Molecular Assesment of Chitinase Activity in Transgenic Wheat

Abstract

Fungal diseases especially rust of common wheat (Triticum aestivum)
and durum (T. turgidum subsp. durum), historically was one of the most destruc- tive wheat diseases. Significant losses occurred in the past when the disease de- veloped into epidemic proportions in wheat crops (Roelfs, 1978). Plants natu- rally respond to fungal attack by a com- plex network of defense mechanisms, which are activated upon perception of a pathogen and designed to limit its pene- tration and development. Defense responses include structural and biochemical responses like reinforcement of the plant cell wall, accumulation of phytox- lexins with microbial toxicity, ribosome- inactivating proteins (RIPs) that inhibit protein synthesis, antimicrobial peptides and the synthesis of other pathogenesis- related (PR) proteins (Yang et al., 1997). Some PR proteins, such as chitinase and glucanases, have hydrolytic activities against structural components of fungal cell walls and may exhibit strong antifun- gal activities in vitro (Schlumbaum et al., 1986; Leah et al., 1991). In vivo, chitin oligomers released from fungal cell walls function as elicitors that stimulate a gen- eral resistance response (Cote and Hahn, 1994). The induction of resistance re- sponses by chitin-derived oligosacchrides has also been described for wheat (Barber et al., 1989).
Chitinase (poly [1,4-N-acetyl-/3- D-glucosaminid] glycan hydrolase, EC3.2.1.14) catalyzes the hydrolysis of chitin polymer in fungal cell walls into N- acetylglucosamine oligomers (Toyoda et al., 1991). Plant chitinases are induced as a result of pathogenic infections as well as by abiotic agents (Lee and Hwang, 1996 and Punja and Zhang, 1993). Chitinases possess anti-fungal activity that causes in vitro lysis of hyphal tips as well as inhibi- tion of spore germination in Alternaria, Fusarium and Trichoderma (Schlumbaum et al., 1986; Mauch et al., 1988). Because of variation in cell wall composition, pa- thogenic fungi differ in their sensitivity towards chitinases. Chitinases are induced locally in the infection sites or accumulate systemically in other tissues following a pathogenic attack (Pan et al., 1992). Chi- tinases also contribute indirectly to the induction of host defense responses. My- celial wall fragments released as a result of chitinase activity may act as elicitors of plant defense mechanisms, i.e., accumula- tion of phenolic compounds, lignification and phytoalexin synthesis (Kurosaki et al., 1988). Nitzsche (1983) reported that chitinase is a possible resistance factor in wheat against yellow rust disease.
Several laboratories have been able to transfer plant- or microbial-derived chitinase genes into plants and develop transgenic crops with enhanced resistance to fungal diseases. These include trans- genic tobacco and canola (Broglie et al., 1991; Terakawa et al., 1997), rice (Nishi- zawa et al., 1999), grapevine (Yamamoto et al., 2000), peanut (Rohini and Sankara,2000), grapevine (Bornhoff et al., 2005), Italian ryegrass (Takahashi et al. 2005) and carrot (Jayaraj and Punja, 2007).
In wheat, Chen et al. (1998) intro- duced rice chitinase gene (chi11) into the spring wheat cultivar ‘Bobwhite’. After inoculation with conidia of F. graminea- rum, the symptoms of scab developed significantly slower in transgenic plants of the T1, T2 and T3 generations than in non-transformed control plants. Oldach et al. (2001) introduced barley class II chiti- nase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, into wheat. They found that the formation of powdery mildew (Erysi- phe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from chitinase II- expressing wheat lines com- pared with nontransgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fun- gal spores used for inoculation. Heterolo- gous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important bio- trophic fungal diseases is inhibited ap- proximately 40 to 50% at an inoculum density of 80 to 100 spores per cm This paper reports the production of two wheat transgenic lines stably ex- pressing one of the pathogen related (PR) proteins, the barley chitinase gene (chi). The plant expression vector pbar- ley/chi/bar is harbouring the chi gene un- der the control of ubi promoter and NOS terminator and the bar gene under the control of 35S promoter and NOS termi- nator. The integration and expression of the transgene(s) were proved using mo- lecular analysis, i.e., PCR, Southern, RT- PCR. The activity of chitinase enzyme on colloidal chitin was measured in the pro- tein extract of the transgenic plants.