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.

Sulphate repression of ssuD-dependent alkanesulphonate-sulphur assimilation in Escherichia coli.

Escherichia coli cells utilize alkanesulphonates including taurine as the sulphur source. We previously reported that when E. coli cells carrying a double deletion in tauD and cysN were inoculated into a taurine-containing minimal medium, they started to grow only after long-term incubation (Nishikawa et al. 2018, Microbiology 164: 1446-1456). We show here that cells that can induce ssuD-dependent alkanesulphonate-sulphur assimilation (SASSA) are essentially rare, but suppressors that can induce SASSA appear during long-term incubation. Mutant cells carrying ΔtauD and ΔcysN, ΔcysC or ΔcysH generated suppressor cells that can induce SASSA at a frequency of about 10-6 in a population. Whereas ΔtauD ΔcysN cells without prior SASSA did not express ssuD even when necessary, the cells with prior SASSA properly expressed ssuD. Whole-genome DNA sequencing of a clone isolated from ΔtauD ΔcysN cells with prior SASSA revealed that the influx of sulphate or thiosulphate may be related to the regulation of SASSA. To clarify whether sulphate or thiosulphate affects the induction of SASSA, the effect of mutations in sbp and cysP, which are responsible for sulphate and thiosulphate uptake with different preferences for substrates, was examined. Only the ΔtauD ΔcysN Δsbp mutant did not show repression of SASSA when no sulphate was added to the medium. When the concentration of the sulphate added was over 10 µM, the Δsbp mutant showed repression of SASSA. Therefore, it was considered that the influx of extracellular sulphate resulted in repression of SASSA.

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 pleioblastus mosaic virus, a distinct member of the genus Potyvirus.

Pleioblastus mosaic virus (PleMV) is a tentative member of the genus Potyvirus in the family Potyviridae and was discovered in bamboo with mosaic symptoms in Tokyo, Japan. Since no information on the genome sequence of PleMV has been reported, its taxonomic position has long been uncertain. Here, we report the first complete genome sequences of two distinct PleMV isolates. Excluding the 3'-terminal poly(A) tail, their genomic RNA sequences consist of 9,634 and 9,643 nucleotides (nt); both contain a large open reading frame (ORF) encoding a polyprotein and a small ORF termed PIPO. The large ORFs of the two isolates share 79.2% and 87.6% sequence identity at the nucleotide (nt) and amino acid (aa) level, respectively, and were found to have the highest nt and aa sequence identity (69.0% and 69.9%) to the potyvirus johnsongrass mosaic virus (JGMV). Phylogenetic analysis showed that PleMV is most closely related to JGMV but forms its own clade. These results suggest that PleMV is a distinct member of the genus Potyvirus.

Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma.

Phytoplasmas are plant pathogenic bacteria that often induce unique phyllody symptoms in which the floral organs are transformed into leaf-like structures. Recently, a novel family of bacterial effector genes, called phyllody-inducing genes (phyllogens), was identified as being involved in the induction of phyllody by degrading floral MADS-domain transcription factors (MTFs). However, the structural characteristics of phyllogens are unknown. In this study, we elucidated the crystal structure of PHYL1OY, a phyllogen of 'Candidatus Phytoplasma asteris' onion yellows strain, at a resolution of 2.4 Å. The structure of PHYL1 consisted of two α-helices connected by a random loop in a coiled-coil manner. In both α-helices, the distributions of hydrophobic residues were conserved among phyllogens. Amino acid insertion mutations into either α-helix resulted in the loss of phyllody-inducing activity and the ability of the phyllogen to degrade floral MTF. In contrast, the same insertion in the loop region did not affect either activity, indicating that both conserved α-helices are important for the function of phyllogens. This is the first report on the crystal structure of an effector protein of phytoplasmas.

Taurine dioxygenase (tauD)-independent taurine assimilation in Escherichia coli.

On the basis of previous studies on taurine assimilation in Escherichia coli, TauD, an iron- and α-ketoglutarate-dependent taurine dioxygenase, has been regarded as an indispensable factor for assimilation. However, we found that tauD-deficient strains did not lose their taurine assimilation ability when there was no deletion of ssuD, which encodes a reduced flavin mononucleotide [FMNH(2)]-dependent alkanesulfonate monooxygenase, which is responsible for the desulfonation of alkanesulfonates. There were no significant differences in lag phase time, growth rate and final growth yield between the tauD-deficient strain and the tauD wild-type strain. Iron increased the growth rate and final growth yield of the ssuD mutant, but not those of the tauD mutant. The double deletion of tauD and ssuD resulted in the loss of the ability to assimilate taurine. When ssuD was artificially expressed in the double-deletion mutant, the mutant recovered its taurine assimilation ability. These findings indicate that there is another taurine assimilation pathway that is dependent on ssuD but independent of tauD.

Complete Genome Sequence of Tea Plant Necrotic Ring Blotch Virus Detected from a Tea Plant in Japan.

We report the complete genome sequence of a Japanese isolate of Tea plant necrotic ring blotch virus (TPNRBV-J). The predicted TPNRBV-J genes have the same organization as those of a Chinese isolate, and the 5' termini of the segments have conserved nucleotide sequences.

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.

Intra-strain biological and epidemiological characterization of plum pox virus.

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.

Complete Genome Sequence of Iris Severe Mosaic Virus Isolated in Japan.

The complete genome sequence of an iris severe mosaic virus isolate (ISMV) from Iris tectorum in Japan was determined for the first time. According to sequence identity analyses, our specimen is closely related to isolates reported from China.

Complete Genome Sequence of a Carrot Torradovirus 1 Isolate, Obtained from Angelica keiskei in Japan.

The complete genome sequence of the first Japanese isolate of carrot torradovirus 1 (CaTV1-J), which infects Angelica keiskei, was determined. This is the first report of a CaTV1 isolate obtained from A. keiskei.

Occurrence of D-histidine residues in antimicrobial poly(arginyl-histidine), conferring resistance to enzymatic hydrolysis.

The antimicrobial peptide poly(arginyl-histidine) is secreted by the ergot fungus Verticillium kibiense. We previously showed that poly(arginyl-histidine) from the fungus inhibits the growth of certain microorganisms more effectively than that chemically synthesized from the L-form of arginine and histidine, implying some substantial differences between the fungal and synthetic peptides. To elucidate what causes such differences, we here investigated the structural features of the fungal peptides. The acid hydrolysates of the fungal peptide contained d-histidine. When synthetic poly(L-arginyl-D-histidine) mimicking the fungal peptide was added to the culture of Salmonella typhimurium together with poly(L-arginyl-L-histidine), poly(L-arginyl-D-histidine) was not easily degraded during the incubation compared with poly(L-arginyl-L-histidine). We concluded that the d-form of histidine residues in the fungal peptide prolongs the life of the peptide leading to the enhancement of antimicrobial activity.

Streptomyces roseoverticillatus produces two different poly(amino acid)s: lariat-shaped gamma-poly(L-glutamic acid) and epsilon-poly(L-lysine).

The poly(amino acid)s gamma-poly(dl-glutamic acid) (gPGA) and epsilon-poly(l-lysine) (ePL) are known to be natural linear poly(amino acid)s secreted by Bacillus spp. and Streptomyces spp., respectively. In this study, a Streptomyces strain producing both ePL and gPGA was identified. Mass spectrometry and other analyses revealed that the gPGA is a mixture of oligomers consisting of 10-13 l-glutamic acid residues linked by isopeptide bonds. In contrast to the known Bacillus gPGA, the glutamic acid oligomers have a cyclodehydrated structure in each molecule. We previously reported that the ePL molecules secreted by the same Streptomyces strain disperse only slightly in an agar culture plate, as though they were larger molecules. This phenomenon is explicable by the observed polyion complex formation between the glutamic acid oligomers and ePLs. The glutamic acid oligomers control the ePL's dispersion, which would also affect the spatial distribution of the ePL's antimicrobial activity. Therefore, gene clustering or common use of the gene was presumed for biosynthesis of the two poly(amino acid)s. However, no gene for biosynthesis of the glutamic acid oligomer was found in the neighbouring region of that for ePL biosynthesis, and the glutamic acid oligomer was produced by a mutant in which the ePL biosynthetic gene was inactivated by gene disruption.

Inhibition of epsilon-poly-L-lysine biosynthesis in Streptomycetaceae bacteria by short-chain polyols.

Antimicrobial epsilon-poly-L-lysine (ePL) is secreted by Streptomycetaceae bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). In this study, by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.

Antimicrobial activity of a chelatable poly(arginyl-histidine) produced by the ergot fungus Verticillium kibiense.

We have recently developed a convenient method of screening a broad range of microorganisms that produce epsilon-poly-L-lysine (M. Nishikawa and K. Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). Using this method, we found an ergot fungus that secretes a charged polypeptide other than epsilon-poly-L-lysine. It was identified as a new species on the basis of its 28S rRNA sequence and was named Verticillium kibiense (formerly Epichloe kibiensis). Peptide sequencing and mass spectrometry revealed that the polypeptide is a linear peptide composed of repeated units of arginyl-histidine. The numbers of repeated units were in most cases five and in some cases four or six. This peptide showed activity against a broad range of bacteria and fungi but lost its activity under conditions of high ionic strength. Zinc and copper ions specifically changed the circular dichroism spectra of the peptide and restored the antimicrobial activity from abrogation under high ionic conditions, although these ions had no reinforcing effect on antimicrobial activity when they were added to solutions at a low ionic strength. The peptide labeled with fluorescein was able to permeate the cell membranes of target microbes, but its ability to permeate cell membranes decreased under conditions of high ionic strength. This decreased ability was partially recovered specifically by the addition of zinc and copper ions. These results indicate that poly(arginyl-histidine) is a cationic polypeptide characterized by specific metal binding and resistance to salts.

Distribution of microbes producing antimicrobial epsilon-poly-L-lysine polymers in soil microflora determined by a novel method.

We developed a simple and sensitive screening method to investigate the distribution of microbes producing an antimicrobial poly(amino acid), epsilon-poly-L-lysine (epsilon-PL), in microflora. An acidic dye, Poly R-478, incorporated in an agar plate detected epsilon-PL producers by electrostatic interaction with the secreted basic polymers. All epsilon-PL producers, isolated after careful and sufficient screening of soil microflora, belonged exclusively to two groups of bacteria of the family Streptomycetaceae and ergot fungi. They were characterized based on the density and diameter of the concentric zone formed by the secreted polymers. The density depended on each isolate. The increase in the diameter of the concentric zone per unit of time varied among isolates and was negatively correlated with the molecular weight. Although the distribution of epsilon-PL producers was extremely limited, their products were structurally varied. The molecular masses of the secreted polymers among the isolates ranged from 0.8 to 2.0 kDa. There were also isolates producing unknown polymers inconsistent with the correlation or producing a mixture of polymers with original and modified structures. A chemically modified polymer was an epsilon-PL derivative, as determined by mass spectrometry. Since the structural variations had no relation to the phylogenetic position of the isolates, it is possible that enzymes involved in the synthesis diversified after putative horizontal transfers of relevant genes.