In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit.

BACKGROUND Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. RESULTS In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.

[1]  K. Hoffmann‐Sommergruber,et al.  Cysteine proteinase inhibitor Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy. , 2010, Molecular nutrition & food research.

[2]  R. Senthilkumar,et al.  Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco. , 2010, Plant biotechnology journal.

[3]  M. Diaz-Mendoza,et al.  Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination1[W] , 2009, Plant Physiology.

[4]  M. Nissen,et al.  Characterization of Solanum tuberosum Multicystatin and Its Structural comparison with Other Cystatins[OA] , 2009, The Plant Cell Online.

[5]  R. Fluhr,et al.  Mechanisms Modulating Postharvest Pathogen Colonization of Decaying Fruits , 2009 .

[6]  I. Díaz,et al.  The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship , 2008, BMC Evolutionary Biology.

[7]  N. Sharma,et al.  Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit. , 2007, Functional plant biology : FPB.

[8]  M. Diaz-Mendoza,et al.  Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases , 2007, FEBS letters.

[9]  C. Tsai,et al.  Purification and Characterization , 2006 .

[10]  Tatiana Giraud,et al.  Partition of the Botrytis cinerea complex in France using multiple gene genealogies , 2005, Mycologia.

[11]  G. Marzban,et al.  Localisation and distribution of the major allergens in apple fruits , 2005 .

[12]  I. Díaz,et al.  The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties. , 2005, Journal of experimental botany.

[13]  E. Sfakiotakis,et al.  Postharvest Biological Control of Botrytis cinerea on Kiwifruit by Volatiles of [Isabella] Grapes. , 2004, Phytopathology.

[14]  R. H. Khan,et al.  Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. , 2004, Archives of biochemistry and biophysics.

[15]  G. Salcedo,et al.  A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests , 1998, Plant Molecular Biology.

[16]  W. Laing,et al.  Purification and characterization of phytocystatins from kiwifruit cortex and seeds. , 2004, Phytochemistry.

[17]  I. Díaz,et al.  Inhibition of plant-pathogenic fungi by the barley cystatin Hv-CPI (gene Icy) is not associated with its cysteine-proteinase inhibitory properties. , 2003, Molecular plant-microbe interactions : MPMI.

[18]  A. Smigocki,et al.  Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the Root-Lesion Nematode. , 2003, Phytopathology.

[19]  L. Beltramini,et al.  A sugarcane cystatin: recombinant expression, purification, and antifungal activity. , 2002, Biochemical and biophysical research communications.

[20]  C. Selitrennikoff.  Antifungal Proteins , 2001, Applied and Environmental Microbiology.

[21]  D. Choi,et al.  A novel proteinase inhibitor gene transiently induced by tobacco mosaic virus infection. , 2000, Biochimica et biophysica acta.

[22]  L. C. Loon,et al.  The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins , 1999 .

[23]  P. Rodríguez-Palenzuela,et al.  Antifungal Activity of a Plant Cystatin , 1999 .

[24]  V. Villeret,et al.  Structural and phylogenetic relationships among plant and animal cystatins. , 1998, Archives of biochemistry and biophysics.

[25]  P. Ranjekar,et al.  Cysteine protease inhibitor from pearl millet: a new class of antifungal protein. , 1998, Biochemical and biophysical research communications.

[26]  S. Boyes,et al.  Actinidin levels in fruit of Actinidia species and some Actinidia arguta rootstock-scion combinations , 1997 .

[27]  C. d’Enfert.  Fungal Spore Germination: Insights from the Molecular Genetics ofAspergillus nidulansandNeurospora crassa , 1997 .

[28]  J. Rotem.  The Genus Alternaria: Biology, Epidemiology, and Pathogenicity , 1997 .

[29]  S. Machida,et al.  Purification and characterization of membrane-bound chitin synthase. , 1993, The Journal of biological chemistry.

[30]  S. Arai,et al.  Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Molecular cloning and expression studies. , 1992, European journal of biochemistry.

[31]  S. Nielsen,et al.  Isolation and partial characterization of a soybean cystatin cysteine proteinase inhibitor of coleopteran digestive proteolytic activity , 1991 .

[32]  M. Daniels,et al.  Evidence for the requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape , 1991 .

[33]  D. Bowles,et al.  Defense-related proteins in higher plants. , 1990, Annual review of biochemistry.

[34]  A. Barrett,et al.  Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. , 1983, The Biochemical journal.

[35]  N. Harboe,et al.  23. Immunization, Isolation of Immunoglobulins, Estimation of Antibody Titre , 1973, Scandinavian journal of immunology. Supplement.

[36]  B. Kassell.  [66] Naturally occurring inhibitors of proteolytic enzymes , 1970 .