Argonaute-independent, Dicer-dependent antiviral defense against RNA viruses.

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.

Continued mycovirus discovery expanding our understanding of virus lifestyles, symptom expression, and host defense.

High-throughput sequencing technologies have greatly expanded the RNA virome in general and have led to an exponential increase in new fungal viruses, also known as mycoviruses. Mycoviruses are omnipresent in fungi and usually induce symptomless infections. Some mycoviruses infecting fungi pathogenic to plants, insects, and mammals are known to modify host virulence positively and negatively and attract particular interests. In addition, fungal viruses continue to provide intriguing research materials and themes that lead to discoveries of peculiar viruses as infectious entities and insights into virus evolution and diversity. In this review, we outline the diversity and neolifestyle of recently discovered fungal RNA viruses, and phenotypic alterations induced by them. Furthermore, we discuss recent advances in research regarding the fungal antiviral defense and viral counterdefense, which are closely associated with host phenotype alterations. We hope that this article will enhance understanding of the interesting and growing fungal virology field.

Exploration of the yadokari/yadonushi nature of YkV3 and RnMBV3 in the original host and a model filamentous fungus.

The yadokari/yadonushi nature is a recently discovered virus lifestyle; "yadokari" refers to the ability of capsidless positive-sense (+) RNA viruses (yadokariviruses) to utilize the capsids of phylogenetically distant double-stranded RNA (dsRNA) viruses possibly as the replication site, while "yadonushi" refers to the ability of dsRNA viruses to provide capsids to yadokariviruses. This virus-virus interaction, however, has been only studied with limited pathosystems. Here, we established a new study model with a capsidless (+)RNA yadokarivirus YkV3 (family Yadokariviridae) and its capsid donor RnMBV3 (family Megabirnaviridae) in the original host fungus Rosellinia necatrix and a model filamentous fungal host Cryphonectria parasitica. YkV3 has a simple genome structure with one open reading frame of 4305 nucleotides encoding a single polyprotein with an RNA-dependent RNA polymerase and a 2A-like self-cleavage peptide domain. Reverse genetics of YkV3 in R. necatrix showed that YkV3 tolerates a nucleotide substitution in the extreme 5'-terminus. The insertion of two termination codons immediately downstream of the 2A-like cleavage site abolished YkV3 viability, suggesting the importance of the C-terminal portion of the polyprotein of unknown function. Transfection of RnMBV3 and YkV3 into an RNA silencing-deficient mutant Δdcl2 of C. parasitica showed the replication competency of both viruses. Comparison between the wild-type and Δdcl2 strains of C. parasitica in virus accumulation suggested that RnMBV3 and YkV3 are susceptible to RNA silencing in C. parasitica. Taken together, we have established a platform to further explore the yadokari/yadonushi nature using genetically manipulable host fungal and virus strains.

ICTV Virus Taxonomy Profile: Hadakaviridae 2023.

The family Hadakaviridae, including the genus Hadakavirus, accommodates capsidless viruses with a 10- or 11-segmented positive-sense (+) RNA genome. Currently known hosts are ascomycetous filamentous fungi. Although phylogenetically related to polymycovirids with a segmented double-stranded RNA genome and certain encapsidated picorna-like viruses, hadakavirids are distinct in their lack of a capsid ('hadaka' means naked in Japanese) and their consequent inability to be pelleted by conventional ultracentrifugation; they show ribonuclease susceptibility in host tissue homogenates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hadakaviridae, which is available at ictv.global/report/hadakaviridae.

ICTV Virus Taxonomy Profile: Yadokariviridae 2023.

The family Yadokariviridae, with the genera Alphayadokarivirus and Betayadokarivirus, includes capsidless non-segmented positive-sense (+) RNA viruses that hijack capsids from phylogenetically distant double-stranded RNA viruses. Yadokarivirids likely replicate inside the hijacked heterocapsids using their own RNA-directed RNA polymerase, mimicking dsRNA viruses despite their phylogenetic placement in a (+) RNA virus lineage. Yadokarivirids can have negative or positive impacts on their host fungi, through interactions with the capsid donor dsRNA viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Yadokariviridae, which is available at ictv.global/report/yadokariviridae.

Three-Layered Complex Interactions among Capsidless (+)ssRNA Yadokariviruses, dsRNA Viruses, and a Fungus.

We have previously discovered a virus neo-lifestyle exhibited by a capsidless positive-sense (+), single-stranded (ss) RNA virus YkV1 (family Yadokariviridae) and an unrelated double-stranded (ds) RNA virus YnV1 (proposed family "Yadonushiviridae") in a phytopathogenic ascomycete, Rosellinia necatrix. YkV1 has been proposed to replicate in the capsid provided by YnV1 as if it were a dsRNA virus and enhance YnV1 replication in return. Recently, viruses related to YkV1 (yadokariviruses) have been isolated from diverse ascomycetous fungi. However, it remains obscure whether such viruses generally show the YkV1-like lifestyle. Here, we identified partner viruses for three distinct yadokariviruses, YkV3, YkV4a, and YkV4b, isolated from R. necatrix that were coinfected with multiple dsRNA viruses phylogenetically distantly related to YnV1. We first established transformants of R. necatrix carrying single yadokarivirus cDNAs and fused them with infectants by single partner candidate dsRNA viruses. Consequently, YkV3 and YkV4s replicated only in the presence of RnMBV3 (family Megabirnaviridae) and RnMTV1 (proposed family "Megatotiviridae"), respectively. The partners were mutually interchangeable between the two YkV4 strains and three RnMTV1 strains but not between other combinations involving YkV1 or YkV3. In contrast to YkV1 enhancing YnV1 accumulation, YkV4s reduced RnMTV1 accumulation to different degrees according to strains. Interestingly, YkV4 rescued the host R. necatrix from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3) or host fungus. Overall, we revealed that while yadokariviruses generally require partner dsRNA viruses for replication, each yadokarivirus partners with a different dsRNA virus species in the three diverse families and shows a distinct symbiotic relation in a fungus. IMPORTANCE A capsidless (+)ssRNA virus YkV1 (family Yadokariviridae) highjacks the capsid of an unrelated dsRNA virus YnV1 (proposed family "Yadonushiviridae") in a phytopathogenic ascomycete, while YkV1 trans-enhances YnV1 replication. Herein, we identified the dsRNA virus partners of three yadokariviruses (YkV3, YkV4a, and YkV4b) with genome organization different from YkV1 as being different from YnV1 at the suborder level. Their partners were mutually interchangeable between the two YkV4 strains and three strains of the partner virus RnMTV1 (proposed family "Megatotiviridae") but not between other combinations involving YkV1 or YkV3. Unlike YkV1, YkV4s reduced RnMTV1 accumulation and rescued the host fungus from impaired growth induced by RnMTV1. YkV3 exerted no apparent effect on its partner (RnMBV3, family Megabirnaviridae) or host fungus. These revealed that while each yadokarivirus has a species-specific partnership with a dsRNA virus, yadokariviruses collectively partner extremely diverse dsRNA viruses and show three-layered complex mutualistic/antagonistic interactions in a fungus.

A novel victorivirus from the phytopathogenic fungus Neofusicoccum parvum.

Neofusicoccum parvum is an important plant-pathogenic ascomycetous fungus that causes trunk diseases in a variety of plants. A limited number of reports on mycoviruses from this fungus are available. Here, we report the characterization of a novel victorivirus, Neofusicoccum parvum victorivirus 3 (NpVV3). An agarose gel dsRNA profile of a Pakistani strain of N. parvum, NFN, showed a band of ~5 kbp that was not detectable in Japanese strains of N. parvum. Taking a high-throughput and Sanger sequencing approach, the complete genome sequence of NpVV3 was determined to be 5226 bp in length with two open reading frames (ORF1 and ORF2) that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP). The RdRP appears to be translated by a stop/restart mechanism facilitated by the junction sequence AUGucUGA, as is found in some other victoriviruses. BLASTp searches showed that NpVV3 CP and RdRP share the highest amino acid sequence identity (80.5% and 72.4%, respectively) with the corresponding proteins of NpVV1 isolated from a French strain of N. parvum. However, NpVV3 was found to be different from NpVV1 in its terminal sequences and the stop/restart facilitator sequence. NpVV3 particles ~35 nm in diameter were partially purified and used to infect an antiviral-RNA-silencing-deficient strain (∆dcl2) of an experimental ascomycetous fungal host, Cryphonectria parasitica. NpVV3 showed symptomless infection in the new host strain.

A new tetra-segmented splipalmivirus with divided RdRP domains from Cryphonectria naterciae, a fungus found on chestnut and cork oak trees in Europe.

Positive-sense (+), single-stranded (ss) RNA viruses with divided RNA-dependent RNA polymerase (RdRP) domains have been reported from diverse filamentous ascomycetes since 2020. These viruses are termed splipalmiviruses or polynarnaviruses and have been characterized largely at the sequence level, but ill-defined biologically. Cryphonectria naterciae, from which only one virus has been reported, is an ascomycetous fungus potentially plant-pathogenic to chestnut and oak trees. We molecularly characterized multiple viruses in a single Portuguese isolate (C0614) of C. naterciae, taking a metatranscriptomic and conventional double-stranded RNA approach. Among them are a novel splipalmivirus (Cryphonectria naterciae splipalmivirus 1, CnSpV1) and a novel fusagravirus (Cryphonectria naterciae fusagravirus 1, CnFGV1). This study focused on the former virus. CnSpV1 has a tetra-segmented, (+)ssRNA genome (RNA1 to RNA4). As observed for other splipalmiviruses reported in 2020 and 2021, the RdRP domain is separately encoded by RNA1 (motifs F, A and B) and RNA2 (motifs C and D). A hypothetical protein encoded by the 5'-proximal open reading frame of RNA3 shows similarity to a counterpart conserved in some splipalmiviruses. The other RNA3-encoded protein and RNA4-encoded protein show no similarity with known proteins in a blastp search. The tetra-segment nature was confirmed by the conserved terminal sequences of the four CnSpV1 segments (RNA1 to RNA4) and their 100% coexistence in over 100 single conidial isolates tested. The experimental introduction of CnSpV1 along with CnFGV1 into a virus free strain C0754 of C. naterciae vegetatively incompatible with C0614 resulted in no phenotypic alteration, suggesting asymptomatic infection. The protoplast fusion assay indicates a considerably narrow host range of CnSpV1, restricted to the species C. naterciae and C. carpinicola. This study contributes to better understanding of the molecular and biological properties of this unique group of viruses.

A second capsidless hadakavirus strain with 10 positive-sense single-stranded RNA genomic segments from Fusarium nygamai.

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.

Molecular Characterization of a Novel Polymycovirus From Penicillium janthinellum With a Focus on Its Genome-Associated PASrp.

The genus Polymycovirus of the family Polymycoviridae accommodates fungal RNA viruses with different genomic segment numbers (four, five, or eight). It is suggested that four members form no true capsids and one forms filamentous virus particles enclosing double-stranded RNA (dsRNA). In both cases, viral dsRNA is associated with a viral protein termed "proline-alanine-serine-rich protein" (PASrp). These forms are assumed to be the infectious entity. However, the detailed molecular characteristics of PASrps remain unclear. Here, we identified a novel five-segmented polymycovirus, Penicillium janthinellum polymycovirus 1 (PjPmV1), and characterized its purified fraction form in detail. The PjPmV1 had five dsRNA segments associated with PASrp. Density gradient ultracentrifugation of the PASrp-associated PjPmV1 dsRNA revealed its uneven structure and a broad fractionation profile distinct from that of typical encapsidated viruses. Moreover, PjPmV1-PASrp interacted in vitro with various nucleic acids in a sequence-non-specific manner. These PjPmV1 features are discussed in view of the diversification of genomic segment numbers of the genus Polymycovirus.

Hadaka Virus 1: a Capsidless Eleven-Segmented Positive-Sense Single-Stranded RNA Virus from a Phytopathogenic Fungus, Fusarium oxysporum.

The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein (proline-alanine-serine-rich protein [PASrp]) in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms.IMPORTANCE Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus Fusarium oxysporum The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses.

ICTV Virus Taxonomy Profile: Megabirnaviridae.

Megabirnaviridae is a family of non-enveloped spherical viruses with dsRNA genomes of two linear segments, each of 7.2-8.9 kbp, comprising 16.1 kbp in total. The genus Megabirnavirus includes the species Rosellinia necatrix megabirnavirus 1, the exemplar isolate of which infects the white root rot fungus (Rosellinia necatrix) to which it confers hypovirulence. Megabirnaviruses are characterized by their bisegmented genome with large 5'-untranslated regions (1.6 kb) upstream of both 5'-proximal coding strand ORFs, and large protrusions on the particle surface. This is a summary of the ICTV Report on the family Megabirnaviridae, which is available at ictv.global/report/megabirnaviridae.This Profile is dedicated to the memory of our valued colleague Professor Said A. Ghabrial.

Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature.

The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5'-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3'-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a -1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the -1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS-polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.

Reverse Genetic Analysis of Antiviral Resistance Signaling and the Resistance Mechanism in Arabidopsis thaliana.

Antiviral RNA silencing and the resistance gene-conferred defense response are major antiviral immune systems in plants. Several of the components involved have been genetically or biochemically identified in Arabidopsis thaliana. One powerful tool to dissect antiviral immune systems involves a reverse genetic approach that analyzes Arabidopsis mutant lines with impaired antiviral defense responses. In particular, to better understand the signaling networks involved in the resistance gene-conferred antiviral response in host plants, establishment of mutant lines carrying the homozygous mutant allele and antiviral resistance gene is required. The information on well-characterized defense-related signaling mutant alleles and the PCR-based genotyping method provided in this chapter allows the efficient selection of Arabidopsis mutant lines that can be used to study antiviral resistance signaling networks and resistance mechanisms.

Molecular and biological characterization of a novel botybirnavirus identified from a Pakistani isolate of Alternaria alternata.

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports a novel double stranded RNA (dsRNA) virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic ascomycetous fungus Alternaria alternata. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA-dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses such as members of the families Totiviridae, Chrysoviridae and Megabirnaviridae. The dsRNA2-encoded protein showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed for the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated its symptomless infection by the virus. Interesting similarities and dissimilarities between AaBbV1 and other previously reported botybirnaviruses are also discussed.

The biological attributes, genome architecture and packaging of diverse multi-component fungal viruses.

Many fungal viruses or mycoviruses have multi-segmented, rather than single-segmented, genomes. This multi-segment nature is frequently possessed by double-stranded RNA viruses, which include members of the Chrysoviridae, Quadriviridae, Megabirnaviridae, Partitiviridae, and Reoviridae families, and unassigned groups. Their genome segments are often packaged separately with the exception of mycoreoviruses, which are multi-segmented but mono-particulate viruses. These multi-segmented fungal dsRNA viruses, as exemplified by reoviruses, have been extensively studied among structural biologists, and contributed to discoveries of novel virion structures. Multi-component systems, interactions of viruses with subviral agents such as satellite and defective RNAs as typified by the yeast killer, and the rule-breaking neo-virus lifestyle exhibited by a capsidless single-stranded RNA virus hosted in an unrelated double-stranded RNA virus are also discussed. Fungal multi-segmented viruses and multicomponent virus systems would continue to provide virologists with interesting future challenges.

Microarray analysis of R-gene-mediated resistance to viruses.

The complex process for host-plant resistance to viruses is precisely regulated by a number of genes and signaling compounds. Thus, global gene expression analysis can provide a powerful tool to grasp the complex molecular network for resistance to viruses. The procedures for comparative global gene expression profiling of virus-resistant and control plants by microarray analysis include RNA extraction, cDNA synthesis, cRNA labeling, hybridization, array scanning, and data mining steps. There are several platforms for the microarray analysis. Commercial services for the steps from cDNA synthesis to array scanning are now widely available; however, the data manipulation step is highly dependent on the experimental design and research focus. The protocols presented here are optimized for analyzing global gene expression during the R gene-conferred defense response using commercial oligonucleotide-based arrays. We also demonstrate a technique to screen for differentially expressed genes using Excel software and a simple Internet tool-based data mining approach for characterizing the identified genes.

Role of intron-mediated enhancement on accumulation of an Arabidopsis NB-LRR class R-protein that confers resistance to Cucumber mosaic virus.

The accumulation of RCY1 protein, which is encoded by RESISTANCE TO CMV(Y) (RCY1), a CC-NB-LRR class R-gene, is tightly correlated with the strength of the resistance to a yellow strain of Cucumber mosaic virus [CMV(Y)] in Arabidopsis thaliana. In order to enhance resistance to CMV by overexpression of RCY1, A. thaliana was transformed with intron-less RCY1 cDNA construct under the control of strong CaMV35S promoter. Remarkably, a relative amount of RCY1 protein accumulation in the transformants was much lower than that in plants expressing genomic RCY1 under the control of its native promoter. To identify a regulatory element of RCY1 that could cause such differential levels of RCY1 accumulation, a series of RCY1 cDNA and genomic RCY1 constructs were transiently expressed in Nicotiana benthamiana leaves by the Agrobacterium-mediated infiltration method. Comparative analysis of the level of RCY1 accumulation in the leaf tissues transiently expressing each construct indicated that the intron located in the RCY1-coding region of genomic RCY1, but not the native RCY1 genomic promoter or the 5'-and 3'-untranslated regions of RCY1, was indispensable for high level RCY1 accumulation. The increased levels of RCY1 accelerated plant disease defense reactions. Interestingly, such intron-mediated enhancement of RCY1 accumulation depended neither on the abundance of the RCY1 transcript nor on the RCY1 specific-intron sequence. Taken together, intron-mediated RCY1 expression seems to play a key role in the expression of complete resistance to CMV(Y) by maintaining RCY1 accumulation at high levels.