Tadaaki Hibi

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens

Abstract The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed.

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase.

Abstract Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R0), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines.

Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea)

Abstract A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance.

Tandem repeat of a transcriptional enhancer upstream of the sterol 14α-demethylase gene (CYP51) in Penicillium digitatum

ABSTRACT We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating theCYP51 gene, which encodes the target enzyme (P45014DM) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance andCYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5′ flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5′ flanking regions of another chitinase gene and a β-glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.

Transgenic chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) expressing a rice chitinase gene shows enhanced resistance to gray mold (Botrytis cinerea)

Abstract Transformation of spray-type chrysanthemum was performed using Agrobacterium tumefaciens strain C58 and MP90 harboring a rice chitinase gene (cDNA clone named: RCC2). Eleven transgenic lines expressing the RCC2 gene were obtained. These lines showed enhanced resistance to gray mold (Botrytis cinerea), although the levels of resistance varied among the transgenic lines. Three higher resistance lines, Y12, Y61 and Y97, showed very slight symptoms against B. cinerea infection and which did not spread even if the incubation period was extended. In these three lines, a higher production of RCC2 protein was detected by enzyme-linked immunosorbent assay (ELISA) compared with non-transgenic plants. These results suggest that the RCC2 gene can be a useful tool to improve resistance to gray mold in chrysanthemum.

Synergistic Antifungal Activity of Chitinolytic Enzymes and Prodigiosin Produced by Biocontrol Bacterium, Serratia marcescens Strain B2 against Gray Mold Pathogen, Botrytis cinerea

Serratia marcescensstrain B2 is an effective biocontrol agent against gray mold of cyclamen, caused by Botrytis cinerea Persoon. Strain B2 has strong antifungal activity against B. cinerea in vitro. The culture filtrate was found to contain different types of chitinolytic enzyme activity, including endochitinase and chitobiase activity. Four chitinolytic enzymes were detected among the extracellular proteins of strain B2. Two major enzymes, a 58-kDa endochitinase and a 98-kDa chitobiase, were purified by chromatography and electrofocusing, respectively. Both enzymes inhibited spore germination of B. cinerea. And a red pigment, prodigiosin, extracted and purified from the bacterial cells, also inhibited spore germination. When prodigiosin and the chitinolytic enzymes were applied in concert, a synergistic inhibitory effect was observed. S. marcescens strain B2 has multiple modes of action against the pathogen.

Detailed analysis of rice chitinase gene expression in transgenic cucumber plants showing different levels of disease resistance to gray mold (Botrytis cinerea)

Abstract The class I chitinase cDNA (RCC2) of rice driven by the CaMV 35S promoter was introduced into cucumber by Agrobacterium-mediated transformation. The transgenic cucumbers showed varying levels of disease resistance to gray mold. We selected three transgenic lines representing different resistance levels, namely the most resistant line (CR32), an intermediate-resistance line (CR3), and a susceptible line (CR20), and studied the correlation between RCC2 expression and disease resistance. ELISA analysis showed that the rice chitinase levels of CR32 and CR3 were higher than that of CR20. RCC2 expression was then examined in more detail using an in situ indirect fluorescent antibody technique and 3-D fluorescence microscopy (CELL Scan system). A homogeneous distribution of cells expressing RCC2 to a high degree was observed in the epidermal and mesophyll cells of CR32 leaves, whereas the intensity of RCC2 expression in CR3 leaves was lower. However, CR3 often showed high expression of RCC2 in parts of leaf tissue. Rice chitinase was localized within cells but was not detected between cells. Optical microscopy of Botrytis cinerea infection behavior revealed that none of the transgenic lines nor any of the non-transgenic lines inhibited the penetration of B. cinerea. However, fungal growth within leaf tissue was suppressed in the resistant lines but not in the non-transformed or CR20 leaf tissue. Thus, we speculate that high expression and intracellular localization of rice chitinase may be involved in enhancing the resistance of transgenic plants to gray mold.

Rice chitinase gene : cDNA cloning and stress-induced expression

Abstract A rice chitinase cDNA clone was isolated using a probe derived from a barley chitinase sequence. The accumulation of the gene transcripts in leaves and suspension cultured cells was also investigated. The cDNA clone had a 1.2 kbp insert containing a single open reading frame of 307 amino acids and showed about 67% homology to the basic chitinases from tobacco and bean. However, the codon usage of the rice chitinase gene was very different from the other known chitinase genes of dicot plants, as expected. DNA blot hybridization analysis suggested that chitinase was encoded by a multigene family of four to six genes. RNA blot hybridization analysis revealed that rice chitinase mRNA levels increased after treating leaves and suspension cultured cells with ethylene or stress-inducings compounds.

Characterization of transgenic rice plants over-expressing the stress-inducible β-glucanase gene Gns1

The Gns1 gene of rice (Oryza sativa L. japonica) encodes 1,3;1,4-β glucanase (EC 3.2.1.73), which hydrolyzes 1,3;1,4-β-glucosidic linkages on 1,3;1,4-β-glucan, an important component of cell walls in the Poaceae family. RNA and protein gel blot analyses demonstrated that blast disease or dark treatment induced the expression of the Gns1 gene. To assess the function of the Gns1 gene in disease resistance, we characterized transgenic rice plants constitutively expressing the Gns1 gene. The introduced Gns1 gene was driven by the CaMV 35S promoter and its products were found in the apoplast and accumulated in up to 0.1% of total soluble protein in leaves. Although transgenic plants showed stunted growth and impaired root formation, fertility, germination, and coleoptile elongation appeared unaffected compared to non-transgenic control plants, indicating that Gns1 does not play a crucial role in rice germination and coleoptile elongation. When transgenic plants were inoculated with virulent blast fungus (Magnaporthe grisea), they developed many resistant-type lesions on the inoculated leaf accompanying earlier activation of defense-related genes PR-1 and PBZ1 than in control plants. Transgenic plants spontaneously produced brown specks, similar in appearance to those reported for an initiation type of disease-lesion-mimic mutants, on the third and fourth leaves and occasionally on older leaves without inoculation of pathogens. Expression of the two defense-related genes was drastically increased after the emergence of the lesion-mimic phenotype.