In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops

Martínez et al. AMB Express 2012, 2:22 http://www.amb-express.com/content/2/1/22 ORIGINAL ARTICLE Open Access In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops Claudia P Martínez1, Claudia Echeverri2, Juan C Florez1, Alvaro L Gaitan2 and Carmenza E Góngora1* Abstract Two genes from Streptomyces albidoflavus, one exochitinase (905-bp) and an endochitinase (1100-bp) were functionally expressed in Escherichia coli in form of a fusion protein with a maltose binding protein (MBP). The goal was to produce and test proteins that inhibit both the coffee berry borer insect Hypothenemus hampei and the coffee rust fungus Hemileia vastatrix. Both recombinant proteins MBP/exochitinase and MBP/endochitinase showed chitinolytic activity. When recombinant purified proteins were added to an artificial coffee-based diet for the coffee berry borer, MBP/exochitinase at a concentration of 0.5% W/W caused delayed growth of larvae and 100% mortality between days 8 and 15, while MBP/endochitinase caused 100% mortality at day 35. H. vastatrix urediniospores presented total cell wall degradation in their germinative tubes within 18 h of exposure to the proteins at enzyme concentrations of 5 and 6 mg ml-1, with exochitinase having the greatest effect. The dual deleterious effect of S. albidoflavus chitinases on two of the most limiting coffee pests worldwide, the coffee borer and the coffee rust, make them potential elements to be incorporated in integrated control strategies. Keywords: Coffee-based artificial diet, Uredinospores, Arrested development, Fungal cell wall, Growth, Mortality Introduction Colombia is one of the most important countries for production of mild coffee (Coffea arabica L.), with over 870,000 Ha planted (Federacafé 2010). The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytidae), is the most significant pest of the Colombian coffee crop, and it is found in all the coffee-growing regions of the world (Benavides et al. 2012; Bustillo 2002). Female insects fly towards coffee beans and bore into them until reaching the seed endocarpium, where they deposit their eggs. After the eggs * Correspondence: 1 Entomology Department, Cenicafé, Federacafe (National Coffee Research Centre - National Federation of Coffee Growers), PlanAlto Km 4 via antigua a Manizales, Chinchina, Caldas, Colombia Full list of author information is available at the end of the article hatch, larvae feed on the seed, causing weight loss in the grain, decreasing quality due to fungal contamination, and the falling of small cherries to the ground (Duque et al. 1997). The coffee leaf rust, caused by the fungus Hemileia vastatrix Berk. and Br. (Uredinales) is the main limiting disease of susceptible Coffea arabica varieties around the world. Although all its life cycle occurs in the leaves, cholorosis and defoliation affect the filling and maturation of coffee beans, reducing bean size and quality. Several rust resistant varieties have been bred in various coffee producing countries, including the Colombia and Castillo varieties (Castillo and Moreno 1988; Alvarado et al. 2005) but the ability of H. vastatrix to overcome such genetic resistance threatens continuously the durability of this control method. To date, the natural resistance © 2012 Martínez et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Martínez et al. AMB Express 2012, 2:22 http://www.amb-express.com/content/2/1/22 to the coffee borer identified in C. arabica germplasm collections is considered weak, and sources of resistance genes against the coffee leaf rust are limited. Both parasites, the coffee berry borer and the coffee rust, contain chitin, an insoluble linear b-1,4 polymer of N-acetyl b-D- glucosamide, that is a structural component present in the cuticle and shells of arthropods and mollusks, in the cell wall of fungi and some algae, and in certain nematode stages (Brydon et al. 1989; Elango et al. 1982; Gooday 1990). In insects, chitin is a significant component of the cuticle, which constitutes part of the exoskeleton, and at least partially lines along internal organs including the digestive tract; in addition it is a structural component of the peritrophic membrane that covers the midgut (Peters 1992). In fungi, a-chitin is an essential component of the structure of the cell wall (Gooday et al. 1992). Chitin can be enzymatically cleaved by two main enzyme classes: chitinases and N-acetyl-b-D-glucosaminidase. Chitinases (E.C. 3.2.1.14) catalyze the hydrolysis of the linear polymer b-1,4 linkages in chitin and chitooligomers resulting in the release of short chitooligomers. Depending on their cleavage pattern chitinases can be divided in endochitinases and exochitinases. Endochitinases degrade chitin from any point along the polymer chain forming random size length products while exochitinases cleave from the non-reduced end and the released product is (GLcNac)2 (Seidl 2008). Fungal chitinases can be divided into three different subgroups, namely, A, B and C, based on the amino acid sequences of their GH 18 modules. These subgroups differ in the architectures of their substrate-binding cleft and, thus, their enzymatic activities (exo vs. endo) and also contain different carbohydrate-binding modules (Gruber et al. 2011; Seidl 2008). Horn et al. (2006) studying the chitinolytic system of the bacterium Serratia marcescens demonstrated another way to classify the enzymatic properties of chitinases by grouping them into processive and non-processive enzymes. Processive chitinases do not release the substrate after hydrolytic cleavage but slide it through the active sitetunnel for the next cleavage step to occur. The presence of a carbohydrate binding domain can enhance processivity, but is not essential for it. Non-processive chitinases dissociate completely from the substrate after hydrolysis. Chitinases are essential to arthropods (Chen 1987) and fungi (Review Seidl 2008; Hartl et al. 2011) to regulate the presence of chitin during growth, development, and differentiation. In insects, chitinases are involved in molting and digestion processes (see review Muthukrishnan et al. 2011), while in fungi, they mainly help to Page 2 of 11 degrade and mobilize organic matter and to interfere with the growth of other fungi. Cuticle in insects is the target site of chitinolytic enzymes and its damage affects survival (Hegedus et al. 2009). Alterations of the peritrophic membrane disturb its selective permeability properties resulting in nutritional imbalances, by affectin (...truncated)