Local Application of Acibenzolar-S-Methyl Treatment Induces Antiviral Responses in Distal Leaves of Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a plant defense mechanism that provides protection against a broad spectrum of pathogens in distal tissues. Recent studies have revealed a concerted function of salicylic acid (SA) and N-hydroxypipecolic acid (NHP) in the establishment of SAR against bacterial pathogens, but it remains unknown whether NHP is also involved in SAR against viruses. We found that the local application of acibenzolar-S-methyl (ASM), a synthetic analog of SA, suppressed plantago asiatica mosaic virus (PlAMV) infection in the distal leaves of Arabidopsis thaliana. This suppression of infection in untreated distal leaves was observed at 1 day, but not at 3 days, after application. ASM application significantly increased the expression of SAR-related genes, including PR1, SID2, and ALD1 after 1 day of application. Viral suppression in distal leaves after local ASM application was not observed in the sid2-2 mutant, which is defective in isochorismate synthase 1 (ICS1), which is involved in salicylic acid synthesis; or in the fmo1 mutant, which is defective in the synthesis of NHP; or in the SA receptor npr1-1 mutant. Finally, we found that the local application of NHP suppressed PlAMV infection in the distal leaves. These results indicate that the local application of ASM induces antiviral SAR against PlAMV through a mechanism involving NHP.

The MAT1 locus is required for microconidia-mediated sexual fertility in the rice blast fungus.

Rice blast fungus (Pyricularia oryzae) is a heterothallic ascomycete that causes the most destructive disease in cultivated rice worldwide. This fungus reproduces sexually and asexually, and its mating type is determined by the MAT1 locus, MAT1-1 or MAT1-2. Interestingly, most rice-infecting field isolates show a loss of female fertility, but the MAT1 locus is highly conserved in female-sterile isolates. In this study, we performed a functional analysis of MAT1 using the CRISPR/Cas9 system in female- and male-fertile isolates and female-sterile (male-fertile) isolates. Consistent with a previous report, MAT1 was essential for sexual reproduction but not for asexual reproduction. Meanwhile, deletion mutants of MAT1-1-1, MAT1-1-2, and MAT1-1-3 exhibited phenotypes different from those of other previously described isolates, suggesting that the function of MAT1-1 genes and/or their target genes in sexual reproduction differs among strains or isolates. The MAT1 genes, excluding MAT1-2-6, retained their functions even in female-sterile isolates, and deletion mutants lead to loss or reduction of male fertility. Although MAT1 deletion did not affect microconidia (spermatia) production, microconidia derived from the mutants could not induce perithecia formation. These results indicated that MAT1 is required for microconidia-mediated male fertility in addition to female fertility in P. oryzae .

Dysfunctional Pro1 leads to female sterility in rice blast fungi.

Although sexual reproduction is widespread in eukaryotes, some fungal species can only reproduce asexually. In the rice blast fungus Pyricularia (Magnaporthe) oryzae, several isolates from the region of origin retain mating ability, but most isolates are female sterile. Therefore, female fertility may have been lost during its spread from the origin. Here, we show that functional mutations of Pro1, a global transcriptional regulator of mating-related genes in filamentous fungi, is one cause of loss of female fertility in this fungus. We identified the mutation of Pro1 by backcrossing analysis between female-fertile and female-sterile isolates. The dysfunctional Pro1 did not affect the infection processes but conidial release was increased. Furthermore, various mutations in Pro1 were detected in geographically distant P. oryzae, including pandemic isolates of wheat blast fungus. These results provide the first evidence that loss of female fertility may be advantageous to the life cycle of some plant pathogenic fungi.

Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

Discovery, Genomic Sequence Characterization and Phylogenetic Analysis of Novel RNA Viruses in the Turfgrass Pathogenic Colletotrichum spp. in Japan.

Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. in Japan. We characterized two novel dsRNA partitiviruses: Colletotrichum associated partitivirus 1 (CaPV1) and Colletotrichum associated partitivirus 2 (CaPV2), as well as two negative single-stranded (ss) RNA viruses: Colletotrichum associated negative-stranded RNA virus 1 (CaNSRV1) and Colletotrichum associated negative-stranded RNA virus 2 (CaNSRV2). Using specific RT-PCR assays, we confirmed the presence of CaPV1, CaPV2 and CaNSRV1 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-264, as well as CaNSRV2 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-288. This is the first time mycoviruses have been discovered in turfgrass pathogenic Colletotrichum spp. in Japan. CaPV1 and CaPV2 are new members of the newly proposed genus "Zetapartitivirus" and genus Alphapartitivirus, respectively, in the family Partitiviridae, according to genomic characterization and phylogenetic analysis. Negative sense ssRNA viruses CaNSRV1 and CaNSRV2, on the other hand, are new members of the family Phenuiviridae and the proposed family "Mycoaspirividae", respectively. These findings reveal previously unknown RNA virus diversity and evolution in turfgrass pathogenic Colletotrichum spp.

Genome editing using a versatile vector-based CRISPR/Cas9 system in Fusarium species.

Fusarium species include important filamentous fungal pathogens that can infect plants, animals, and humans. Meanwhile, some nonpathogenic Fusarium species are promising biocontrol agents against plant pathogens. Here, we developed a genome editing technology using a vector-based CRISPR/Cas9 system for Fusarium oxysporum f. sp. lycopersici (Fol). This optimized CRISPR/Cas9 system, harboring an endogenous U6 small nuclear RNA promoter for the expression of single-guide RNA and an endogenous H2B nuclear localization signal for the localization of Cas9, enabled efficient targeted gene knock-out, including in the accessory chromosomal regions in Fol. We further demonstrated single crossover-mediated targeted base editing and endogenous gene tagging. This system was also applicable for genome editing in F. oxysporum f. sp. spinaciae and F. commune without any modifications, suggesting that this CRISPR/Cas9 vector has a potential application for a broad range of researches on other Fusarium species.

Differentiation of the Pea Wilt Pathogen Fusarium oxysporum f. sp. pisi from Other Isolates of Fusarium Species by PCR.

Pea wilt disease, caused by the soilborne and seedborne fungal pathogen Fusarium oxysporum f. sp. pisi (Fop), first appeared in Japan in 2002. We herein investigated the molecular characteristics of 16 Fop isolates sampled from multiple locations and at different times in Japan. The 16 isolates were divided into three clades in molecular phylogenic ana-lyses based on both the TEF1α gene and the rDNA-IGS region. All of the Fop isolates harbored a PDA1 gene, which encodes the cytochrome P450 pisatin demethylase (Pda1), and also carried one or both of the SIX6 and SIX13 genes, which encode secreted in xylem (Six) proteins. Other forms of F. oxysporum and other species of Fusarium did not carry these sets of genes. Based on these results, a PCR method was developed to identify Fop and differentiate it from other forms and non-pathogenic isolates of Fusarium spp. We also demonstrated that the PCR method effectively detected Fop in infected pea plants and infested soils.

Unique Terminal Regions and Specific Deletions of the Segmented Double-Stranded RNA Genome of Alternaria Alternata Virus 1, in the Proposed Family Alternaviridae.

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35-193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1-4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36-50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35-193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.

Acibenzolar-S-methyl-mediated restriction of loading of plantago asiatica mosaic virus into vascular tissues of Nicotiana benthamiana.

Long-distance movement via vascular tissues is an essential step for systemic infection by plant viruses. We previously reported that pre-treatment of Nicotiana benthamiana with acibenzolar-S-methyl (ASM) both suppressed the accumulation of plantago asiatica mosaic virus (PlAMV) in inoculated leaves and delayed the long-distance movement to uninoculated upper leaves. These two effects occurred independently of each other. However, it remained unclear where and when the viral long-distance movement is inhibited upon ASM treatment. In this study, we found that ASM treatment restricted the loading of GFP-expressing PlAMV (PlAMV-GFP) into vascular tissues in the inoculated leaves. This led to delays in viral translocation to the petiole and the main stem, and to untreated upper leaves. We used cryohistological fluorescence imaging to show that ASM treatment affected the viral localization and reduced its accumulation in the phloem, xylem, and mesophyll tissues. A stem girdling experiment, which blocked viral movement downward through phloem tissues, demonstrated that ASM treatment could inhibit viral systemic infection to upper leaves, which occurred even with viral downward movement restricted. Taken together, our results showed that ASM treatment affects the loading of PlAMV-GFP into the vascular system in the inoculated leaf, and that this plays a key role in the ASM-mediated delay of viral long-distance movement.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity.

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

Two Novel Endornaviruses Co-infecting a Phytophthora Pathogen of Asparagus officinalis Modulate the Developmental Stages and Fungicide Sensitivities of the Host Oomycete.

Two novel endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) were found in isolates of a Phytophthora pathogen of asparagus collected in Japan. A molecular phylogenetic analysis indicated that PEV2 and PEV3 belong to the genus Alphaendornavirus. The PEV2 and PEV3 genomes consist of 14,345 and 13,810 bp, and they contain single open reading frames of 4,640 and 4,603 codons, respectively. Their polyproteins contain the conserved domains of an RNA helicase, a UDP-glycosyltransferase, and an RNA-dependent RNA polymerase, which are conserved in other alphaendornaviruses. PEV2 is closely related to Brown algae endornavirus 2, whereas PEV3 is closely related to Phytophthora endornavirus 1 (PEV1), which infects a Phytophthora sp. specific to Douglas fir. PEV2 and PEV3 were detected at high titers in two original Phytophthora sp. isolates, and we found a sub-isolate with low titers of the viruses during subculture. We used the high- and low-titer isolates to evaluate the effects of the viruses on the growth, development, and fungicide sensitivities of the Phytophthora sp. host. The high-titer isolates produced smaller mycelial colonies and much higher numbers of zoosporangia than the low-titer isolate. These results suggest that PEV2 and PEV3 inhibited hyphal growth and stimulated zoosporangium formation. The high-titer isolates were more sensitive than the low-titer isolate to the fungicides benthiavalicarb-isopropyl, famoxadone, and chlorothalonil. In contrast, the high-titer isolates displayed lower sensitivity to the fungicide metalaxyl (an inhibitor of RNA polymerase I) when compared with the low-titer isolate. These results indicate that persistent infection with PEV2 and PEV3 may potentially affect the fungicide sensitivities of the host oomycete.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Spray Application of Nonpathogenic Fusaria onto Rice Flowers Controls Bakanae Disease (Caused by Fusarium fujikuroi) in the Next Plant Generation.

Bakanae disease, caused by Fusarium fujikuroi, is an economically important seed-borne disease of rice. F. fujikuroi is horizontally transmitted to rice flowers and vertically transmitted to the next generation via seeds. The fungus induces typical symptoms such as abnormal tissue elongation and etiolation. Sanitation of seed farms and seed disinfection are the only effective means to control bakanae disease at present; however, the efficacy of these methods is often insufficient. Therefore, alternative and innovative control methods are necessary. We developed a novel method for applying nonpathogenic fusaria as biocontrol agents by spraying spore suspensions onto rice flowers to reduce the incidence of seed-borne bakanae. We visualized the interaction between Fusarium commune W5, a nonpathogenic fusarium, and Fusarium fujikuroi using transformants expressing two different fluorescent proteins on/in rice plants. W5 inhibited hyphal extension of F. fujikuroi on/in rice flowers and seedlings, possibly by competing with the pathogen, and survived on/in rice seeds for at least 6 months.IMPORTANCE We demonstrated that a spray treatment of rice flowers with the spores of nonpathogenic fusaria mimicked the disease cycle of the seed-borne bakanae pathogen Fusarium fujikuroi and effectively suppressed the disease. Spray treatment of nonpathogenic fusaria reduced the degree of pathogen invasion of rice flowers and vertical transmission of the pathogen to the next plant generation via seeds, thereby controlling the bakanae disease. The most promising isolate, F. commune W5, colonized seeds and seedlings via treated flowers and successfully inhibited pathogen invasion, suggesting that competition with the pathogen was the mode of action. Seed-borne diseases are often controlled by seed treatment with chemical fungicides. Establishing an alternative method is a pressing issue from the perspectives of limiting fungicide resistance and increasing food security. This work provides a potential solution to these issues using a novel application technique to treat rice flowers with biocontrol agents.

Mutations Found in the Asc1 Gene That Confer Susceptibility to the AAL-Toxin in Ancestral Tomatoes from Peru and Mexico.

Tomato susceptibility/resistance to stem canker disease caused by Alternaria alternata f. sp. lycopersici and its pathogenic factor AAL-toxin is determined by the presence of the Asc1 gene. Several cultivars of commercial tomato (Solanum lycopersicum var. lycopersicum, SLL) are reported to have a mutation in Asc1, resulting in their susceptibility to AAL-toxin. We evaluated 119 ancestral tomato accessions including S. pimpinellifolium (SP), S. lycopersicum var. cerasiforme (SLC) and S. lycopersicum var. lycopersicum "jitomate criollo" (SLJ) for AAL-toxin susceptibility. Three accessions, SP PER018805, SLC PER018894, and SLJ M5-3, were susceptible to AAL-toxin. SLC PER018894 and SLJ M5-3 had a two-nucleotide deletion (nt 854_855del) in Asc1 identical to that found in SLL cv. Aichi-first. Another mutation (nt 931_932insT) that may confer AAL-toxin susceptibility was identified in SP PER018805. In the phylogenetic tree based on the 18 COSII sequences, a clade (S3) is composed of SP, including the AAL-toxin susceptible PER018805, and SLC. AAL-toxin susceptible SLC PER018894 and SLJ M5-3 were in Clade S2 with SLL cultivars. As SLC is thought to be the ancestor of SLL, and SLJ is an intermediate tomato between SLC and SLL, Asc1s with/without the mutation seem to have been inherited throughout the history of tomato domestication and breeding.

Population Structure of Double-Stranded RNA Mycoviruses That Infect the Rice Blast Fungus Magnaporthe oryzae in Japan.

Various viruses infect Magnaporthe oryzae (syn. Pyricularia oryzae), which is a well-studied fungus that causes rice blast disease. Most research has focused on the discovery of new viruses and the hypovirulence-associated traits conferred by them. Therefore, the diversity and prevalence of viruses in wild fungal populations have not been explored. We conducted a comprehensive screening of M. oryzae mycoviruses from various regions in Japan using double-stranded RNA (dsRNA) electrophoresis and RT-PCR assays. We detected three mycoviruses, Magnaporthe oryzae virus 2 (MoV2), Magnaporthe oryzae chrysovirus 1 (MoCV1), and Magnaporthe oryzae partitivirus 1 (MoPV1), among 127 of the 194 M. oryzae strains screened. The most prevalent virus was MoPV1 (58.8%), which often co-infected in a single fungal strain together with MoV2 or MoCV1. MoV2 and MoCV1 were found in 22.7 and 10.8% of strains, respectively, and they were usually distributed in different regions so that mixed-infection with these two mycoviruses was extremely rare. The predominance of MoPV1 in M. oryzae is supported by significant negative values from neutrality tests, which indicate that the population size of MoPV1 tends to increase. Population genetic analyses revealed high nucleotide diversity and the presence of phylogenetically diverse subpopulations among the MoV2 isolates. This was not the case for MoPV1. Furthermore, studies of a virus-cured M. oryzae strain revealed that MoV2 does not cause any abnormalities or symptoms in its host. However, a leaf sheath inoculation assay showed that its presence slightly increased the speed of mycelial growth, compared with virus-free mycelia. These results demonstrate that M. oryzae in Japan harbors diverse dsRNA mycovirus communities with wide variations in their population structures among different viruses.

Fusarium diseases of cultivated plants, control, diagnosis, and molecular and genetic studies.

Fusarium diseases are significant hindrances to food plant production and are very difficult to control, especially soilborne diseases caused by F. oxysporum. First I outline the Fusarium diseases and introduce examples of the recent outbreak of Fusarium diseases in Japan. Then I summarize my studies on (1) the control of Fusarium diseases by biological agents and by inducing resistance to diseases in plants, (2) the specific detection of forms and races in F. oxysporum using immunological measures and molecular measures based on phylogeny and pathogenicity-determining genes, and (3) molecular and genetic studies on Fusarium diseases, including evolutionary, genetic, and genomic analyses of the emergence and divergence of forms and races in F. oxysporum.

High-Quality Draft Genome Sequence of Fusarium oxysporum f. sp. cubense Strain 160527, a Causal Agent of Panama Disease.

Fusarium oxysporum f. sp. cubense is the causal agent of banana Fusarium wilt, also known as Panama disease. Here, we present a high-quality genome sequence of F. oxysporum f. sp. cubense strain 160527. The genome assembly is composed of 12 contigs with a total assembly length of 51,139,495 bp (N 50 contig length, 4,884,632 bp).

Acibenzolar-S-Methyl Restricts Infection of Nicotiana benthamiana by Plantago Asiatica Mosaic Virus at Two Distinct Stages.

Plant activators, including acibenzolar-S-methyl (ASM), are chemical compounds that stimulate plant defense responses to pathogens. ASM treatment inhibits infection by a variety of plant viruses, however, the mechanisms of this broad-spectrum and strong effect remain poorly understood. We employed green fluorescent protein (GFP)-expressing viruses and Nicotiana benthamiana plants to identify the infection stages that are restricted by ASM. ASM suppressed infection by three viral species, plantago asiatica mosaic virus (PlAMV), potato virus X (PVX), and turnip mosaic virus (TuMV), in inoculated cells. Furthermore, ASM delayed the long-distance movement of PlAMV and PVX, and the cell-to-cell (short range) movement of TuMV. The ASM-mediated delay of long-distance movement of PlAMV was not due to the suppression of viral accumulation in the inoculated leaves, indicating that ASM restricts PlAMV infection in at least two independent steps. We used Arabidopsis thaliana mutants to show that the ASM-mediated restriction of PlAMV infection requires the NPR1 gene but was independent of the dicer-like genes essential for RNA silencing. Furthermore, experiments using protoplasts showed that ASM treatment inhibited PlAMV replication without cell death. Our approach, using GFP-expressing viruses, will be useful for the analysis of mechanisms underlying plant activator-mediated virus restriction.