associated domain of RAD23. Our findings indicate that phyllogen functionally mimics ubiquitin as a mediator between MTF and RAD23.

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.

Complete genome sequence of a new orthotospovirus associated with ringspot in Fatsia japonica.

Fatsia japonica is an evergreen shrub native to Japan. For decades, virus-like ringspot symptoms have been observed on leaves of F. japonica in Japan; however, previous attempts to identify the causal agents have been unsuccessful. In this study, we detected an orthotospovirus-like sequence in symptomatic F. japonica plants using RNA sequencing analysis. The complete nucleotide sequences of the L, M, and S segments of the virus were determined using conventional sequencing strategies. The virus had a typical orthotospovirus genome structure, and the putative nucleocapsid protein showed the highest sequence identity to that of groundnut chlorotic fan-spot virus, with 83.7% identity at the amino acid level (which is below the 90% species demarcation cutoff for the genus Orthotospovirus). Although we could not confirm the pathogenicity of the virus in F. japonica due to difficulties associated with mechanical inoculation, its association with the observed symptoms was suggested by the fact that the virus was detected only in symptomatic leaf areas. Based on these results, we consider this virus, which we have named "Fatsia japonica ringspot-associated virus" (FjRSaV), to be the first representative of a new orthotospovirus species, for which we propose the binomial "Orthotospovirus fatsiae".

Complete genome sequence of viola mottle virus, revealing its synonymous relationship to tulip virus X.

Viola mottle virus (VMoV) was discovered in Viola odorata showing symptoms of reduced growth, leaf mottling, and whitish stripes on flowers in northern Italy in 1977. This virus has been provisionally classified as a member of the genus Potexvirus based on its morphological, serological, and biological characteristics. However, since genetic information of VMoV has never been reported, the taxonomic status of this virus is unclear. Here, we report the first complete genome sequence of VMoV to clarify its taxonomic position. Its genomic RNA is 6,052 nucleotides long, excluding the 3'-terminal poly(A) tail, and has five open reading frames (ORFs) typical of potexviruses. Among potexviruses, VMoV showed the most similarity to tulip virus X (TVX) with 81.1-81.2% nucleotide and 90.4-90.7% amino acid sequence identity in ORF1 and 82.9-83.5% nucleotide and 93.2-95.2% amino acid sequence identity in ORF5. These values are much higher than the species demarcation threshold for the genus. Phylogenetic analysis also indicated that VMoV is nested within the clade of TVX isolates. These data demonstrate that VMoV and TVX are members of the same species.

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.

Complete Genome Sequence of Clover Yellow Mosaic Virus Isolated from White Clover in Japan.

Clover yellow mosaic virus (ClYMV) infecting white clover was isolated in Japan, and the complete genome sequence was determined.

Complete Genome Sequence of Mirabilis Crinkle Mosaic Virus Isolated from Pokeweed in Japan.

The complete genome sequence of a pokeweed (Phytolacca americana L.) isolate of mirabilis crinkle mosaic virus (MiCMV) in Japan was determined.