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.