Hideki Takahashi

The control of seedborne rice seedling diseases in the seed beds is important to avoid epidemics in rice nurseries and paddies, which may result in severe yield loss. Recently, irradiation with plasma containing electrons, creating positive or negative ions and neutral species, has been shown to have an antimicrobial effect, probably via generation of reactive oxygen species. This study examines whether two seedborne rice seedling diseases, bakanae disease caused by the fungal pathogen Fusarium fujikuroi , and bacterial seedling blight caused by Burkholderia plantarii , are suppressed by irradiation of infected rice seeds with atmospheric plasma. Seed germination and seedling growth were not inhibited in plasma‐treated healthy seeds. When F. fujikuroi ‐infected rice seeds were irradiated with plasma after being immersed in sterile distilled water, bakanae disease severity index and the percentage of plants with symptoms were reduced to 18.1% and 7.8% of non‐irradiated control, respectively, depending on the duration of plasma irradiation. The bacterial seedling blight disease index was also reduced by plasma irradiation in vacuum‐inoculated seeds to 38.6% of the non‐irradiated control, and in infected seeds harvested from spray‐inoculated heads of rice plants to 40.1% of the control. Therefore, plasma irradiation seems to be effective in controlling two independent seedborne rice seedling diseases.

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 ( PIP1 ) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum ‐inoculated stem tissues of quantitatively resistant tomato cultivar LS ‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3 , which is required for the Cf‐2 ‐mediated hypersensitive response ( HR ) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP 1 in R. solanacearum ‐inoculated LS ‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS ‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS ‐89 or Ponderosa were infiltrated with 10 ⁶ cells mL ⁻¹ R. solanacearum , cell death appeared in LS ‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS ‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR ‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS ‐89 is a true HR , and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS ‐89 to R. solanacearum .

Most of the reported dominant disease-resistance genes in plants, R genes, encode NB-LRR immune receptors. Plant genomes carry many NB-LRR type R genes that recognize specific pathogens and induce resistance against them. Thus, this immune system in plants is thought to perform similar functions as the adaptive immune system in animals. In this review, we provide an overview of the resistance mechanisms, evolution, and agricultural applications of R genes against plant viruses. We also introduce recent advances in research into the regulatory mechanisms of R gene expression, focusing on regulation by microRNAs and introns. One of the most intriguing phenomena that occur following R gene-mediated recognition of viruses is programmed cell death around the initial infection site, although its significance in the survival strategies of plants remains to be elucidated. We discuss the possible benefits for plants of inducing such programmed cell death based on our empirical observations and some hypotheses from an ecological point of view.

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.

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.

RCY1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (LRR) class R protein, confers the hypersensitive response (HR) to a yellow strain of Cucumber mosaic virus (CMV[Y]) in Arabidopsis thaliana. Nicotiana benthamiana transformed with hemagglutinin (HA) epitope-tagged RCY1 (RCY1-HA) also exhibited a defense response accompanied by HR cell death and induction of defense-related gene expression in response to CMV(Y). Following transient expression of RCY1-HA by agroinfiltration, the defense reaction was induced in N. benthamiana leaves infected with CMV(Y) but not in virulent CMV(B2)-infected N. benthamiana leaves transiently expressing RCY1-HA or CMV(Y)-infected N. benthamiana leaves transiently expressing HA-tagged RPP8 (RPP8-HA), which is allelic to RCY1. This result suggests that Arabidopsis RCY1-conferred resistance to CMV(Y) could be reproduced in N. benthamiana leaves in a gene-for-gene manner. Expression of a series of chimeric constructs between RCY1-HA and RPP8-HA in CMV(Y)-infected N. benthamiana indicated that induction of defense responses to CMV(Y) is regulated by the LRR domain of RCY1. Interestingly, in CMV(Y)-infected N. benthamiana manifesting the defense response, the levels of both RCY1 and chimeric proteins harboring the RCY1 LRR domain were significantly reduced. Taken together, these data indicate that the RCY1-conferred resistance response to CMV(Y) is regulated by an LRR domain-mediated interaction with CMV(Y) and seems to be tightly associated with the degradation of RCY1 in response to CMV(Y).

The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp .

Phytophthora infestans, the cause of potato and tomato late blight disease, produces INF1 elicitin, a 10 kDa extracellular protein. INF1 induces a hypersensitive response (HR) and systemic acquired resistance in species of the Nicotiana genus and a few other genera. We analysed the response of tomato to INF1 and INF1S3, which has a Cys to Ser substitution at position 3 of the processed protein and therefore lacks HR induction activity in tobacco. No HR cell death was induced in either INF1‐ or INF1S3‐treated tomato leaves. The expression of salicylic acid (SA)‐responsive PR‐1a(P6) and PR‐2a genes was not induced by treatment with either INF1 or INF1S3. However, the expression of jasmonic acid (JA)‐responsive PR‐6 encoding proteinase inhibitor II, LeATL6 encoding ubiquitin ligase E3, and LOX‐E encoding lipoxygenase, was up‐regulated in tomato leaves treated with INF1 but not in those treated with INF1S3. Their induction was completely compromised in INF1‐treated jai1‐1 mutant tomato, in which the JA signalling pathway is impaired. The accumulation of ethylene (ET) and the expression of ET‐responsive genes were also induced in tomato by INF1 but not INF1S3 treatment. The activation of JA and ET‐mediated signals but not the SA‐mediated signalling in INF1‐treated tomato was also demonstrated by global gene expression analysis. INF1‐treated tomatoes, but not those treated with INF1S3, exhibited resistance to bacterial wilt disease caused by Ralstonia solanacearum. Thus, INF1 seems to induce resistance to bacterial wilt disease in tomato and activate JA‐ and ET‐mediated signalling pathways without development of HR cell death.