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Transgenic Approaches to Combat Fusarium Head Blight in Wheat and Barley.

FUSARIUM HEAD BLIGHT has caused devastating losses to wheat, durum, and barley growers and industry in the USA, particularly in the 1990s (McMullen et al., 1997). Researchers searching for resistance have found a limited number of barley and wheat genotypes that may provide partial resistance to the Fusarium species that cause FHB (Rudd et al., 2001). No sources of immunity to the disease have been identified (Gocho and Hirai, 1987; McKendry et al., 1999; Prom et al., 1997). The resistance that has been identified is under the control of multiple genes, creating a challenge for wheat and barley breeders to develop resistant cultivars that meet quality and agronomic standards.

Recent advances have made possible the insertion of individual genes into cereals including wheat and barley. A variety of antifungal genes have been isolated, and some of their products have been shown to inhibit Fusarium growth in vitro and in planta. The development of transgenic wheat and barley expressing these genes may help in the fight against FHB. This paper reviews transformation methods used in wheat and barley, some of the challenges to achieve successful transformation, prospects for transgene-mediated disease resistance, current research on candidate antifungal genes and promoters, and strategies for optimal antifungal activity.

Transformation Methods

The first reports of successful production of fertile transgenic barley were published in 1994. These experiments used particle bombardment (biolistics) to insert the genes into barley tissue (Ritala et al., 1994; Wan and Lemaux, 1994). Since then, a number of laboratories have reported transformation of barley with selectable genes such as bar for resistance to the herbicide bialaphos [L-2-amino-4-((hydroxy)methyl)(phosphinoyl)-butyryl-L-alanyl-L-alanine], nptII for resistance to the antibiotics kanamycin and G418, and visual reporter genes such as uidA for [Beta]-glucuronidase (GUS) expression, using a variety of target tissues (reviewed by Lemaux et al., 1999). In the last few years, research has moved beyond developing methods using selectable and screenable genes to inserting genes of interest into barley (Lemaux et al., 1999).

Tingay et al. (1997) developed an Agrobacterium-mediated method for transforming immature barley embryos, recovering plants from 54 independently transformed cell lines. Two advantages of the Agrobacterium-mediated method compared with the particle bombardment method are insertion of fewer copies of the transgenes in stably transformed plants and the preferential integration of the transgenes into transcriptionally active chromosome regions (Lemaux et al., 1999).

The first report of successful production of fertile transgenic wheat plants was by Vasil et al. (1992). They used the biolistic method to introduce DNA into 5- to 7-mo-old embryogenic callus cultures and successfully regenerated plants containing, expressing and transmitting bar, the selectable marker gene encoding resistance to the phosphinothricin- (L-PPT) based herbicide, BASTA (glufosinate). Within the next 2 yr, several independent laboratories, beginning with Weeks et al. (1993), published variations of the method that is still most widely used today for wheat transformation (Becket et al., 1994; Nehra et al., 1994; Vasil et al., 1993). This method targets embryogenic cells derived from the scutellum of immature embryos of highly regenerable hexaploid wheat genotypes such as `Bob-white', `Pavon', `Fielder', and `Florida'. In these especially responsive varieties, scutellar cells readily form embryogenic callus within a few days of culture in the presence of the auxin 2,4-dichlorophenoxy acetic acid (2,4-D). Regeneration, even after bombardment and selection, is not limiting for transformation efficiency. More recently, durum wheat has been transformed using similar methods (Bommineni et al., 1997; He et al., 1999). Immature inflorescences of wheat also have been successfully transformed by the biolistic method (Rasco-Gaunt and Barcelo, 1999).

Several selection agent/marker gene combinations have been used to identify wheat transformants. The herbicides bialaphos, BASTA, and L-PPT inhibit glutamine synthetase, and are still the most commonly used in conjunction with the bar gene encoding phosphinothricin acetyl transferase. The drawback in using these selection agents is the large number of plantlets, between 50 and 95% (Nehra et al., 1994; Altpeter et al., 1996a; Ortiz et al., 1996; Barro et al., 1998; Witrzens et al., 1998), that grow even though they have not integrated the marker gene. These so-called "escapes" can be eliminated by molecular analyses later, but only after much time and effort has been expended in their culture and regeneration. Other selection agents-genes that have been used to identify wheat transformants are glyphosate-gox + CP4 (Zhou et al., 1995), G418 or geneticin-nptII (Nehra et al., 1994; Wirtzens et al., 1998; Barro et al., 1998), paromomycin-nptII (Wirtzens et al., 1998), and hygromycin-hpt (Ortiz et al., 1996). Similar transformation efficiencies (1-2%), but fewer escapes (20-50%), were found in laboratories comparing phosphinothricin or bialaphos to G418 selection (Nehra et al., 1994; Barro et al., 1998; Witrzens et al., 1998). The recovery of transgenic plants by means of glyphosate was relatively low (0.15%) compared with that reported by the same laboratory using bialaphos (10-15%) or G418 (1-3%), but no escapes were found using glyphosate (Zhou et al., 1995). Ortiz et al. (1996) recovered twice as many transgenic plants using hygromcyin compared with L-PPT selection, but with a similar percentage of escapes.

Thus far, there has been only one published report of using Agrobacterium to transform wheat (Cheng et al., 1997). Transformants were selected on G418 and showed a higher percentage of single copy transgenes (35%) than those obtained by the biolistics method (17%) and a higher percentage of coexpression of co-transformed unselected genes (98% vs. 42-62%). The transformation efficiency of 1.12% was 10-fold lower than that reported by the same laboratory using particle bombardment. Another method for introducing genes into wheat used pollen tubes to ferry DNA into developing styles (Chong et al., 1998).

Challenges in Transforming Bread Wheat, Durum, and Barley

Genotype Effects on Plant Regeneration

A basic requirement for high throughput transformation is a high rate of green plant regeneration from tissue culture. These rates vary widely among barley genotypes, and even for single-plant-derived lines grown from the same seed lot of a cultivar (Dahleen and Bregitzer, 1999). Six-rowed malting cultivars developed for the Upper Midwest USA do not perform well in tissue culture, and some are incapable of regenerating green plants on the standard media of Murashige and Skoog (1962) (Bregitzer, 1992). North American two-rowed cultivars regenerate more green plants (Baillie et al., 1993), but have poor regeneration rates compared with European cultivars such as Golden Promise on standard media (Dahleen and Bregitzer, 1999). Almost all transformation attempts in the USA use Golden Promise because the likelihood of recovering transformed plants is greatly increased. Use of a model cultivar for transformation will require multiple cycles of backcrossing to transfer the transgene into germplasm containing the 40 or more agronomic and quality traits required for commercial barley cultivars. Recent improvements in tissue culture media (Bregitzer et al., 1998; Cho et al., 1998) and transformation techniques (Zhang et al., 1999) may make transformation of commercial barley cultivars practical.

The responsiveness of wheat genotypes to in vitro culture also varies widely (reviewed by Maheshwari et al., 1995). Most genotypes are not as amenable to tissue culture manipulation as the cultivars first used for transformation. Nehra et al. (1994) found that many less tractable Canadian genotypes would form embryogenic callus if the scutellum was cultured in the absence of the embryo axis. This practice also has been adopted by the Barcelo-Lazzeri (DuPont) group who have transformed several commercially grown European cultivars (Sparks et al., 1998; Rasco-Gaunt et al., 1999). Other "non-model" genotypes that have been transformed include `Chinese Spring' (Takumi and Shimada, 1996), and several cultivars from German (Iser et al., 1999) and Australian (Witrzens et al., 1998) breeding programs. Since the ability to form regenerable callus is the limiting factor for recovery of transformed plants of these genotypes, it becomes important to optimize, as much as possible, other parameters in the transformation protocol (e.g., Rasco-Guant et al., 1999).

Factors Affecting Efficiency

There are a number of factors that affect efficiency of transformation in wheat and barley (Lemaux et al., 1999). These include the proportion of cells that receive DNA and survive, the number of expressing transformed cells that can divide and multiply to form callus, the number of transformed cells that integrate and express the selectable marker gene and the gene of interest, and the number of calli that regenerate green plants. All of these factors contribute to the low efficiencies often reported for wheat and barley transformation. In the 23 barley projects reviewed by Lemaux et al. (1999), the effective transformation frequency (no. of independent events/no. of targets) ranged from 0 to 5.6%, with effective transformation frequencies less than 1% for 10 of the projects. The low effective transformation frequencies obtained mean that time and resources are wasted selecting and maintaining tissues that will never regenerate green plants. Lemaux et al. (1999) reviewed improvements in tissue culture techniques, media composition, and targeted tissues that may help increase transformation efficiency.

Efficiencies for the particle bombardment method in the first sets of published wheat transformation experiments ranged from 0.1 to 2.5% and typically averaged around 1%; that is, one transgenic plant was recovered per 100 embryos bombarded. Those averages usually included experiments in which no transgenic plants were recovered. Even the same group of experimenters performing the same protocol can recover several transformants in some experiments and none in others (e.g., Weeks et al., 1993; Nehra et al., 1994; Uze et al., 1999). The efficiency is important because the high degree of variability in gene expression that occurs among different transformants (position effects) means that several independent transformants must be isolated for each gene construct of interest. Thus, a low efficiency limits the number of different genes that can be studied by a single investigator in a given period of time.

Recently, a number of laboratories have developed improvements in the speed and/or efficiency of recovering transgenic wheat plants. A significant improvement in efficiency was achieved (Altpeter et al., 1996a; Blechl and Anderson, 1996; Rasco-Gaunt et al., 1999) by placing the target material on media with elevated osmotic potential before and after bombardment, as originally done in maize (Zea mays L.) by Vain et al. (1993). The health of the plants that serve as sources of the immature embryos is believed to be a critical factor, and some laboratories have taken steps to make the growing environment of the source plants as consistent as possible (Zhou et al., 1995; Altpeter et al., 1996a). Various groups have made improvements in their efficiencies by adjustments in hormones and other media constituents, and in bombardment and culture conditions (Altpeter et al., 1996a; Barro et al., 1998; Rasco-Gaunt et al., 1999). Even with these improvements, typical wheat transformation efficiencies remain in the range of a few percent, too low for routine transformation of genotypes recalcitrant to tissue culture.

Calculation of these efficiencies is based on the number of plants that receive and express the selectable marker genes. Also of interest for wheat and barley improvement efforts are the percentage of transformants that receive and express cotransformed genes whose products are not subject to direct selection. Changing the properties of the plants by expression of the cotransformed "genes of interest" is the aim of most transformation experiments. In the majority of cases, cotransformed genes cointegrate with marker genes into a single locus, even though the genes are usually cobombarded as separate plasmids. Among wheat transformation experiments using particle bombardment, cotransformation frequencies can be as high as 88% (7/8 plants) (Fettig and Hess, 1999), while coexpression frequencies for nonmarker genes in the first generation range from 83% (10/12 plants) (Stoger et al., 1999a) to 40% (8/20 plants) (Altpeter et al., 1996b). Transgene expression, particularly of marker genes, is sometimes reduced or silenced during transmission between generations (see below).

Somaclonal Variation

Current transformation techniques in wheat and barley use tissue culture systems that involve disorganized cell growth, such as passage through a callus phase before plant regeneration. This callus phase usually induces somaclonal variation, i.e., mutations caused by the tissue culture process. Somaclonal variation found in wheat and barley regenerants include gross chromosomal changes such as aneuploidy, breakage, and re-arrangements (Karp and Maddock, 1984), and changes in gene expression caused by point mutations, altered methylation patterns, or other modifications (Karp and Lazzeri, 1992; Maheshwari et al., 1995). The majority of changes caused by tissue culture are undesirable for breeding improved cultivars, and include reductions in test weight, 1000-kernel weight, percent plump seed, and yield (Baillie et al., 1992; Bregitzer and Poulson, 1995). Bregitzer et al. (1998) examined somaclonal variation in the progeny of transgenic Golden Promise barley and found that the transformation process (particle bombardment) appeared to increase variation above the level induced by the tissue culture process alone. Typical changes included reduced yield, seed size, and height. The presence of somaclonal variation in transgenic plants means that extensive field testing will be needed, and hybridization may be required to develop transgenic cultivars that meet the appropriate agronomic and quality requirements. A recent report (Lemaux et al., 1999) indicates that improvements in tissue culture and transformation techniques and targets may reduce the amount of somaclonal variation generated, decreasing the time needed for transgenic cultivar development.

Somaclonal variation also occurs among regenerants of wheat (reviewed by Maheshwari et al., 1995). In addition to the types of changes listed for barley above, variations have also been noted in the presence or absence of awns, tiller number, glume and grain color (Larkin et al., 1984), gliadin storage proteins (Larkin et al., 1984; Maddock et al., 1985), grain number per spike, grain hardness and protein content, flour yield, and mixograph characteristics (Ryan et al., 1987). The extent of variability is partially a function of genotype (Carver and Johnson, 1989) and time in culture (Hartmann et al., 1989). Since these studies did not include the cultivars and exact culture conditions commonly used for transformation, it is not clear what impact somaclonal variation will have on the results of wheat transformation experiments.

Transgene Inheritance and Stability of Expression

Since wheat and barley transformation methods have been practiced for only a few years and in only a few laboratories, studies of long-term inheritance of transgenes are rare. From the data reported thus far, it appears that most transgenes are transmitted following Mendelian rules of inheritance. In one particularly detailed study, Cannell et al. (1999) reported that inheritance and expression of two marker transgenes was fairly stable in five of six lines they followed through the [T.sub.3]. They noted that while bar gene expression remained at its original levels, uidA gene expression declined overall in each succeeding generation. One line transmitted the transgene to only two of 36 [T.sub.1] progeny, but one of these progeny exhibited normal inheritance in subsequent generations. Occasionally, transgenes can be lost during transmission. Iser et al. (1999) observed the loss of transgenes between the [T.sub.0] and [T.sub.1] in three of their 16 lines. All other lines showed normal inheritance of both genotype and phenotype. Srivastava et al. (1996) reported that one of six lines lost bar-uidA transgenes between the [T.sub.2] and [T.sub.3]. Although the transgene loci in the other five lines were inherited normally, changes in the bar transgene structure were noted. By the [T.sub.2], four lines had lost GUS activity although no changes in the uidA transgene structure were detected.

The decline or loss of gene expression from apparently unrearranged, once-active transgenes is called "gene silencing." This phenomenon may account for the observation that wheat transformants often exhibit lower than expected transmittance of transgene-encoded phenotypes, particularly from the [T.sub.0] to the [T.sub.1]. For example, 5 of 21 [T.sub.0] plants of Altpeter et al. (1996a) had lower than the expected proportion (75%) of bialaphos resistant [T.sub.1] progeny. About 30% of Agrobacterium-transformed wheat lines exhibited significantly less than 3:1 ratios in paromomycin-resistant:sensitive progeny (Cheng et al., 1997). In these and most other cases, it is the phenotype conferred by the marker transgene (e.g., herbicide or antibiotic resistance or GUS activity) rather than the DNA itself whose inheritance is followed. When the presence of the DNA is assayed, it is found that loss of the transgene DNA (see above) is much rarer than loss of expression. Silencing can occur as a result of either selfing (as in the cases above) or crossing. Demeke et al. (1999) found that of two homozygous lines uniformly expressing uidA through the [T.sub.5], one exhibited gene silencing in some progeny derived from a cross of [T.sub.4] plants to the original nontransformed cultivar.

It appears that some gene constructs are more prone to silencing than others. Chen et al. (1998) observed that all 12 of their lines containing rice chitinase transgenes driven by the Cauliflower Mosaic Virus 35S promoter exhibited either a reduction or complete absence of expression in the [T.sub.1], even though there were no detectable changes in transgene structure. The same group noted that in another transformant containing four different transgenes integrated at a single locus, the two genes driven by the 35S promoter were silenced in the [T.sub.1], while the two driven by maize Ubi1 promoter remained active (Chen et al., 1999). Stoger et al. (1999b) found that uidA transgenes driven by the maize Ubi1 promoter remained active over four generations. He et al. (1999) reported that although two of three of their lines exhibited silencing of marker genes (uidA in one, and both uidA and bar in another), expression of the HMW-glutenin genes in these same lines was stable over three generations. Stable expression of wheat HMW-glutenin transgenes over three generations also has been reported by Blechl and Anderson (1996) and Altpeter et al. (1996b). As further understanding of the mechanisms and triggers of gene silencing is developed, it may become possible to decrease its occurrence by altering the DNA elements in transformation vectors.

Thus far, it does not seem as if either somaclonal variation or occasional abnormalities in transgene inheritance and expression will limit the usefulness of genetic transformation of wheat and barley. However, the occurrence of these phenomena means that several different transgenic events need to be generated and characterized for each construct to ensure that at least one useful transformed line will be obtained.

Prospects for Transgene-Mediated Disease Resistance in Wheat and Barley

Both Triticum aestivum and Hordeum vulgare are capable of mounting defense responses, best characterized by interactions with the powdery mildew pathogen Erysiphe graminis DC [= Blumeria graminis (DC) E.O. Speer] (Schweizer et al., 1989; Gregersen et al., 1997), and the stem rust pathogen Puccinia graminis Pers.:Pers. (Munch-Garthoff et al., 1997). In these cases, resistance is governed by gene-for-gene interactions, and is accompanied by the hypersensitive response and the induction of mRNAs encoding pathogenesis-related (PR) proteins. Foliar application of benzothiadiazole induces resistance to powdery mildew in wheat, which is accompanied by the accumulation of several classes of PR protein mRNAs (Gorlach et al., 1996). This defense system is not effective against F. graminearum although increases in steady-state levels of some PR protein transcripts are detected in pathogen-challenged wheat florets (Pritsch et al., 2000). It is possible that the Fusarium pathogen has evolved mechanisms for evasion of or tolerance to the wheat and barley defense systems. To achieve some degree of protection against the FHB pathogen, native wheat and barley PR proteins may need to be engineered to accumulate earlier, more abundantly, faster, or in tissues other than currently occurs in infected plants. Alternatively, heterologous genes for proteins with better anti-Fusarium activity need to be introduced.

Candidate Antifungal Proteins

Resistance to FHB might be achieved by the expression of several classes of candidate proteins, each with a distinct mode of antifungal action. For instance, proteins that (i) degrade structural components of the fungal pathogen, such as its cell wall or membranes, (ii) interfere with biochemical and metabolic processes in the pathogen, including protein synthesis and the accumulation of DON, (iii) bolster the host defense response system, or (iv) interfere with fungal pathogenesis, all provide strategies for transgene-mediated resistance. Proteins with undesirable side effects, including mammalian toxicity, allergenicity, and negative impact on product yield and quality, should be excluded. In the following paragraphs, the potential of each of these classes of proteins for fighting Fusarium infection is considered.

Cell wall-degrading proteins have been grouped into classes on the basis of their biochemical and structural properties. Both endo- and exochitinases appear to be effective in the hydrolysis of fungal cell wall chitin. Class I chitinases have chitinolytic activity against bacterial cell walls and are known to bind chitin directly. By contrast, the Class II chitinases do not act on bacterial cell walls and lack chitin binding activity; they are postulated to play a role in production of fungal elicitors that trigger the host defense response (Graham and Sticklen, 1994; Fritig et al., 1998). Of the five major classes of [Beta]-glucanases, only the [Beta]-1,3-glucanases have been shown to exhibit antifungal activity (Simmons, 1994). In general, basic chitinases and glucanases (Class I) accumulate in the vacuole, whereas the acidic isoforms (Class II) are extracellular, with some exceptions (e.g., Wu et al., 1994; Graham and Sticklen, 1994). Sela-Buurlage et al. (1993) and others observed that the specific activities of Class I chitinases and glucanases are higher than those of the Class II enzymes, but implications of this for engineering FHB resistance remain to be determined. Cell walls of Fusarium species appear to be relatively refractory to the action of cell wall-degrading enzymes, possibly because of their high protein and high chitin content (Sivan and Chet, 1989; Barbosa and Kemmelmeier, 1993), and clustering of acetylated glucosamine residues (Fukamizo et al., 1992). However, activity against various Fusarium species has been obtained with chitinases and glucanases in vitro (Mauch et al., 1988; Melchers et al., 1994; Yun et al., 1996) and in vivo (Jongedijk et al., 1995). These enzymes act preferentially on the tips of growing fungal hyphae (Broekaert et al., 1988; Collinge et al., 1993). Thus, chitinases and [Beta]-1,3-glucanases, possibly in conjunction with proteases, might be effective against F. graminearum, if expressed in the appropriate organs and subcellular compartments within the host. A number of laboratories in the USA are testing chitinase and glucanase genes from rice (Oryza sativa L.), barley, alfalfa (Medicago sativa L.), and Fusarium venenatum Nirenberg, a close relative of F. graminearum, in the fight against head blight (Table 1).
Table 1. Anfifungal and antitoxin genes targeted for insertion into
wheat and barley to help combat Fusarium head blight.

Laboratory location      Crop         Genes (source)

USDA-ARS, Fargo, ND      barley       Tri101 (Fusarium), PDR5
                                        (yeast)
USDA-ARS, Aberdeen, ID   barley       tlp-1, tlp-2 (oat)
USDA-ARS, Albany, CA     wheat        Tri101 (Fusarium), PDR5
                                        (yeast), leaf tlp-1 (wheat),
                                        endochitinase, exochitinase,
                                        glucanase (Fusarium)
USDA-ARS, Madison, WI    barley       permatin (oat), seed
                                        hordothionin (barley)
Univ. MN, St. Paul, MN   barley and   [Alpha]-thionin (wheat), leaf
                           wheat        tlp-1 (wheat), acid
                                        glucanase (alfalfa),
                                        chitinase (barley and rice),
                                        class [Beta]II-1,3-glucanase
                                        (barley), PR5/tlp
                                        (arabidopsis), tlp-1, tlp-4
                                        (oat), zeamatin (maize),
                                        type 1 RIP (barley)
Univ. WI, Madison, WI    wheat        permatin (oat), seed
                                        hordothionin (barley), NPR1
                                        (Arabidopsis)
Univ. NE, Lincoln, NE    wheat        lactoferrin (mammal), oxalyl-
                                        CoA-decarboxylase
                                        (bacterium), IAP
                                        (baculovirus), ced-9 (C.
                                        elegans)
Kansas State Univ.,
  Manhattan, KS          wheat        tlp (rice), chitinase (rice,
                                        barley), glucanase (rice,
                                        barley)


Thionins, thaumatin-like proteins, osmotins and other classes of small proteins that disrupt fungal membranes also are promising antifungal agents. Each class of polypeptides has distinct structural features (Vigers et al., 1992; Broekaert et al., 1997). Thionins are basic, cysteine-rich polypeptides encoded by a large multigene family, have broad-spectrum antimicrobial activity, and occur in a wide range of plant species. The antimicrobial activity of the thionins is attributed to their abilities to create pores in fungal membranes, to disrupt signal transduction pathways via interactions with membrane phospholipids and phosphoinositides, or to modulate enzyme activities by reducing disulfide bonds. Constitutive over-expression of an endogenous thionin of Arabidopsis reduced the growth of F. oxysporum Schlechtend.:Fr, hyphae and reduced damage to inoculated leaves (Epple et al., 1997). Strong in vitro activities of a radish (Raphanus sativus L.) thionin against F. culmorum (Wm. G. Sm.) Sacc. (Terras et al., 1996) and a seed thionin from barley against F. graminearum (Skadsen et al., 1999a,b) have been reported.

Thaumatin-like proteins comprise a class of polypeptides that share homology to thaumatin, the sweet protein from Thaumatococcus danielli (Bennett) Benth. Thaumatin-like proteins share amino acid sequence identity with permatins, zeamatins, and the PR-5 class of pathogenesis-related proteins. A barley permatin and other related proteins were found to have glucanase activity in vitro (Grenier et al., 1999), suggestive of two modes of action for some thaumatin-like proteins. Expression of specific thaumatin-like proteins in response to fungal pathogens has been reported for a number of plant species, including oat (Avena sativa L.) (Lin et al., 1996) and wheat (Rebmann et al., 1991). A recent report of Chen et al. (1999), that FHB damage was delayed in wheat plants expressing a rice thaumatin-like protein, indicates that this class of proteins holds promise for engineered resistance. Transformation of wheat and barley with genes encoding thionins, thaumatin-like proteins and permatins from oat, wheat, rice, and maize is under way in several laboratories (Table 1).

Ribosome-inactivating proteins (RIPs) are polypeptides that bind to fungal ribosomes and thereby interfere with fungal protein synthesis (Logemann et al., 1992). Constitutive expression of a barley RIP in wheat and barley is being engineered in one laboratory (Table 1).

Some flavonoids have been shown to inhibit Fusarium growth in vitro. Skadhauge et al. (1997) examined barley mutants lacking different enzymes in the flavonoid pathway. Seed of one mutant that accumulated dihydroquercetin in testa cells showed a marked reduction in Fusarium colonization in vitro. However, the mutant was susceptible to FHB in field trials (B. Steffenson, 2000, personal communication). McKeehen et al. (1999) found that two other phenolic compounds, ferulic acid and p-coumaric acid, reduced the growth of F. graminearum and F. culmorum in vitro, and that ferulic acid accumulated at slightly higher levels in kernels of Fusarium-tolerant cultivars of wheat than in kernels of susceptible cultivars. Since phenolic acids in wheat kernels are mainly associated with the cell wall, they might also fortify the host cell wall against pathogen invasion. Further manipulation of the flavonoid pathway using transformation technology may increase intermediate compounds to levels that provide field resistance for FHB.

Antimicrobial activity has been reported for a mammalian protein, lactoferrin, and its cleavage product, lactoferricin. Tobacco (Nicotiana tabacum L.) plants expressing human lactoferrin showed delayed leaf wilt induced by the bacterial pathogen Ralstonia solanacearum Smith, and synthetic bovine lactoferricin was active against two other bacterial pathogens of plants in in vitro assays (Zhang et al., 1998). The antifungal activity of lactoferricin is now being tested on F. graminearum in wheat (Table 1).

During the course of infection, some strains of F. graminearum produce DON, a trichothecene mycotoxin that primarily affects smooth muscle function, and therefore poses a health hazard to humans and monogastric animals (Busby and Wogan, 1981). There is strong evidence that DON is a virulence factor that enhances disease severity in maize and wheat (Desjardins et al. 1996; Proctor et al., 1995, 1997). Two promising candidate antifungal proteins that confer tolerance to DON in yeast (Adam and Lemmens, 1996; McCormick et al., 1999) have been engineered for expression in wheat and barley (T. Hohn, unpublished). The first of these, 3-OH trichothecene acetyltransferase, encoded by FsTRI101 from F. sporotrichioides Sherb., converts DON to a less toxic acetylated form (McCormick et al., 1999). The acetyltransferase is required for trichothecene biosynthesis, rather than for detoxification of DON, within the fungus. The ATP-binding cassette transporter encoded by the Saccharomyces cerevisiae PDR5 gene (Balzi et al., 1994) acts as a efflux transporter, shunting DON across the plasma membrane from the interior of the cell. Constructs containing these genes are being introduced into wheat and barley by several laboratories (Table 1). A synthetic peptide that competes with DON for binding to a DON monoclonal antibody has recently been reported (Yuan et al., 1999). This peptide has no deleterious effects on bone marrow erythrocytes or protein synthesis, and therefore has promise as a possible DON antagonist. An alternative strategy has been developed to modify host plant ribosomes, the primary sites of DON cytotoxicity (McLaughlin et al., 1977). Expression of a ribosomal protein with reduced affinity for DON conferred tolerance in stably transformed maize calli (Harris et al., 1998).

Components of defense response signal transduction pathways from plants and nonplant eukaryotes also are being recruited for use against the FHB pathogens (Table 1). High-level expression of the Arabidopsis NPR1 gene, a positive regulator of the systemic acquired resistance signaling pathway, conferred enhanced resistance to a bacterial and fungal pathogen of Arabidopsis (Cao et al., 1998). It is hoped that NPR1 and/or its rice homolog, NH1, will be effective against Fusarium if highly expressed in wheat. The Caenorhabditis elegans ced-9 (Hengartner et al., 1992), and insect baculovirus IAP (inhibitor of apoptosis) gene products (reviewed by Devereux and Reed, 1999) act to block proteins that promote apoptosis, or programmed cell death pathways, in a wide range of organisms. If constitutively expressed in transgenic wheat plants, these positive regulators of cell survival might arrest the necrosis that accompanies F. graminearum infection.

Enhanced resistance to F. graminearum also might be achieved by reducing the action of possible pathogenic determinants, such as oxalic acid, which has a role in the pathogenicity of Sclerotinia, Rhizoctonia, and other fungi (Dutton and Evans, 1996). Although it is not known if oxalic acid is involved in Fusarium pathogenesis, expression of oxalyl-CoA-decarboxylase, which metabolizes oxalic acid, might be helpful in providing resistance against the fungus by alteration of pH, chelation and/or neutralization.

Effective anti-Fusarium activity may require expression of two or more transgenes, each encoding proteins with distinct modes of antifungal action. The synergistic action of glucanases and chitinases in conferring resistance to several kinds of fungal pathogens has been widely reported (reviewed by Graham and Stricklen, 1994; Van Loon, 1997). The anti-Fusarium activities of thionins from wheat and barley were enhanced synergistically by the action of a 2S albumin seed storage protein in vitro (Terras et al., 1993). In one case, a barley Class II chitinase in combination with a barley ribosome inactivating protein inhibited R. solani Kuhn infection (Jach et al., 1995). Likewise, the effect of glucanases might be enhanced by the action of some thaumatin-like proteins (Grenier et al., 1999).

With the exception of those targeted against DON, most of the antifungal proteins described above have the potential to be active against fungi other than F. graminearum. Thus, it would be useful to test the transgenic plants containing these proteins against other wheat and barley pathogens.

Promoters

The effectiveness of an antifungal protein in planta will be determined in part by its expression levels in the crucial host tissues, and by the timing of its expression such that suitable levels accumulate before the host becomes most vulnerable to infection. For initial constructs, expression governed by strong promoters with widespread (constitutive) activity offers the best chance for protection against head blight, where multiple tissues appear to be vulnerable and the mechanisms of host-pathogen interaction are still poorly understood. At present, constitutive promoters, including those of the cauliflower mosaic virus 35S transcript (Ou-Lee et al., 1986; Battraw and Hall, 1990), the rice Actin-1 gene (Zhang et al., 1991), and the maize Polyubiquitin-1 gene (Christensen and Quail, 1996), are widely used for expression of candidate antifungal genes in wheat and barley. A novel constitutive promoter from a badnavirus of sugarcane (Saccharum officinarum L.) and banana (Musa acuminta Colla), designated ScBV, has been reported to function in the glume, lemma, palea and other floret organs of oat (Tzafrir et al., 1998), and represents a promising molecular resource for the expression of candidate antifungal protein genes in the corresponding organs of wheat and barley. The promoter of the adenine methyltransferase gene from the algal virus PBCV-1 is active in the leaf, stem and floral parts of transgenic tobacco plants, and confers transient chloramphenicol acetyltransferase activity in rice protoplasts and calli derived from wheat microspores (Mitra and Higgins, 1994). Antifungal constructs containing constitutive promoters have the added advantage that they may be active against other cereal pathogens that attack plant organs and at developmental stages other than those infected by Fusarium.

Eventually, organ- or tissue-specific promoters will be needed to limit antifungal protein expression to only susceptible parts of the plant, to minimize the metabolic drain on the host associated with constitutive over-expression, to reduce the exposure of the pathogen to the antifungal proteins when host resistance is not needed, and to help garner public acceptance. At a minimum, expression in the glume and lemma is desired for both barley and wheat, because these organs comprise the outer-most protective barrier encasing the reproductive organs. Ingress of Fusarium hyphae, visualized with the jellyfish green fluorescent protein, indicates that points of entry or proliferation of the pathogen in barley include the stigmatic and ovary epithelial hairs of the extruded seed tip, and the pericarp (Skadsen et al., 1999a). In wheat, the stomata of the glume provide primary routes for invasion of the spike, and hyphae proliferate in glume parenchyma cells (Pritsch et al., 2000). These studies suggest that additional expression in these host tissues will be beneficial. The promoter of a floret-expressed gene from barley is now in use to direct expression of antifungal genes in the lemma, palea and pericarp (Skadsen et al., 1999b). Wheat is most vulnerable to FHB at anthesis, probably because of the availability of compounds in the pollen that stimulate fungal growth (Strange et al., 1974). Isolation of new floret-specific promoters of wheat could be undertaken by several approaches, including conventional screens for genes differentially expressed in the organs of interest, promoter trapping strategies, and emerging microarray technologies.

Further Engineering of Genes for Optimal Antifungal Activity

Even when regulated by a strong promoter, transcripts of antifungal protein genes can contain features that reduce mRNA stability (De Rocher et al., 1998), especially if the gene originates from a heterologous source. Such features include (i) an unfavorable start codon context (Luehrsen and Walbot, 1994); (ii) unfavorable nucleotide composition of the 5' untranslated region; (iii) gene- or host-specific codon preferences (Lonsdale et al., 1998); (iv) the absence of elements that are required for proper mRNA processing, such as positioning of the 3' end (Joshi, 1987); and (v) the presence of cryptic regulatory elements that result in intron splicing, premature polyadenylation, or other undesirable processing events (Haseloff et al., 1997; Iannacone et al., 1997). Overall and local GC contents also can be important as they affect codon frequencies and discrimination between exon and intron sequences. Coding sequences rich in A and T may harbor cryptic intron splicing signals (Ko et al., 1998) and transcriptional terminators. Any of these features can be barriers to the accumulation of biologically active antifungal proteins to levels high enough for effective resistance.

Some candidate proteins may be more effective if secreted into the extracellular space, while others may be better targeted to the vacuole or cytoplasm to be released upon cell lysis, depending on the protein's antifungal activities, the pathogen's infection site, and the ramification of the fungus during pathogenesis. As our understanding of the F. graminearum infection process increases, and host-based factors important in scab pathogenesis are elucidated, we will be better able to refine the expression of anti-Fusarium proteins for maximum efficacy.

Challenges and Future Directions

Although many, if not all, plants express glucanases, chitinases, and other classes of pathogenesis-related proteins, each protein appears to be effective against a specific pathogen or subset of pathogens (Graham and Stricklen, 1994). The differential activities of the chitinases have been attributed to inherent properties of the enzymes (Sela-Buurlage et al., 1993; Brunner et al., 1998), to differences in cell wall architecture among the fungi (Sivan and Chet, 1989; Van Loon, 1997), or to other factors. The development of in vitro or transient assays will greatly aid in focusing efforts on proteins with activity against F. graminearum and F. culmorum.

Anti-Fusarium proteins with potential for further development may be identified in biocontrol agents, cereals (Caruso et al., 1996; Joshi et al., 1998), noncereals (Tailor et al., 1997), and insects (deLucca et al., 1997; Cavallarin et al., 1998). A search is in progress for new genes from wheat that confer tolerance to trichothecenes (N. Alexander, S. McCormick, and G. Muehlbauer, unpublished). Additional candidate proteins might also include wheat phytoalexin biosynthetic enzymes (Du et al., 1998), and other components of the host defense pathway (e.g., Shirasu et al., 1999; Zhou et al., 1998). For more effective and durable activity against FHB, transgenes with different modes of action may eventually need to be combined with one another. The most promising candidates also may be combined with partial FHB resistance loci identified in conventional field tests of wheat and barley. The parallel and synergistic efforts in traditional breeding and biotechnology should provide wheat and barley growers and industries with FHB resistant cultivars that meet their agronomic and quality needs.

ACKNOWLEDGMENTS

The authors thank Drs. N. Alexander, S. Baenziger, P. Bregitzer, L. Harris, T. Hohn, H. Kaeppler, G. Muehlbauer, S. Muthukrishnan, and R. Skadsen for providing information about their ongoing research, and Drs. D. Horvath and A. Cenci for reviewing the manuscript.

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Abbreviations: FHB, Fusarium head blight; DON, deoxynivalenol.

Lynn S. Dahleen,(*) Patricia A. Okubara, and Ann E. Blechl

L.S. Dahleen, USDA-Agricultural Research Service, Red River Valley Agric. Res. Center, Fargo, ND 58105; P.A. Okubara and A.E. Blechl, USDA-ARS, Western Regional Research Center, Albany, CA 94710-1105. Received 28 March 2000. (*) Corresponding author (dahleenl@fargo.ars.usda.gov).
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Author:Dahleen, Lynn S.; Okubara, Patricia A.; Blechl, Ann E.
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Date:May 1, 2001
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