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Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

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Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3-β-glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3-β-glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3-β-glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3-β-glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3-β-glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3-β-glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3-β-glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3-β-glucanase are encoded by gene families of considerable complexity.

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References

  1. Bedbrook J: A plant nuclear DNA preparation procedure. Plant Mol Biol Newsl 2: 24 (1981).

    Google Scholar 

  2. Bol JF, Linthorst HJM, Cornelissen BJC: Plant pathogenesis-related proteins induced by virus infection. Annu Rev Phytopath 28: 113–138 (1990).

    Google Scholar 

  3. Boller T: Hydrolytic enzymes in plant disease resistance. In: Kosuge T, Nester EW (eds) Plant-Microbe Interactions, vol 2: Molecular and Genetic Perspectives, pp. 385–413. Macmillan, New York (1987).

    Google Scholar 

  4. Boller T: Ethylene and the regulation of antifungal hydrolases in plants. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, vol. 5, pp. 145–174. Oxford University Press, Oxford (1988).

    Google Scholar 

  5. Brederode FT, Linthorst HJM, Bol JF: Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding. Plant Mol Biol 17: 1117–1125 (1991).

    Google Scholar 

  6. Broekaert W, Lee H-I, Kush A, Chua N-H, Raikhel N: Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis). Proc Natl Acad Sci USA 87: 7633–7637 (1990).

    Google Scholar 

  7. Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R: Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254: 1194–1197 (1991).

    Google Scholar 

  8. Broglie KE, Gaynor JJ, Broglie RM: Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris. Proc Natl Acad Sci USA 83: 6820–6824 (1986).

    Google Scholar 

  9. Castresana C, deCarvalho F, Gheysen G, Habets M, Inzé D, VanMontagu M: Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia β-1,3-glucanase gene. Plant Cell 2: 1131–1143 (1990).

    Google Scholar 

  10. Chen EY, Seeburg PH: Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4: 165–170 (1985).

    Google Scholar 

  11. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K: Plant chitinases. Plant J 3: 31–40 (1993).

    Google Scholar 

  12. deJong AJ, Cordewener J, LoSchiavo F, Terzi M, Vandekerckhove J, vanKammen A, deVries SC: A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4: 425–433 (1992).

    Google Scholar 

  13. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).

    Google Scholar 

  14. Edington BV, Lamb CJ, Dixon RA: cDNA cloning and characterization of a putative 1,3-β-D-glucanase transcript induced by fungal elicitor in bean cell suspension cultures. Plant Mol Biol 16: 81–94 (1991).

    Google Scholar 

  15. Fritzemeier K-H, Cretin C, Kombrink E, Rohwer F, Taylor J, Scheel D, Hahlbrock K: Transient induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in potato leaves infected with virulent and avirulent races of Phytophthora infestans. Plant Physiol 85: 34–41 (1987).

    Google Scholar 

  16. Gaynor JJ: Primary structure of an endochitinase mRNA from Solanum tuberosum. Nucl Acids Res 16: 5210 (1988).

    Google Scholar 

  17. Gubler U, Hoffmann BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).

    Google Scholar 

  18. Hahlbrock K, Scheel D: Biochemical responses of plants to pathogens. In: Chet I (ed) Innovative Approaches to Plant Disease Control, pp. 229–254. John Wiley, New York (1987).

    Google Scholar 

  19. Hedrick SA, Bell JN, Boller T, Lamb CJ: Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection. Plant Physiol 86: 182–186 (1988).

    Google Scholar 

  20. Høj PB, Hartman DJ, Morrice NA, Doan DNP, Fincher GB: Purification of (1 → 3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone. Plant Mol Biol 13: 31–42 (1989).

    Google Scholar 

  21. Joosten MHAJ, deWit PJGM: Identification of several pathogenesis-related proteins in tomato leaves inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-β-glucanases and chitinases. Plant Physiol 89: 945–951 (1989).

    Google Scholar 

  22. Knogge W, Kombrink E, Schmelzer E, Hahlbrock K: Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants. Planta 171: 279–287 (1987).

    Google Scholar 

  23. Kombrink E, Beerhues L, Garcia-Garcia F, Hahlbrock K, Müller M, Schröder M, Witte B, Schmelzer E: Expression patterns of defense-related genes in infected and uninfected plants. In: Fritig B, Legrand M (eds) Mechanisms of Plant Defense Responses, pp. 236–249. Kluwer Academic Publishers, Dordrecht (1993).

    Google Scholar 

  24. Kombrink E, Hahlbrock K, Hinze K, Schröder M: Molecular responses of potato to infection by Phytophthora infestans. In: Smith CJ (ed) Biochemistry and Molecular Biology of Plant-Pathogen Interactions, pp. 237–254. Oxford University Press (1991).

  25. Kombrink E, Schröder M, Hahlbrock K: Several ‘pathogenesis-related’ proteins in potato are 1,3-β-glucanases and chitinases. Proc Natl Acad Sci USA 85: 782–786 (1988).

    Google Scholar 

  26. Leah R, Tommerup H, Svendsen I, Mundy J: Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem 266: 1564–1573 (1991).

    Google Scholar 

  27. Linthorst HJM, Melchers LS, Mayer A, vanRoekel JSC, Cornelissen BJC, Bol JF: Analysis of gene families encoding acidic and basic β-1,3-glucanases of tobacco. Proc Natl Acad Sci USA 87: 8756–8760 (1990).

    Google Scholar 

  28. Lois R, Dietrich A, Hahlbrock K, Schulz W: A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements. EMBO J 8: 1641–1648 (1989).

    Google Scholar 

  29. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

    Google Scholar 

  30. Margis-Pinheiro M, Metz-Boutigue MH, Awade A, deTapia M, leRet M, Burkard G: Isolation of a complementary DNA encoding the bean PR4 chitinase: an acidic enzyme with an amino-terminus cysteine-rich domain. Plant Mol Biol 17: 243–253 (1991).

    Google Scholar 

  31. Mauch F, Hadwiger LA, Boller T: Antifungal hydrolases in pea tissue. I. Purification and characterization of two chitinases and two 1,3-β-glucanases differentially regulated during development and in response to fungal infection. Plant Physiol 87: 325–333 (1988).

    Google Scholar 

  32. Mauch F, Mauch-Mani B, Boller T: Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and 1,3-β-glucanase. Plant Physiol 88: 936–942 (1988).

    Google Scholar 

  33. MeinsJr F, Neuhaus J-M, Sperisen C, Ryals J: The primary structure of plant pathogenesis-related glucanohydrolases and their genes. In: Boller T, Meins F (eds) Genes Involved in Plant Defense, pp. 245–282. Springer-Verlag, Wien (1992).

    Google Scholar 

  34. Memelink J, Linthorst HJM, Schilperoort RA, Hoge JHC: Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns. Plant Mol Biol 14: 119–126 (1990).

    Google Scholar 

  35. Metraux JP, Burkhart W, Moyer M, Dincher S, Middlesteadt W, Williams S, Payne G, Carnes M, Ryals J: Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proc Natl Acad Sci USA 86: 896–900 (1989).

    Google Scholar 

  36. Murray NE: Phage lambda and molecular cloning. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA (eds) Lambda II, pp. 395–432. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983).

    Google Scholar 

  37. Neuhaus J-M, Ahl-Goy P, Hinz U, Flores S, MeinsJr F: High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol Biol 16: 141–151 (1991).

    Google Scholar 

  38. Neuhaus J-M, Sticher L, MeinsJr F, Boller T: A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88: 10362–10366 (1991).

    Google Scholar 

  39. Ori N, Sessa G, Lotan T, Himmelhoch S, Fluhr R: A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass. EMBO J 9: 3429–3436 (1990).

    Google Scholar 

  40. Payne G, Ahl P, Moyer M, Harper A, Beck J, MeinsJr F, Ryals J: Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco. Proc Natl Acad Sci USA 87: 98–102 (1990).

    Google Scholar 

  41. Pierpoint WS, Jackson PJ, Evans RM: The presence of a thaumatin-like protein, a chitinase and a glucanase among the pathogenesis-related proteins of potato (Solanum tuberosum). Physiol Mol Plant Path 36: 325–338 (1990).

    Google Scholar 

  42. Samac DA, Hironaka CM, Yallaly PE, Shah DM: Isolation and characterization of the genes encoding basic and acidic chitinase in Arabidopsis thaliana. Plant Physiol 93: 907–914 (1990).

    Google Scholar 

  43. Schlumbaum A, Mauch F, Vögeli U, Boller T: Plant chitinases are potent inhibitors of fungal growth. Nature 324: 365–367 (1986).

    Google Scholar 

  44. Schröder M: Abwehrreaktionen von Solanum tuberosum L. gegen Phytophthora infestans (Mont.) de Bary: Untersuchungen zur biologischen Funktion, subzellulären Lokalisation und Expression von PR-Proteinen. Ph.D. thesis, Universität zu Köln (1990).

  45. Schröder M, Hahlbrock K, Kombrink E: Temporal and spatial patterns of 1,3-β-glucanase and chitinase induction in potato leaves infected by Phytophthora infestans. Plant J 2: 161–172 (1992).

    Google Scholar 

  46. Shinshi H, Mohnen D, MeinsJr F: Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci USA 84: 89–93 (1987).

    Google Scholar 

  47. Shinshi H, Wenzler H, Neuhaus J-M, Felix G, Hofsteenge J, MeinsJr F: Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-β-1,3-glucanase. Proc Natl Acad Sci USA 85: 5541–5545 (1988).

    Google Scholar 

  48. Sperisen C, Ryals J, Meins F: Comparison of cloned genes provides evidence for intergenomic exchange of DNA in the evolution of a tobacco glucan endo-1,3-β-glucosidase gene family. Proc Natl Acad Sci USA 88: 1820–1824 (1991).

    Google Scholar 

  49. Sticher L, Hofsteenge J, Neuhaus J-M, Boller T, MeinsJr F: Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase. Plant Physiol 101: 1239–1247 (1993).

    Google Scholar 

  50. Takeuchi Y, Yoshikawa M, Takeba G, Tanaka K, Shibata D, Horino O: Molecular cloning and ethylene induction of mRNA encoding a phytoalexin elicitor-releasing factor, β-1,3-endoglucanase, in soybean. Plant Physiol 93: 673–682 (1990).

    Google Scholar 

  51. Taylor JL, Fritzemeier K-H, Häuser I, Kombrink E, Rohwer F, Schröder M, Strittmatter G, Hahlbrock K: Structural analysis and activation by fungal infection of a gene encoding a pathogenesis-related protein in potato. Mol Plant-Microbe Interact 3: 72–77 (1990).

    Google Scholar 

  52. vonHeijne G: Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133: 17–21 (1983).

    Google Scholar 

  53. Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Métraux J-P, Ryals JA: Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3: 1085–1094 (1991).

    Google Scholar 

  54. Witte B: Chitinasen in Kartoffel: Charakterisierung verschiedener Isoformen und Untersuchungen zu ihrer Expression. Ph.D. thesis, Universität zu Köln (1991).

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Beerhues, L., Kombrink, E. Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato. Plant Mol Biol 24, 353–367 (1994). https://doi.org/10.1007/BF00020173

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