Immunomicroscopic anaysis of infection process of plant pathogen

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07456031
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 植物保護
• Research Institution: KYOTO PREFECTURAL UNIVERSITY
• Principal Investigator: HORINO Osamu KYOTO PREFECTURAL UNIVERSITY,Laboratory of Plant Pathology, Faculty of Agriculture, Professor, 農学部, 教授 (10209306)
• Co-Investigator(Kenkyū-buntansha): TSUGE Seiji KYOTO PREFECTURAL UNIVERSITY,Laboratory of Plant Pathology, Faculty of Agricultu, 農学部, 助手 (10254319) ; KUBO Yasuyuki KYOTO PREFECTURAL UNIVERSITY,Laboratory of Plant Pathology, Faculty of Agricultu, 農学部, 助教授 (80183797)
• Project Period (FY): 1995 – 1996
• Keywords: Xanthomonas / Colletotrichum / Immunoelectronmicroscopy / Polysaccharide / Avirulent gene / Melanin / Appressoria

Research Abstract

Some plant-pathogenic bacteria including Xanthomonas oryzae pv.oryzae have been shown to have hypersensitive reaction and pathogenicity (hrp) genes which were involved in pathogenicity for host plants and inducing hypersensitive reaction for nonhost plants. Although a hrpXo locus of Xanthomonas oryzae pv.oryzaeis suggested to encode a protein which is a regulator for other hrp genes by its homology with other bacterial genes, the details remain unknown. In this study antibody reacting with the hrpXo product was obtained and using this antibody expression and localization of the protein in host plants (rice) and non-host plants (cow pea) invaded with X.o.pv.oryzae was examined. As antigen a part of the protein encoded on hrpXo was expressed by the in vitro expression system using Escherichia coli. Immuno-microscopicobservation using the antibody obtained showed that hrpXo product localized in the bacterial cells specifically in the vessels of rice leaves infected with X.o.pv.oryzae. No specific signal was detected in the cell incubated on the medium, suggesting that this protein expressed specifically during infection on plants. In the cow pea leaves inoculated with X.o.pv.oryzae, bacterial cells were shown to be enveloped by fibrillar and granular material extruded from the host cell walls by the electron microscopic observation 7 hours after inoculation, following bacterial degradation. Necrotic legions were formed 12 hours after inoculation. hrpXo product was, however, already expressed and accumulated in 3 hours after inoculation. A mutant which deleted hrpXo region was shown to loose pathogenicity for rice and not to induce hypersensitive reaction for cow pea. Results described above show that hrpXo product of X.o.pv.oryzae was indispensable for pathogenicity for host plants and for induction ability of hypersensitive reaction for nonhost plants, and support the hypothesis that this product regulate other hrp genes of this pathogen. In the experiment using a plant pathogenic fungus, Colletotrichum lagenarium, polyclonal antibodies were prepared against melanin biosynthetic enzymes, trihydroxynaphthalene reductase and scytalone dehydratase which were purified from E.coli.expressing those recombinant enzymes. Immuno electron microscopc observation was performed to examine localization of those melanin biosynthetic enzymes.