Sugihiro Ando

Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants.

Increase in BrAO1 gene expression and aldehyde oxidase activity during clubroot development in Chinese cabbage (Brassica rapa L.)

SUMMARY In clubroot disease, gall formation is induced by infection with the obligate biotroph Plasmodiophora brassicae due to increased levels of auxins and cytokinins. Because aldehyde oxidase (AO) may be involved in auxin biosynthesis in plants, we isolated two AO genes (BrAO1 and BrAO2) from Chinese cabbage (Brassica rapa ssp. pekinensis cv. Muso), which are the most similar to AAO1 among Arabidopsis AO genes, and examined their expressions during clubroot development. The expression of BrAO1 was enhanced in inoculated roots from 15 days post-inoculation (dpi) when visible clubroots were still undetectable. Thereafter, BrAO1 expression increased with clubroot development compared with uninoculated roots, although BrAO2 expression was repressed. In situ hybridization revealed that BrAO1 was strongly expressed in tissues that were invaded by immature plasmodia at 35 dpi, suggesting that BrAO1 expression was enhanced by the pathogen in order to establish its pathogenesis. In addition, we detected AO activity, as evidenced by the occurrence of at least six bands (BrAO-a to BrAO-f) in the roots of Chinese cabbage using an active staining method with benzaldehyde and indlole-3-aldehyde as the substrate. Coincidental with BrAO1 expression, the signals of BrAO-a and BrAO-d increased with inoculation by P. brassicae during clubroot development compared with healthy roots, resulting in an increase in total AO activity. By contrast, the band BrAO-b decreased post-inoculation, in parallel with the expression of BrAO2. The other bands of activity were not clearly influenced by the infection. Based on these results, we discuss the involvement of AO in auxin-overproduction during clubroot development in Chinese cabbage.

Transcriptional profile of tomato roots exhibiting Bacillus thuringiensis-induced resistance to Ralstonia solanacearum.

Key messageActivation of SA-dependent signaling pathway and suppression of JA-dependent signaling pathway seem to play key roles inB. thuringiensis-induced resistance toR. solanacearumin tomato plants.AbstractBacillus thuringiensis, a well-known and effective bio-insecticide, has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. Treatment of tomato roots with a filter-sterilized cell-free filtrate (CF) of B. thuringiensis systemically suppresses bacterial wilt caused by Ralstonia solanacearum through systemic activation of the plant defense system. Comparative analysis of the expression of the Pathogenesis-Related 1(P6) gene, a marker for induced resistance to pathogens, in various tissues of tomato plants treated with CF on their roots suggested that the B. thuringiensis-induced defense system was activated in the leaf, stem, and main root tissues, but not in the lateral root tissue. At the same time, the growth of R. solanacearum was significantly suppressed in the CF-treated main roots but not in the CF-treated lateral roots. This distinct activation of the defense reaction and suppression of R. solanacearum were reflected by the differences in the transcriptional profiles of the main and lateral tissues in response to the CF. In CF-treated main roots, but not CF-treated lateral roots, the expression of several salicylic acid (SA)-responsive defense-related genes was specifically induced, whereas jasmonic acid (JA)-related gene expression was either down-regulated or not induced in response to the CF. On the other hand, genes encoding ethylene (ET)-related proteins were induced equally in both the main and lateral root tissues. Taken together, the co-activation of SA-dependent signaling pathway with ET-dependent signaling pathway and suppression of JA-dependent signaling pathway may play key roles in B. thuringiensis-induced resistance to R. solanacearum in tomato.

RCY1-Mediated Resistance to Cucumber mosaic virus Is Regulated by LRR Domain-Mediated Interaction with CMV(Y) Following Degradation of RCY1

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).

Microbial community profiles in intercellular fluid of rice

Plants harbor microorganisms that are thought to stimulate plant defense systems or promote plant growth. Individual species in these intercellular microbial communities are often not sufficiently abundant to be easily described, although some endophytic microorganisms amenable to culture have been characterized. To better understand the microbial population of plants, we collected intercellular fluid (IF) from leaf blades and sheaths of rice and subsequently isolated DNA from the IF. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S and 18S rDNA fragments amplified from IF DNA by PCR indicated that these band patterns were distinguishable from those of a leaf surface-wash fluid (SF). Analysis of a set of rDNA fragments amplified from IF DNA of rice with different genotypes, paddies or growth stages for the primary survey of overall microbial community in the IF suggested that this approach is suitable for analyzing microbial diversity in the IF from various plant samples. Actually, comparative analysis of amplified rDNA fragments of rice and other five plant species indicated that the microbial diversity in IF is likely to vary substantially among plant species. We can also use sequence analysis of 16S rDNA fragments amplified from rice IF DNA to identify species including unculturable bacteria and proteobacteria and Xanthomonas and 18S rDNA fragments to identify Tilletiaria anomala, Tilletia iowensis, Ustilago maydis and unculturable eukaryotes. Thus, IF DNA analysis seems to be a good tool to further study the microbial ecology of plants.

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.

Suppressive Potential of Paenibacillus Strains Isolated from the Tomato Phyllosphere against Fusarium Crown and Root Rot of Tomato

The suppressive potentials of Bacillus and Paenibacillus strains isolated from the tomato phyllosphere were investigated to obtain new biocontrol candidates against Fusarium crown and root rot of tomato. The suppressive activities of 20 bacterial strains belonging to these genera were examined using seedlings and potted tomato plants, and two Paenibacillus strains (12HD2 and 42NP7) were selected as biocontrol candidates against the disease. These two strains suppressed the disease in the field experiment. Scanning electron microscopy revealed that the treated bacterial cells colonized the root surface, and when the roots of the seedlings were treated with strain 42NP7 cells, the cell population was maintained on the roots for at least for 4 weeks. Although the bacterial strains had no direct antifungal activity against the causal pathogen in vitro, an increase was observed in the antifungal activities of acetone extracts from tomato roots treated with the cells of both bacterial strains. Furthermore, RT-PCR analysis verified that the expression of defense-related genes was induced in both the roots and leaves of seedlings treated with the bacterial cells. Thus, the root-colonized cells of the two Paenibacillus strains were considered to induce resistance in tomato plants, which resulted in the suppression of the disease.

Magnaporthe oryzae Glycine-Rich Secretion Protein, Rbf1 Critically Participates in Pathogenicity through the Focal Formation of the Biotrophic Interfacial Complex

Magnaporthe oryzae, the fungus causing rice blast disease, should contend with host innate immunity to develop invasive hyphae (IH) within living host cells. However, molecular strategies to establish the biotrophic interactions are largely unknown. Here, we report the biological function of a M. oryzae-specific gene, R equired-for-Focal- B IC- F ormation 1 (RBF1). RBF1 expression was induced in appressoria and IH only when the fungus was inoculated to living plant tissues. Long-term successive imaging of live cell fluorescence revealed that the expression of RBF1 was upregulated each time the fungus crossed a host cell wall. Like other symplastic effector proteins of the rice blast fungus, Rbf1 accumulated in the biotrophic interfacial complex (BIC) and was translocated into the rice cytoplasm. RBF1-knockout mutants (Δrbf1) were severely deficient in their virulence to rice leaves, but were capable of proliferating in abscisic acid-treated or salicylic acid-deficient rice plants. In rice leaves, Δrbf1 inoculation caused necrosis and induced defense-related gene expression, which led to a higher level of diterpenoid phytoalexin accumulation than the wild-type fungus did. Δrbf1 showed unusual differentiation of IH and dispersal of the normally BIC-focused effectors around the short primary hypha and the first bulbous cell. In the Δrbf1-invaded cells, symplastic effectors were still translocated into rice cells but with a lower efficiency. These data indicate that RBF1 is a virulence gene essential for the focal BIC formation, which is critical for the rice blast fungus to suppress host immune responses.

Survey of the response of 82 domestic landraces of Zea mays to cucumber mosaic virus (CMV) reveals geographical region-related resistance to CMV in Japan

Zea mays has been historically imported to Japan via two independent geographical routes: into southern Japan by trading with Europe in the 16th century and into northern Japan by import from North America in the 19th century. Breeding to genetically improve on quality traits and high yields has led to the current domestic landraces in each region. In a survey of 82 domestic landraces, nine out of 38 landraces originating from southern Japan showed complete immunity to cucumber mosaic virus yellow strain (CMV(Y)) without the formation of necrotic local lesions (NLLs). In contrast, three out of 44 landraces originating from northern Japan developed NLLs, but revealed no systemic spread of the virus. Due to the absence of good documentation on NLL formation in Z. mays, the response of domestic landraces Aso-1 and Aso-3, originating from Ibaraki in northern Japan, to a challenge with CMV(Y) and CMV(Ma-1) was further analysed. Aso-3 only formed NLL in response to CMV(Y) but not to CMV(Ma-1). Moreover, in CMV(Y)-inoculated Aso-3, virus spread was restricted to the primary infection site and the expression of defence-related genes was up-regulated, whereas Aso-1 became systemically infected with either CMV(Y) or CMV(Ma-1). The response of Aso-3 to CMV(Y) was inherited as a single dominant trait. Together, these results pointed towards the induction of hypersensitive response (HR)-mediated resistance to CMV(Y) in Aso-3. Although HR-mediated resistance to viruses has been studied mainly in dicots, the pathosystem CMV-Z. mays may provide a model to investigate HR-mediated resistance to viruses in monocot plants.

Atmospheric-pressure plasma irradiation can disrupt tobacco mosaic virus particles and RNAs to inactivate their infectivity

Low-temperature atmospheric-pressure air plasma is a source of charged and neutral gas species. In this study, N-carrying tobacco plants were inoculated with plasma irradiated and non-irradiated tobacco mosaic virus (TMV) solution, resulting in necrotic local lesions on non-irradiated, but not on irradiated, TMV-inoculated leaves. Virus particles were disrupted by plasma irradiation in an exposure-dependent manner, but the viral coat protein subunit was not. TMV RNA was also fragmented in a time-dependent manner. These results indicate that plasma irradiation of TMV can collapse viral particles to the subunit level, degrading TMV RNA and thereby leading to a loss of infectivity.