A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination
1 Embrapa Recursos Genéticos e Biotecnologia. Parque Estação Biológica - PqEB - Av. W5 Norte (final). Postal box 02372 - Brasília, DF- 70770-917 - Brasil
2 Embrapa Soja. Rod. Carlos João Strass - Distrito de Warta. Postal box 231 - Londrina, PR- 86001-970 - Brasil
3 Embrapa Cerrados. BR 020 Km 18. Postal box: 08223 - Planaltina, DF- 73310-970 - Brasil
4 Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília (UnB). Campus Universitário Darcy Ribeiro, DF - 70910-900, Brasília
5 School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK
BMC Biotechnology 2011, 11:14 doi:10.1186/1472-6750-11-14Published: 7 February 2011
Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris.
A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%.
Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.