Antifungal proteins

Many of the genes induced by the plant disease-resistance responses described in (Genes in the defence against fungi) encode proteins with antifungal activity, which probably have an important role against fungal infection. The AFP strategy involves the constitutive expression in transgenic plants of genes encoding proteins with a fungitoxic or fungistatic capacity. This is an extension of the paradigm that has worked so well for insect control genes based on insecticidal proteins from Bacillus thuringiensis (see below). Table 4.1 presents an overview of the reports on transgenic plants obtained using this approach.

Plant defence mechanisms are usually accompanied by the expression of a large set of genes termed PR. At least ten families of PRPs have been identified (Dempsey et al. 1998) (Table 4.2) and the two most prominent members have been the hydrolytic enzymes chitinase and β-1,3 glucanase, which are capable of degrading the major cell wall constituents (i.e. chitin and β-1,3 glucan) of most filamentous fungi. Expression of either enzyme individually in a number of plants has conferred resistance against a particular pathogen, but constitutive coexpression of both enzymes confers even higher levels of resistance (Table 4.1), suggesting a synergistic interaction between the two enzymes. Other genes coding for different PRPs have yielded comparable results. Constitutive expression in tobacco of PR1a, a protein with unknown biological function, increased resistance to two oomycete pathogens (Alexander et al. 1993). However, fungal resistance is significantly enhanced when more than one gene is employed. Future studies are necessary to identify different combinations of PR proteins that might confer effective broad-spectrum protection.

	Table 4.1 Fungal resistance in transgenic plants
	Plant		Transgene(s)		Pathogen		Reference
	Alfalfa		Peanut Resveratrol synthase		Phoma medicaginis		Hipskind and Paiva 2000
	Brassica napus		Bean chitinase		Rhizoctonia solani		Broglie et al. 1991
			Tomato/tobacco Chitinase		Cylindrosporium conc.		Grison et al. 1996
					Sclerotinia sclerotiorum		Grison et al. 1996
					Phoma lingam		Grison et al. 1996
	Carrot		Tobacco Chitinase + β-1,3 Glucanase		Alternaria dauci		Melchers and Stuiver 2000
			Tobacco AP24		Alternaria radicina		Melchers and Stuiver 2000
					Cercospora carotae		Melchers and Stuiver 2000
					Erysiphe heracleı		Melchers and Stuiver 2000
	Potato		AP24		Phytophthora infestans		Liu et al. 1994
			Glucose oxidase		Phytophthora infestans		Wu et al. 1995
					Verticillium dahliae		Wu et al. 1995
			Osmotin		Phytophthora infestans		Li et al. 1999
			Tobacco class II catalase		Phytophthora infestans		Yu et al. 1999
			Aly AFP		Verticillium sp.		Liang et al. 1998
			Soybean β-1,3-Glucanase		Phytophthora infestans		Borkowska et al. 1998
			PR-1a, SAR 8.2		Peronospora tabacina		Alexander et al. 1993
	Tobacco				Phytophthora parasitica, Pythium sp.		Alexander et al. 1993
			Class III Chitinase		Phytophthora parasitica		Alexander et al. 1993
			Class I Chitinase		Rhizoctonia solani		Alexander et al. 1993
			Bean Chitinase		Rhizoctonia solani		Broglie et al. 1991
			Barley RIP		Rhizoctonia solani		Logemann et al. 1992
			Serratia marcescens Chitinase		Rhizoctonia solani		Logemann et al. 1992
			Barley Chitinase + β-1, 3 Glucanase		Rhizoctonia solani		Jach et al. 1995
			Barley Chitinase + RIP		Rhizoctonia solani		Jach et al. 1995
			Rice Chitinase + Alfalfa Glucanase		Cercospora nicotianae		Zhu et al. 1994
			Sarcotoxin IA		Rhizoctonia solani		Mitsuhara et al. 2000
					Pythium aphanidermatum		Mitsuhara et al. 2000
			Pseudomonas syringae hrmA		Phytophthora parasitica		Shen et al. 2000
			Oxalate decarboxylase		Sclerotinia sclerotiorum		Kesarwani et al. 2000
			Radish AFP		Alternaria longipes		Terras et al. 1995
	Tomato		Tobacco Chitinase + β-1, 3 Glucanase		Fusarium oxysporum		Jongedijk et al. 1995
	Rice		Rice Chitinase		Rhizoctonia solani		Lin et al. 1995
	Wheat		Aly AFP		Fusarium sp.		Liang et al. 1998

	Table 4.2 Pathogenesis-related proteins in plants
	PR protein family		Enzymatic activity		Target in pathogen
	PR-1		Unknown		Membrane?
	PR-2		1, 3-β-glucanase		Cell wall glucan
	PR-3		Endochitinase		Cell wall chitin
	PR-4		Endochitinase		Cell wall chitin
	PR-5		Osmotin-like		Membrane
	PR-6		Proteinase inhibitor		Proteinase
	PR-7		Proteinase		Unknown
	PR-8		Endochitinase		Cell wall chitin
	PR-9		Peroxidase		Plant cell wall
	PR-10		RNAase?		Unknown
	PR-11		Endochitinase		Cell wall chitin
	Unclassified		α-Amylase		Cell wall α-glucan
			Polygalacturonase		Unknown
			inhibitor protein (PGIP)		Polygalacturonase

In addition to PR proteins, a broad family of small, cysteine-rich AFP has been characterised (Broekaert et al. 1997). This family include plant defensins, thionins, lipid transfer proteins (LTP) and hevein- and knottin-type peptides and have been shown to posses antifungal activity in vitro against a broad spectrum of fungal pathogens (Broekaert et al. 1995; 1997). They seem to inhibit fungal growth by permeabilisation of fungal membranes (Thevissen et al. 1999). Plant defensins have been isolated from seeds of a variety of plants and shown to be induced upon pathogen infection in an SA-independent manner (Terras et al. 1998). They have been used to confer resistance against Alternaria longipes in tobacco (Broekaert et al. 1995). Thionins and LTP are also induced in an SAindependent manner by infection of a variety of plant pathogens in many plant tissues (Broekaert et al. 1997). Overexpression of an endogenous thionin gene in Arabidopsis conferred protection against Fusarium oxysporum (Epple et al. 1997) whereas expression of a barley non-specific LTP in Arabidopsis and tobacco conferred enhanced resistance to the bacterial pathogen Pseudomonas syringae (Molina and Garcia-Olmedo 1997). In the only example where heveinand knottin-type peptides were overexpressed in transgenic plants, the resultant tobacco plants were not any more resistant to infection by Alternaria longipes than control plants (De Bolle et al. 1993). This might be explained by the high susceptibility of the antifungal activity of hevein- and knottin-type peptides to the presence of inorganic cations (De Bolle et al. 1993).

Finally, there are several AFP that do not fall into any of the classes described above. For example, the H₂O₂-producing enzyme oxalate oxidase has been shown to accumulate in barley infected by Erysiphe gramminis (Zhou et al. 1995). Interestingly, Wu et al. (1995) demonstrated that constitutive expression of another H₂O₂-producing enzyme, glucose oxidase provided disease resistance to a range of plant pathogens, including Phytophtora infestans, Erwinia carotovora and Verticillium wilt disease.

Most of the reports described above are based exclusively on observations of the increased fungal resistance of transgenic plants tested in climate-controlled growth chambers or greenhouse facilities. The challenge now is to translate such results into a significant outcome in the field.