Reiko Tomita

Involvement of hydrogen peroxide in leaf abscission signaling, revealed by analysis with an in vitro abscission system in Capsicum plants

Although auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion- and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H(2)O(2)) is involved in abscission signaling. Microscopic analyses revealed continuous H(2)O(2) production by AZ cells. H(2)O(2) scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H(2)O(2) promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H(2)O(2) production, whereas the expression of ethylene-responsive genes was unaffected by both H(2)O(2) and an H(2)O(2) inhibitor. These results indicated that H(2)O(2) acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H(2)O(2) production at the AZ, which preceded leaf abscission. These results indicate that H(2)O(2) has roles in leaf abscission associated with ethylene both in vitro and in planta.

Cooperative effect of two amino acid mutations in the coat protein of Pepper mild mottle virus overcomes L3-mediated resistance in Capsicum plants

We found that an L3 resistance-breaking field isolate of Peppermildmottlevirus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L3-harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L3-mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L3-mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L3-mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L3-mediated resistance.

Reactive oxygen species in leaf abscission signaling

Reactive oxygen species (ROS) are produced in response to many environmental stresses, such as UV, chilling, salt, and pathogen attack. These stresses also accompany leaf abscission in some plants, however, the relationship between these stresses and abscission is poorly understood. In our recent report, we developed an in vitro abscission system that reproduces stress-induced pepper leaf abscission in planta. Using this system, we demonstrated that continuous production of hydrogen peroxide (H2O2) is involved in leaf abscission signaling. Continuous H2O2 production is required to induce expression of the cell wall-degrading enzyme, cellulase, and functions downstream of ethylene in abscission signaling. Furthermore, enhanced production of H2O2 occurs at the execution phase of abscission, suggesting that H2O2 also plays a role in the cell-wall degradation process. These data suggest that H2O2 has several roles in leaf abscission signaling. Here, we propose a model for these roles. Addendum to: Sakamoto M, Munemura I, Tomita R, Kobayashi K. Involvement of hydrogen peroxide in leaf abscission signaling revealed by analysis with an in vitro abscission system in Capsicum plants. Plant J 2008; doi: 10.1111/j.1365-313X.2008.03577.x

Recombinant plant dsRNA-binding protein as an effective tool for the isolation of viral replicative form dsRNA and universal detection of RNA viruses

The isolation of viral replicative form (RF) double-stranded RNA (dsRNA) is a classic technique for plant virus detection when the virus species cannot be predicted from disease symptoms. However, the method has not been very widely used, most likely because dsRNA isolation using CF-11 cellulose is laborious and time-consuming. Here we report an alternative tool, a recombinant plant dsRNA-binding protein, to isolate dsRNA. This tool enables us to isolate viral RF dsRNA in an hour from either extracted nucleic acids or crude detergent extracts. Combining this technique with sequence-non-specific reverse transcription, PCR amplification, cloning, and sequencing, a variety of viruses were efficiently detected using a single set of reagents and procedures.

Establishment of an agroinoculation system for broad bean wilt virus 2

We determined the complete nucleotide sequence of a broad bean wilt virus 2 (BBWV-2) isolate from gentian in Japan. The full-length RNA1 and RNA2 sequences, excluding poly(A) tails, were 5955 and 3600 nucleotides long, respectively. Analysis indicated that, in contrast to other BBWV-2 isolates, the 5’ end of both RNA1 and RNA2 starts with a GUU sequence. We successfully inoculated Nicotiana benthamiana with BBWV-2 by infiltrating a mixed suspension of two Agrobacterium tumefaciens clones carrying binary vectors with the full-length RNA1 and RNA2 sequences. This is the first report on the efficient, easy and high-throughput use of agroinoculation for generating BBWV-2 infections.

Amino acids in Tobamovirus coat protein controlling pepper L1a gene-mediated resistance

In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L(1a) can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV-J) and Paprika mild mottle virus Japanese strain (PaMMV-J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 °C. Interestingly, L(1a) gene-mediated resistance against TMGMV-J, but not PaMMV-J, is retained at 30 °C. This observation led us to speculate that L(1a) can discriminate between CPs of TMGMV-J and PaMMV-J. In this study, we aimed to determine the region(s) in CP by which L(1a) distinguishes TMGMV-J from PaMMV-J. By using chimeric CPs consisting of TMGMV-J and PaMMV-J, we found that the chimeric TMGMV-J CP, whose residues in the β-sheet domain were replaced by those of PaMMV-J, lost its ability to induce L(1a) gene-mediated resistance at 30 °C. In contrast, the chimeric PaMMV-J CP with the β-sheet domain replaced by TMGMV-J CP was able to induce L(1a) gene-mediated resistance at 30 °C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the β-sheet domain were involved in both the induction of L(1a) gene-mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV-J and PMMoV indicated that amino acids within the β-sheet domain alone were not sufficient for the induction of L(1a) gene-mediated resistance by TMGMV-J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L(1a) gene-mediated resistance.

A protein containing an XYPPX repeat and a C2 domain is associated with virally induced hypersensitive cell death in plants.

In this study, we characterized a Capsicum hypersensitive response (HR)‐associated gene, SS52, which encodes a protein that contains an N‐terminal C2 domain and a C‐terminal XYPPX repeat. Expression analyses revealed that SS52 and its homologue in Arabidopsis were induced by infection with incompatible viruses, indicating the conserved function of this gene. SS52 was not induced by treatment with defense‐related hormones, but was induced by abiotic stresses, including wounding. Overexpression of SS52 in tobacco plants suppressed the spread of HR cell death and restricted the spread of an incompatible virus from local lesions. Collectively, the results suggest that SS52 negatively regulates plant HR cell death.

Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as “DECS-C,” is a powerful method for detecting novel plant viruses.

Prevalence and genetic diversity of an unusual virus associated with Kobu-sho disease of gentian in Japan.

Gentian Kobu-sho-associated virus (GKaV) is a recently discovered novel virus from Kobu-sho (a hyperplastic or tumorous disorder)-affected Japanese gentians. To obtain insight into GKaV transmission and pathogenesis, the genetic diversity of the virus in the putative helicase and RNA-dependent RNA polymerase coding regions was studied. The extent of GKaV sequence diversity within single host plants differed within samples and between viral genomic regions. Phylogenetic analysis of 30 Kobu-sho-affected samples from different production areas and host cultivars revealed that GKaV populations have diverged as they became prevalent in different geographical regions. The diversification of GKaV was shown to be driven by geographical isolation rather than host adaptation; however, no geographical patterns were found. Therefore, it was not feasible to trace the pathway of GKaV spread.

Oat retrotransposon OARE-1 is activated in both compatible and incompatible interactions with pathogenic fungi

Transcriptional activity of the retrotransposon OARE-1 was monitored in oat leaves infected with pathogenic fungi. The transcription of OARE-1 was upregulated in both incompatible and compatible interactions between an oat cultivar and Magnaporthe grisea isolates but more extensively in the compatible. OARE-1 was also strongly activated in a Pc2/vb-carrying oat line inoculated with Helminthosporium victoriae. The upregulation in the Pc2/Vb-carrying oat line was reproduced by treatment with victorin C, the major compound of the host-specific toxin produced by the fungus. These results suggest that OARE-1 is responsive to various signals or stresses associated with compatibility and incompatibility.