Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification.

Magnaporthe oryzae chrysovirus 1 (MoCV1) is a mycovirus with a dsRNA genome that infects the rice blast fungus Magnaporthe oryzae and impairs its growth. To date, MoCV1 has only been found in Vietnamese isolates of M. oryzae, and the distribution of this virus in M. oryzae isolates from other parts of the world remains unknown. In this study, using a one-step reverse transcription PCR (RT-PCR) assay, we detected a MoCV1-related virus in M. oryzae in Japan (named MoCV1-AK) whose sequence shares considerable similarity with that of the MoCV1 Vietnamese isolate. To establish a system for a comprehensive survey of MoCV1 infection in the field, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for direct detection of the virus. The sensitivity of the RT-LAMP assay was at least as high as that of the one-step RT-PCR assay. In addition, we detected MoCV1-AK in M. oryzae-infected oatmeal agar plates and lesions on rice leaves using the RT-LAMP assay without dsRNA extraction, by simple sampling with a toothpick. Preliminary screening of MoCV1 in Japanese M. oryzae isolates indicated that MoCV1 is currently distributed in rice fields in Japan. Our results provide a first example of the application of RT-LAMP for the detection of mycoviruses, which will accelerate surveys for mycovirus infection.

Enhanced defense responses in Arabidopsis induced by the cell wall protein fractions from Pythium oligandrum require SGT1, RAR1, NPR1 and JAR1.

The cell wall protein fraction (CWP) is purified from the non-pathogenic biocontrol agent Pythium oligandrum and is composed of two glycoproteins (POD-1 and POD-2), which are structurally similar to class III elicitins. In tomato plants treated with CWP, jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways are activated, and resistance to Ralstonia solanaceraum is enhanced. To dissect CWP-induced defense mechanisms, we investigated defense gene expression and resistance to bacterial pathogens in Arabidopsis thaliana ecotype Col-0 treated with CWP. When the leaves of Col-0 were infiltrated with CWP, neither visible necrosis nor salicylic acid (SA)-responsive gene (PR-1 and PR-5) expression was induced. In contrast, JA-responsive gene (PDF1.2 and JR2) expression was up-regulated and the resistance to R. solanaceraum and Pseudomonas syringae pv. tomato DC3000 was enhanced in response to CWP. Such CWP-induced defense responses were completely compromised in CWP-treated coi1-1 and jar1-1 mutants with an impaired JA signaling pathway. The induction of defense-related gene expression after CWP treatment was partially compromised in ET-insensitive ein2-1 mutants, but not in SA signaling mutants or nahG transgenic plants. Global gene expression analysis using cDNA array also suggested that several other JA- and ET-responsive genes, but not SA-responsive genes, were up-regulated in response to CWP. Further analysis of CWP-induced defense responses using another eight mutants with impaired defense signaling pathways indicated that, interestingly, the induction of JA-responsive gene expression and enhanced resistance to two bacterial pathogens in response to CWP were completely compromised in rar1-1, rar1-21, sgt1a-1, sgt1b (edm1) and npr1-1 mutants. Thus, the CWP-induced defense system appears to be regulated by JA-mediated and SGT1-, RAR1- and NPR1-dependent signaling pathways.

Influence of the terminal left domain on horizontal and vertical transmissions of tomato planta macho viroid and potato spindle tuber viroid through pollen.

Viroids can be transmitted vertically and/or horizontally by pollen. Tomato planta macho viroid (TPMVd) has a high rate of horizontal transmission by pollen, whereas potato spindle tuber viroid (PSTVd) does not. To specify the domain(s) involved in horizontal transmission, four viroid chimeras were created by exchanging the terminal left (TL) and/or pathogenicity (P) domains between PSTVd and TPMVd. PSTVd-based chimeras containing TPMVd-TL and P, or TPMVd-TL alone, displayed a high rate of horizontal transmission. TPMVd-based chimeras containing PSTVd-TL and P lost infectivity, and those containing PSTVd-TL alone displayed a low rate of horizontal transmission. In addition, the vertical transmission rate was also higher in the mutants containing TPMVd-TL than in the others. These findings indicate that the sequences or structures in the TL and P (although the role is limited) domains are important not only for horizontal but also for vertical transmission by pollen.

Magnaporthe oryzae chrysovirus 1 strain D confers growth inhibition to the host fungus and exhibits multiform viral structural proteins.

A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1-4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis.

High level expression of a virus resistance gene, RCY1, confers extreme resistance to Cucumber mosaic virus in Arabidopsis thaliana.

A coiled coil-nucleotide binding site-leucine rich repeat-type resistance gene, RCY1, confers resistance to a yellow strain of Cucumber mosaic virus, CMV(Y), in Arabidopsis thaliana ecotype C24. Resistance to CMV(Y) in C24 is accompanied by a hypersensitive response (HR) that is characterized by the development of necrotic local lesions at the primary infection sites. To further study the HR and resistance to CMV(Y) in ecotype Col-0, which is susceptible to CMV(Y), Col-0 were transformed with RCY1. Systemic spread of CMV(Y) was completely suppressed in RCY1-transformed Col-0 (Col::pRCY1 lines 2 to 6), whereas virulent strain CMV(B2) spread and multiplied systemically in these transgenic lines similar to that in wild-type Col-0. Interestingly, the resistant phenotype of Col::pRCY1 varied among the lines. In lines 3 and 6, in which levels of RCY1 transcript were similar to that in wild-type C24, the HR and resistance to CMV(Y) was induced. Line 4, which expresses moderately elevated levels of RCY1 transcript, exhibited moderately enhanced resistance compared with that in C24 or line 3. In contrast, lines 2 and 5, which highly overexpress the RCY1 gene, did not exhibit either visible lesions or a micro-HR on the inoculated leaves. Moreover, virus coat protein was not detected in either inoculated or noninoculated upper leaves of these two lines, suggesting that extreme resistance (ER) to CMV(Y) was induced by high levels of expression of RCY1. Furthermore, in transgenic lines expressing hemagglutinin (HA) epitope-tagged RCY1 (Col::pRCY1-HA), high levels of accumulation of RCY1-HA protein were also correlated with the ER phenotype. Global gene expression analysis in line 2, which highly overexpresses RCY1, indicated that expression of several defense-related genes were constitutively elevated compared with wild-type Col-0. Despite this, line 2 did not have enhanced resistance to other avirulent and virulent pathogens. Take together, constitutive accumulation of high levels of RCY1 protein appears to regulate the strength of RCY1-conferred resistance in a gene-for-gene manner and implies that ER and HR-associated resistance differ only in the strength of resistance.

The LeATL6-associated ubiquitin/proteasome system may contribute to fungal elicitor-activated defense response via the jasmonic acid-dependent signaling pathway in tomato.

The expression of LeATL6, an ortholog of Arabidopsis ATL6 that encodes a RING-H2 finger protein, was induced in tomato roots treated with a cell wall protein fraction (CWP) elicitor of the biocontrol agent Pythium oligandrum. The LeATL6 protein was expressed as a fusion protein with a maltose-binding protein (MBP) in Escherichia coli, and it catalyzed the transfer of ubiquitin to the MBP moiety on incubation with ubiquitin, the ubiquitin-activating enzyme E1, and the ubiquitin-conjugating enzyme E2; this indicated that LeATL6 represents ubiquitin ligase E3. LeATL6 expression also was induced by elicitor treatment of jail-1 mutant tomato cells in which the jasmonic acid (JA)-mediated signaling pathway was impaired; however, JA-dependent expression of the basic PR-6 and TPI-1 genes that encode proteinase inhibitor II and I, respectively, was not induced in elicitor-treated jail-1 mutants. Furthermore, transient overexpression of LeATL6 under the control of the Cauliflower mosaic virus 35S promoter induced the basic PR6 and TPI-1 expression in wild tomato but not in the jail-1 mutant. In contrast, LeATL6 overexpression did not activate salicylic acid-responsive acidic PR-1 and PR-2 promoters in wild tomato. These results indicated that elicitor-responsive LeATL6 probably regulates JA-dependent basic PR6 and TPI-1 gene expression in tomato. The LeATL6-associated ubiquitin/proteasome system may contribute to elicitor-activated defense responses via a JA-dependent signaling pathway in plants.

The Natural Occurrence of Two Distinct Begomoviruses Associated with DNAbeta and a Recombinant DNA in a Tomato Plant from Indonesia.

ABSTRACT Two begomoviruses (Java virus-1 and Java virus-2), two satellite DNAs (DNAbeta01 and DNAbeta02), and a recombinant DNA (recDNA) were cloned from a single tomato plant from Indonesia with leaf curl symptoms, and the role of these satellite DNAs in the etiology of begomovirus disease was investigated. The genome organizations of the two viruses were similar to those of other Old World monopartite begomoviruses. Comparison of the sequences with other begomoviruses revealed that Java virus-1 was a newly described virus for which the name Tomato leaf curl Java virus (ToLCJAV) is proposed. Java virus-2 was a strain of Ageratum yellow vein virus (AYVV) (AYVV-[Java]). ToLCJAV or AYVV-[Java] alone did not induce leaf curl symptoms in tomato plants. However, in the presence of DNAbeta02, both ToLCJAV and AYVV-[Java] induced leaf curl symptoms in tomato plants. In the presence of DNAbeta01, these viruses induced mild leaf curl symptoms in tomato plants. The recDNA had a chimeric sequence, which arose from recombination among ToLCJAV, AYVV-[Java], DNAbeta01, and DNAbeta02; it was replicated only in the presence of AYVV-[Java] in tomato plants.

Beta-cyanoalanine synthase as a molecular marker for induced resistance by fungal glycoprotein elicitor and commercial plant activators.

ABSTRACT The biocontrol agent Pythium oligandrum produces glycoprotein elicitor in the cell wall fraction, designated CWP, and induces resistance to a broad range of pathogens. To understand the mechanism of CWP-induced resistance to pathogens, gene expression at the early stage of CWP treatment in tomato roots was analyzed using a cDNA array. At 4 h after CWP treatment, 144 genes were up-regulated and 99 genes were down-regulated. In the 144 up-regulated genes, nine genes exhibited about eightfold increased expression. Analysis of the response of these nine genes to three commercial plant activators indicated that a high level of one gene, beta-cyanoalanine synthase gene (LeCAS) encoding hydrogen cyanide (HCN) detoxification enzyme, was stably induced in tomato roots by such treatment. However, expression of LeCAS was not significantly induced in tomato roots at 4 h by abiotic stresses, whereas only a very low level of induction of such expression by cold stress was observed. This LeCAS expression was also induced after exogenous treatment with a low level of 1-amino-cyclopropane-1-carboxylate as the precursor of ethylene, but not with either salicylic acid or methyl jas-monate. The induction of LeCAS expression in CWP-treated and plant activator-treated roots is likely to be caused by the detoxification of HCN during ethylene production. Transient activation of LeCAS expression caused by ethylene production in tomato roots may be a general phenomenon in fungal elicitor-induced and synthetic plant activator-induced resistance. LeCAS seems to be useful for screening possible novel plant activators for plant protection against pathogens.

Enhanced resistance to Cucumber mosaic virus in the Arabidopsis thaliana ssi2 mutant is mediated via an SA-independent mechanism.

The Arabidopsis thaliana SSI2 gene encodes a plastid-localized stearoyl-ACP desaturase. The recessive ssi2 mutant allele confers constitutive accumulation of the pathogenesis-related-1 (PR-1) gene transcript and salicylic acid (SA), and enhanced resistance to bacterial and oomycete pathogens. In addition, the ssi2 mutant is a dwarf and spontaneously develops lesions containing dead cells. Here, we show that the ssi2 mutant also confers enhanced resistance to Cucumber mosaic virus (CMV). Compared with the wild-type plant, viral multiplication and systemic spread were diminished in the ssi2 mutant plant. However, unlike the ssi2-conferred resistance to bacterial and oomycete pathogens, the ssi2-conferred enhanced resistance to CMV was retained in the SA-deficient ssi2 nahG plant. In addition, SA application was not effective in limiting CMV multiplication and systemic spread in the CMV-susceptible wild-type plant. The acd1, acd2, and cpr5 mutants which, like the ssi2 mutant, accumulate elevated SA levels, constitutively express the PR-1 gene, spontaneously develop lesions containing dead cells, and are dwarfs, are, however, fully susceptible to CMV. Our results suggest that dwarfing, cell death, and constitutive activation of SA signaling are not important for the ssi2-conferred enhanced resistance to CMV. However, the sfd1 and sfd4 mutations, which affect lipid metabolism, suppress the ssi2-conferred enhanced resistance to CMV, thus implicating a lipid or lipids in the ssi2-conferred resistance to CMV. Interestingly, the ssi2-conferred resistance to CMV was compromised in the ssi2 eds5 plant, suggesting the involvement of an SA-independent, EDS5-dependent mechanism in the ssi2-conferred resistance to CMV.

Single amino acid alterations in Arabidopsis thaliana RCY1 compromise resistance to Cucumber mosaic virus, but differentially suppress hypersensitive response-like cell death.

Resistance to an yellow strain of Cucumber mosaic virus [CMV(Y)] in Arabidopsis thaliana ecotype C24 is conferred by the CC-NBS-LRR type R gene, RCY1. RCY1-conferred resistance is accompanied by a hypersensitive response (HR), which is characterized by the development of necrotic local lesion (NLL) at the site of infection that restricts viral spread. To further characterize the role of RCY1 in NLL formation we have identified six recessive CMV(Y)-susceptible rcy1 mutants, four of which contain single amino acid substitutions in RCY1: rcy1-2 contains a D to N substitution in the CC domain, rcy1-3 and rcy1-4 contain R to K and E to K substitutions, respectively, in the LRR domain, and rcy1-6 contains a W to C substitution in the NBS domain. The rcy1-5 and rcy1-7 contain nonsense mutations in the LRR and NBS domains, respectively. Although the virus systemically spread in all six rcy1 mutants, HR-associated cell death was differentially induced in these mutants. In comparison to the wild type C24 plant, HR was not observed in the CMV(Y)-inoculated leaves of the rcy1-3, rcy1-5, rcy1-6 and rcy1-7 mutants. In contrast, delayed NLL development was observed in the virus inoculated leaves of the rcy1-2 and rcy1-4 mutants. In addition, necrosis accompanied by elevated accumulation of PR gene transcript also appeared in the non-inoculated leaves of the rcy1-2 and rcy1-4 mutants. Trans-complementation was observed between the rcy1-2 and rcy1-4 alleles; in F1 plants derived from a cross between rcy1-2 and rcy1-4, HR associated cell death was accelerated and systemic spread of the virus was partially suppressed than in the homozygous rcy1-2 and rcy1-4 plants. Our results suggest that the CC, NBS and LRR domains of RCY1 are required for restriction of virus spread but differentially impact the induction of HR-like cell death. Furthermore, these results also predict inter-molecular interaction involving RCY1 in Arabidopsis resistance to CMV(Y).

The genome organization of pea stem necrosis virus and its assignment to the genus Carmovirus.

The complete sequence of the single-stranded, positive-sense RNA genome of pea stem necrosis virus (PSNV) has been determined. The 4,048-nucleotide genome contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 25-kD protein (p25). If the amber termination codon is read through, the ORF produces a read-through protein of 84 kD (p84). Two small, centrally located ORFs encoded a 7-kD protein (p7) and a 6-kD protein (p6), respectively. The 38-proximal ORF encodes a 38-kD (p38) capsid protein. Comparison of the genome organization with that of other viruses justifies the assignment of PSNV to the genus Carmovirus.

Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments.

A cDNA from deep water rice treated with ethylene, encoding an ethylene receptor homologous to Arabidopsis thaliana ETR2 and EIN4, was isolated using differential display and RACE techniques. The cDNA (2880 bp), corresponding to the Os-ERL1 gene (Oryza sativa ETHYLENE RESPONSE 2 like 1; GenBank accession number AB107219), contained an open reading frame of 2289 bp coding for 763 amino acids. The protein Os-ERL1 has 50% and 52% similarity to Arabidopsis ETR2 and EIN4, respectively. The Os-ERL1 gene was up-regulated by flooding, and by treatment with ethylene and gibberellin. These results suggest that deep water rice responds to flooding by increasing the number of its ethylene receptors.

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus.

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.

Antagonistic interactions between the SA and JA signaling pathways in Arabidopsis modulate expression of defense genes and gene-for-gene resistance to cucumber mosaic virus.

Gene-for-gene resistance to a yellow strain of cucumber mosaic virus [CMV(Y)] is conferred by the dominant RESISTANCE to CMV(Y) (RCY1) allele in the Arabidopsis thaliana ecotype C24. RCY1-conferred resistance to CMV(Y) and expression of the Pathogenesis-related 1 (PR-1) and PR-5 genes are partially compromised by the eds5 mutation and the nahG transgene that block accumulation of salicylic acid (SA). In contrast, the RCY1-conferred resistance to CMV(Y) is not affected by the jasmonic acid (JA)-insensitive coi1 and jar1 mutations. Interestingly, we report here that in contrast to the eds5 RCY1 plant, the eds5 coi1 RCY1 double-mutant plant exhibited a higher level of resistance to CMV(Y). Presence of the coi1 mutant allele also restored the CMV(Y)-activated expression of the PR-1 and PR-5 gene in the eds5 coi1 RCY1 plant. In contrast to the PR-1 and PR-5 genes, expression of the JA-dependent PLANT DEFENSIN 1.2 (PDF1.2) and HEVEIN-LIKE PROTEIN (HEL) genes was elevated in the CMV(Y)-inoculated leaves of the eds5 RCY1 plant, but not in the virus-inoculated leaves of the wild-type RCY1 and coi1 RCY1 plants. We propose that antagonistic interactions between the SA and JA signaling mechanisms modulate defense gene expression and the activation of RCY1-conferred gene-for-gene resistance to CMV(Y).

RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene, conferring resistance to cucumber mosaic virus requires salicylic acid, ethylene and a novel signal transduction mechanism.

The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently.