Transgenic crop resistance to bacteria

Share Embed


Descrição do Produto

ELSEVIER

Field Crops Research 45 (1996) 85-97

Field Crops _ Research

Transgenic crop resistance to bacteria N.J. Panopoulos a,b,*, E. Hatziloukas a, A.S. Afendra a a Department of Environmental Science, Policy and Management, University ~fCalifornia, Berkeley, CA 94720, USA b Institute of Molecular Biology and Biotechnology, Foundation.for Research and Technology - Hellas, and Department of Biology, Universi~ of Crete, Heraklion, Greece

Abstract The advent of molecular genetics not only made possible analysis of disease mechanisms at a level of resolution not previously possible, but also provided the necessary tools to genetically engineer into plants new capabilities for self-defence against pathogens. In the case of bacterial pathogens, the study of genes and mechanisms of pathogenesis and natural or induced plant resistance, and parallel work with antibacterial proteins from various sources, have provided a basis for implementing a range of molecular strategies to introduce novel forms of transgenic resistance in plants. These approaches fall in three basic categories: (a) introduction of bacterial avirulence genes; (b) incorporation of pathogen-derived genes for resistance to bacterial phytotoxins; and (c) expression of antibacterial proteins from plants, insects, or bacteriophages as bactericidal or bacteriolytic agents. If the preliminary laboratory results hold during field evaluations, ultimately, a broad range of genes and engineering strategies can be envisioned that singly or, more likely, in combination, should provide effective and durable resistance to bacterial pathogens. Keywords: Biotechnology; Disease control; Resistance; Disease; Review; Strategy; Transgenic plants

1. I n t r o d u c t i o n Several bacterial pathogens are responsible for significant economic damage to crop plants. The severity and significance o f bacterial disease epidemics usually relate to (a) many environmental factors, including moisture and rainfall patterns in the regions where the crops are grown, (b) vector and inoculum sources and survival, and (c) other biotic and abiotic factors that affect host susceptibility and disease cycles in general. Although the economic importance of bacterial disease varies widely with region, crop, and socioeconomic conditions,

Corresponding author. Correspondence address: Department of Biology, University of Crete, Heraklion, Greece.

some pathogens cause economically important diseases worldwide and are difficult to control by present methods. Some examples are P s e u d o m o n a s s o l a n a c e a r u m , causing wilt diseases on solanaceous plants, the soft-rot caused by Erwinia carotovora ssp. carotovora, X a n t h o m o n a s campestris pv. citri, the agent of citrus blight, and ice-nucleating strains of P s e u d o m o n a s syringae, which increase the susceptibility of many crops to frost. The availability o f genetic resistance in commercial varieties varies widely with crop and pathogen. Furthermore, changing cultural practices, including the release of new cultivars, expansion of crops to new areas or marginal lands, introduction o f new pathogens to areas where they were previously absent, and the emergence o f new f o r m s / s t r a i n s of pathogens are a frequent cause

0378-4290/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0378-4290(95)00098-4

86

N•J. Panopoulos et al. / Field Crops Research 45 (1996) 8 5 - 9 7

-- ~

~

"~ --

•- ~ . . ~

~

"~

~

=

~ .~ ..~

. ~

"~ "~

~

o

N,.L Panopoulos et a l . / Field Crops Research 45 (1996) 85-97

of new bacterial disease problems. Sanitation measures, certification of propagative stocks and availability, economics and efficacy of bactericide applications are important determining factors in disease control. For many diseases of worldwide importance there is no effective chemical control. Furthermore, the registration of new bactericides is becoming increasingly difficult for economic and environmental reasons. The emergence of genetic biotechnology raises hopes that effective, durable, environmentally friendly and economically feasible resistance to bacterial diseases through genetic modification of plants can be devised and implemented in both high-intensity and low-input cropping systems. This paper provides a brief review of the current state of activity and examples of transgenic resistance to bacterial diseases, along with the conceptual background upon which they are based.

2. Experimental and conceptual approaches to transgenic resistance Most plants display one or more forms of natural resistance to bacteria, as they do to other pathogens. Any given plant is resistant to most bacterial pathogens; most diseases are tissue or organ specific; and disease symptoms are often nonsystemic but localized in nature (many diseases are of the local lesion type, even though the surrounding tissue is usually infectible). These forms of resistance undoubtedly are complex and diverse, both in terms of the kinds of genes and the types of underlying mechanisms. In theory, a susceptible plant could be modified genetically in ways that mimic the fundamental processes underlying these natural forms of resistance. These ideas are conceptually simple, but require specific molecular engineering strategies that cannot be implemented with our current level of knowledge of the genetics and molecular physiology of natural resistance in plants. However, recent progress in various aspects of pathogenesis and host resistance provides basis for optimism that various forms of self-defense against pathogens may be ultimately engineered in crop plants. The approaches that have been tried experimentally thus far (see Table 1 for a list of case studies) to

87

engineer bacterial disease resistance in plants can be classified into three general categories. One approach that may become very popular in the near future, embodies the concept of 'strategic cell death' and would entail the use of genetic cassettes encoding a two-component 'sensor-actuator' system, consisting of an avirulence gene, or both avirulence and corresponding resistance genes and an appropriate promoter that would be activated early and locally after infection. Functional activation of this cassette in the plant would initiate the process of hypersensitive necrosis (HR) associated with many forms of resistance in nature (De Witt, 1992). Another approach exploits the concept of 'pathogen-derived' resistance (Sanford and Johnston, 1985), which has been broadly and successfully tested with plant viruses. In the case of bacteria, the same basic rationale applies as in viruses, but different types of genes are used. The two examples that have been pursued thus far involve transfer of genes encoding phytotoxin-insensitive cellular targets (De la Fuente-Martinez et al., 1992; Hatziloukas and Panopoulos, 1992) or enzymes that convert the toxin to a biologically inactive form (Anzai et al., 1989). This approach is analogous in concept to the transgenic modification of plants for resistance to herbicides. A conceptually different approach involves the transfer of genes that encode certain enzymes and polypeptides from various organisms, or synthetic genes encoding such peptides, that are lethal to bacteria or inhibit their growth (Trudel et al., 1992; Carmona et al., 1993; During, 1993; Jaynes et al., 1993). By expression of these genes in transgenic plants, the ability of the pathogen to multiply, or its toxin to damage the plant, is negated or reduced. This report presents an overview of reported attempts to engineer transgenic resistance to bacteria in plants along with some background on the underlying bacterial virulence factors and the genetics and mechanisms of bacterial pathogenesis.

3. Bacterial virulence, avirulence and pathogenicity genes Infection of plants by potential pathogens results either in development of disease ('compatible interactions') or in the inhibition of pathogen multiplica-

88

N.J. Panopoulos et al.// Field Crops Research 45 (1996) 8 5 - 9 7

tion ('incompatible interactions'). In compatible interactions, the pathogenicity/virulence traits of the pathogen are actively expressed and plant resistance responses either remain quiescent or may be actively suppressed by the pathogen. The incompatible interaction often (but not always) leads to limited local cell necrosis at the site of pathogen entry at some early stage postinoculation (hypersensitive reaction, HR; Klement, 1988). This necrosis is generally coincident with the activation of a range of biochemical and other responses in the plant that are associated with the phenotypic expression of disease resistance. These responses lead to inhibition of pathogen multiplication a n d / o r systemic spread. A large number of factors that are involved in the compatible response have been identified for different bacterial plant pathogens. Among these are phytotoxins, plant growth hormones, bacterial exopolysaccharides, enzymes that degrade polysaccharides, proteins or other macromolecular constituents of plant cell walls (Yoder, 1980; Kotoujansky, 1987; Daniels et al., 1988; Gross, 1991; Boucher et al., 1992; Leigh and Coplin, 1992; Surico and Iakobelis, 1992). These are referred to collectively as 'virulence' or 'pathogenicity' factors. The distinction between the two terms is that the former are not absolutely essential for, but mainly increase the pathogenic potential, while the latter are necessary for any disease to occur. It should be noted that plant pathogenic bacteria are extracellular parasites and do not need to penetrate living plant in order to cause disease (this is also true in Agrobacterium infections, but with the distinction that the formative phase of crown gall symptoms involves the continued expression of T-DNA genes within the host rather than in the pathogen). Bacterial genes involved in the biosynthesis of these factors have been cloned from several pathogens and characterized at various levels of molecular detail. Different virulence/pathogenicity factors have different degrees of proven importance for particular pathogens, pathogen groups, or disease types. For example, pectolytic enzymes are produced by many bacterial pathogens and assume greater importance in diseases known as soft rots than in other diseases. Similarly, toxins and plant growth hormones are produced by several pathogens but assume varying importance in different disease types.

Among the pathogenicity-related genes whose functions are still poorly understood at the biochemical level are the hrp and the avr genes. The former ('harp', hypersensitive reaction and pathogenicity; Lindgren et al., 1986; reviewed in Willis et al., 1991b) are broadly distributed among Gram-negative phytopathogenic bacteria and have the unique property of being necessary for both compatible and incompatible interactions. These genes encode: (a) regulatory proteins that are necessary for the expression of other hrp and aur genes (see below); (b) proteins, termed 'harpins', that elicit necrotic reactions on plants, but for unknown reasons are necessary for pathogenesis; and (c) components of an export apparatus that mediates the secretion of harpins. The aur ('avirulence') genes relate genetically and functionally to so-called 'R' genes, also known as the 'vertical resistance' genes of plants against pathogens. The aur and R genes were initially defined through classical genetic analysis with fungal pathogens and their hosts (Flor, 1971) as 'Mendelian' characters that control the race/cultivar specificity in plant-pathogen interactions. They exhibit strict 'specificity' and functional correspondence, usually against single genes in the pathogen (gene-for-gene hypothesis). During the last decade, the original tenets of this hypothesis were proven to hold for bacterial pathogens of plants, which had not been possible to investigate through classical genetics. About twenty such genes have now been cloned, nearly all from members of two taxonomic groups of plant pathogenic bacteria, the fluorescent Pseudomonads and the Xanthomonads (Vivian and Mansfield, 1993). Bacterial aur genes are necessary for the activation of resistance responses in plants that carry 'functionally correspondent' R genes, which enable the plant to recognize the presence of the aur gene or its product. A given pathogen may contain one or more avirulence genes, each blocking the progression of disease development on particular plants depending on whether or not these carry the appropriate R genes. The R / a u r gene interactions thus determine host/pathogen specificity at the cultivar/race (strain) level, as predicted by the gene-forgene hypothesis. An important conceptual breakthrough from this line of research was the finding that analogous interactions between a u r / R genes/products are involved in the expression of at

N.£ Panopoulos et al. / Field Crops Research 45 (1996) 85-97

least some categories of resistance between a given plant and its heterologous pathogens ('non-host' resistance).

4. Transgenic plants expressing avirulence bacterial genes As stated in the introduction, the majority of plants are naturally resistant to a given pathogen and the majority of pathogens have a limited host range. The most extensively studied host range determinants are the avr genes, avr-mediated resistance is manifested usually in the form of HR, less often without visible necrosis. The biochemical mechanisms by which avr genes and their products act in the plant are not entirely clear. A basic question is whether avr gene products are catalytic proteins (i.e., function as enzymes), synthesizing elicitors of the defense response, or function in other ways, as many interpretations of the gene-for-gene based models have suggested. Convincing evidence for enzymatic function has been obtained only in the case of the AvrD protein (product of the avrD gene from P. syringae pv. tomato) that directs the synthesis of a low molecular weight necrosis elicitor. This elicitor consists of two related, C-glycosyl lipid rlactones, given the trivial names syringolides 1 and 2 (Midland et al., 1993; Smith et al., 1993). It is assumed that the AvrD protein catalyses the first committed step leading to syringolide synthesis, but this activity has not been demonstrated in vitro. It is important to note that both the avrD gene itself and its cognate elicitor(s) demonstrate the same specificity of necrosis induction. The concept of De Witt (1992) to engineer disease resistance in plants envisions the introduction of a two-component genetic cassette consisting of: (a) a 'sensor' gene (e.g., an avr gene) under the control of a plant promoter that would be activated during pathogenesis; (b) an 'effector' gene (e.g., the corresponding R gene). Upon pathogen attack, the avr and R gene products would interact to activate HR. This concept embodies the idea of 'strategic cell death' proposed for genetic ablation in animals. To be effective against a pathogen and nonlethal to the plant, one or both genes would need to be expressed as a specific response to infection but not constitu-

89

tively, as this would be lethal to the plant. Candidate promoters would be sought among the several plant genes that are known to be expressed upon pathogen attack, such as those encoding pathogenesis-related proteins or phytoalexin biosynthetic enzymes. These promoters may need to be properly chosen or finely tuned, because at least some of them can be expressed under different conditions of stress or plant developmental stages, and would desirably be activated by a wide range of pathogens. Recently, a simpler version of this concept was experimentally tested in a bacterial model disease system. Two avirulence genes, avrD from P. s. tomato and avrRxv from X. campestris pv. vesicatoria were transferred and expressed in transgenic tobacco plants (Huang et al., 1993), resulting in varying levels of resistance to two bacterial (P. solanacearum and P. syringae pv. tabaci) and a fungal pathogen of tobacco (Alternaria alternata). In theory, expression of avr genes that elicit HR-type resistance responses in a functional form in a plant that possesses the corresponding R gene, would be expected to be lethal. Whether such genes are present or absent can be established by infecting the plant with compatible bacteria that carry the avr gene in question on a recombinant plasmid. Such experiments were carried out for avrD and tobacco, by using P. syringae pv. tabaci (which lacks avrD and avrRxv) as the compatible pathogen, and indicated that this plant does not possess an R gene with recognitional specificity for avrD (N.T. Keen, pets. commun.). The avrD and avrRxv genes in the transgenic plants were expressed by three different plant promoters: the phenylalanine ammonia iyase (PAL); the chalcone synthetase (CHS); and the proteinase inhibitor II. All these are activated after pathogen infection or physical damage. The avrD transgenic calli and plants regenerated from them showed no necrosis and appeared normal in all respects. However, 50% of the avrRxv transgenic calli showed massive necrosis. Plants regenerated from transgenic calli showed elevated constitutive expression of PAL, CHS, and other defense response genes, and severe inhibition of pathogen growth. This work is at a preliminary stage and many important details about the mechanisms, heritability of the reported resistance, and possible generality need to be clarified. It opens exciting new possibilities for potential trans-

90

N.J. Panopoulos et a l . / Field Crops Research 45 (1996) 85-97

genic resistance to bacterial and fungal pathogens, however, which undoubtedly will be further explored in the future.

5. Transgenic defense based on resistance to bacterial phytotoxins

Microbial phytotoxins are classified according to (i) their host range, as host specific and nonhost specific, and (ii) their contribution to the phytopathogenic process, as pathogenicity or virulence factors. Bacterial phytotoxins are usually nonhost specific and act as virulence factors in the development of the respective disease (Mitchell, 1984). Their main contribution is believed to be the enhancement of the bacterial population buildup and pathogen persistence in planta (Gross, 1991). In some instances, the production of bacterial phytotoxins has been viewed as a fortuitous event unrelated to the phytopathogenesis, much like the production of various secondary metabolites, having some kind of biological activity. The isolation of T o x - mutants from different pathovars of Pseudomonas syringae (e.g., pv. phaseolieola (Patil et al., 1974; Peet et al., 1986), pv. tabaci (Barta et al., 1993), or pv. coronafaciens (Kinscherf et al., cited in Willis et al., 1991a)), which retain their pathogenic capacity, seems to support this hypothesis. In contrast to the above views, there is both theoretical and experimental evidence contradicting the idea that bacterial phytotoxins are mere virulence factors or fortuitous products: (i) the long-term coevolution of plants with their pathogens has probably contributed significantly to the acquisition and maintenance of the capacity to synthesize toxins. This becomes apparent when considering the wide variety of chemical forms of toxins produced by very similar groupings of organisms (e.g. Pseudomonads), as would be expected in a case of co-evolution of pathogen and plant in different ecological niches (Mitchell, 1984). (ii) The presence of the genetic information and biosynthetic machinery to carry out such a complex synthesis (as in the case of many toxins), implies the presence of positive selection (Vining, 1990), otherwise the growth disadvantage resulting from the energy waste of this process would finally lead to the elimination of the production. This

becomes even more obvious by considering the additional energy investment required for the maintenance/development of regulatory or, especially, self-protection mechanisms, which probably have to be redundant (Durbin and Langston-Unkefer, 1988) in order to avoid suicide, in case the main protection mechanism fails for any reason. Plant transformation with bacterial genes that confer autoimmunity in the bacterium against its own toxin conforms to the concept of 'pathogen-derived' resistance (Sanford and Johnston, 1985). There are two examples of experiments involving pathogen-derived toxin resistance gene transfer to import toxin immunity into plants. The first concerns tabtoxin and the second phaseolotoxin resistance. 5.1. Tabtoxin: resistance based on a detoxifying enzyme derived from the toxin producer Tabtoxin is a biologically inactive dipeptide pretoxin secreted by several phytopathogenic Pseudomonas, including P. syringae pv. tabaci (tobacco wildfire pathogen), P. syringae pv. coronafaciens (halo blight of oat), P. syringae pv. garcae (bacterial scorch of coffee), P. syringae isolate BR2 (bean wildfire pathogen), and P. syringae isolate 0152 (soybean wildfire) (Mitchell, 1991), and consists of the unusual amino acid tabtoxinine-/3-1actam (T/3L) linked (predominantly) to a threonine residue. Tabtoxin itself is not toxic to plants, but one of its hydrolysis products, the T/3 L, which is released by the action of aminopeptidases (of either bacterial or plant origin), is the bioactive form of this toxin (Uchytil and Durbin, 1980). T i l L is a general inhibitor of glutamine synthetase (GS) from plants, fungi and bacteria, including the enzyme from one of the producer organisms that has been tested. The phytotoxic activity of T/3 L in plants results from the irreversible inhibition of GS, which, under light conditions, leads to accumulation of NH~- to levels that are toxic to plant leaves. TIlL induces chlorosis in leaves and disrupts the thylakoid membranes in chloroplasts, causing uncoupling of photophosphorylation (Turner and Debbage, 1982). Genetic analysis indicates that the significance of tabtoxin in the pathogenicity of the producing bacteria differs with the producer a n d / o r its host(s). Thus, mutational loss of tabtoxin production eliminates the character-

N.J. Panopoulos et al. / Field Crops Research 45 (1996) 85-97

istic chlorosis, but not the lesion-forming ability of P. syringae pv. tabaci on tobacco; however, such mutations lead to complete loss of pathogenicity of the bean wildfire strain BR2 on bean (Kinscherf et al., cited in Willis et ai., 1991a). All genes necessary for tabtoxin biosynthesis and one or more genes for resistance to T i l L are located in a ~ 25-kb chromosomal region of the P. syringae pv. tabaci genome (Willis et al., 1991a). At least three mechanisms have been proposed for T i l L resistance in the producers: (i) adenylation of the resident GS; (ii) a il-lactamase activity that was detected in Tox ÷ Tox r (TilL-resistant) but not in T o x - Tox s (TilL-sensitive) P. syringae pv. tabaci; and (iii) a tabtoxin-specific acetyl transferase cloned from P. syringae pv. tabaci encoded that is encoded by the ttr locus (reviewed in Willis et al., 1991a). The first approach to obtaining tabtoxin resistance in plants (tobacco) was Carison's selection of resistant regenerants in a mutagenized population of haploid tobacco tissue-culture cells, using the tabtoxin analogue methionine sulfoximine (MSO) as a selective agent (Carlson, 1973). Plants regenerated from MSO r calli were still sensitive to bacterial lesion formation, but did not form the chlorotic halos normally associated with infection by P. syringae pv. tabaci. In a more recent approach, Anzai et al. (1989) cloned this locus from P. syringae pv. tabaci, fused to the 35S promoter from cauliflower mosaic virus (CaMV), and transferred into tobacco plants via Agrobacterium-mediated transformation. Transgenic plants expressing the highest level of ttr transcripts were resistant to TilL-induced chlorosis and, surprisingly, to the infection by P. syringae pv. tabaci as well. This was an unexpected result, because T o x - mutants of this bacterium produce disease lesions, albeit without chlorosis (Willis et al., 1991a). This is also true in P. syringae pv. coronafaciens, unlike P. syringae BR2, where T o x - mutants no longer produce water-soaking symptoms (Kinscherf et al., cited in Willis et al., 1991a). Other approaches for creating plants that are resistant to T i l L might be possible. The study of Marek and Dickson (1987), carried out in Saccharomyces cerevisiae, indicated that cloning of the GS structural gene (as well as of three additional DNA sequences, which are unrelated to the GS gene or to each other) on multicopy plasmids leads to an increased resis-

91

tance to MSO and tabtoxin. Overexpression of genes encoding herbicide-target enzymes in plants often gives herbicide resistance. In the case of GS, transgenic tobacco plants that expressed the GS gene from alfalfa under the 35S promoter were 20-fold more resistant in vitro to the GS-inhibitor herbicide, L-phosphinotricin (the active ingredient in BASTA).

5.2. Phaseolotoxin: resistance based on a toxin-insensitiue target enzyme deriued from the producer The second relevant example of pathogen-derived phytotoxin resistance involves phaseoiotoxin, which is produced only by the bean halo blight pathogen Pseudomonas syringae pv. phaseolicola. Phaseolotoxin is a nonribosomally synthesized tripeptide, N ~(N'-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine (Moore et al., 1984) that inhibits reversibly the enzyme ornithine carbamoyltransferase (OCTase; Patil et al., 1970). A hydrolysis product of phaseoiotoxin, octicidine [N~-(N'sulphodiaminophosphinyl) ornithine], generated through the action of aminopeptidases, is a much more potent, irreversible inhibitor of the OCTase (Mitchell and Bielski, 1977; Templeton et al., 1985). Upon infection of the bean plants with P. syringae pv. phaseolicola, the phaseolotoxin/octicidine produced inactivates the host plant OCTase(s). The inhibition causes accumulation of ornithine (a precursor of arginine biosynthesis) and, often (but not always), arginine depletion, but it is not known if this biochemical lesion is solely responsible for the other secondary symptoms of the disease, which include local and systemic chlorosis, disruption of the apical dominance, growth retardation and systemic movement of the pathogen in the plant. The genes necessary for phaseolotoxin production by P. syringae pv. phaseolicola are localized on the chromosome (Quigley et al., 1985) and form a contiguous cluster, approximately 22 kb in length, termed tox or pht cluster (Peet et al., 1986; Zhang et al., 1993). The tox region is part of a ~ 28-kb region that is completely absent from closely related pathovars of P. syringae (Takikawa, Hatziloukas and Panopoulos, unpublished data). Adjacent to the tox cluster is the argK gene, which encodes the only known OCTase that is resistant to phaseolotoxin/octicidine inhibition. All other OCTases tested thus far

92

N.J. Panopoulos et al./ Field Crops Research 45 (1996) 85-97

(a total of 20), including a duplicate OCTase from P. syringae pv. phaseolicola (the product of argF; Peet and Panopoulos, 1987; Hatziloukas and Panopoulos, in prep.) are sensitive to the toxin. ArgK is the only resistance factor identified to date that confers effective resistance against phaseolotoxin. The deduced sequence of ArgK reveals unique substitutions of several amino acid residues, relative to other OCTases, including two of the residues found in the binding site of carbamoylphosphate (Mosqueda et al., 1990; Hatziloukas and Panopoulos, 1992), which is one of the two substrates of OCTase. This is presumed to be the binding site for phaseolotoxin/octicidine. ArgK binds the inhibitors inefficiently and reversibly (Templeton et al., 1985), presumably because of these substitutions. Two different groups had used the same strategy to generate toxin-resistant transgenic plants (De la Fuente-Martinez et ai., 1992; Hatziloukas and Panopoulos, 1992). In both cases, the gene argK was fused to the transit peptide of the gene small subunit of ribulose biphosphate carboxylase (rbcS), to target the resulting chimeric protein RbcS::ArgK into the chloroplasts, where the plant OCTases are located and citrulline biosynthesis takes place (Shargool et al., 1988). The resulting construct was subcloned into a binary vector and used to transform tobacco plants through a standard Agrobacteriummediated procedure. Transgenic plants expressing the bacterial enzyme showed a 2- to 10-fold increase in OCTase activity, compared to the untransformed plants, and virtual resistance to phaseolotoxin/octicidine, based on both in vitro (enzymatic) and in vivo (leaf chlorosis) assays. The pH-activity profile obtained by Hatziloukas and Panopoulos (1992) indicated that the bacterial and plant enzymes may form heterotrimers. A significantly higher content of arginine and proline in the transgenics was reported by De la Fuente-Martinez et al. (1992), presumably reflecting degradation of excess arginine in the mitochondria. These authors also reported that toxintreated areas of the transgenic plants did not show any change in chlorophyll content compared to untreated areas, unlike control plants, which showed a 30% decrease in treated vs. untreated areas. Furthermore, transgenic tobacco seemed to be less susceptible to systemic infection by P. syringae pv. phaseolicola strain TEXCOCO (an unusual strain of pv.

phaseolicola claimed to be pathogenic on tobacco). Finally, Hatziloukas and Panopoulos (1992) showed that the transgene can be transferred by cross-pollination to other tobacco cultivars, indicating normal heritability of the transgene.

6. Transgenic defense based on antibacterial proteins

Antibacterial peptides and proteins have been isolated from many different organisms; they include, among others, the cecropins, attachins and diptericins from insects (Hultmark et al., 1983; Lee et al., 1983; Boman and Hultmark, 1987; Hoffmann and Hoffmann, 1990), the magainins from amphibians (Zasloff, 1987), the defensins from mammalian polymorphonuclear lymphocytes (Selsted et al., 1985; Lehrer et al., 1991), the lysozymes from various organisms and phages (Joll~s and Joll~s, 1984), the thionins of cereals (Garcia-Olmedo et al., 1989) and a variety of other defense-related proteins from higher plants (Bowles, 1990). Work carried out in a number of laboratories indicates that several of these have significant antimicrobial potential when they are expressed in transgenic plants. The following examples illustrate the diversity of available possibilities.

6.1. Lysozymes Lysozymes are a ubiquitous class of antibacterial proteins, found in many plants, in insects, birds and other terrestrial and marine animals, in bacteriophages, and in some fungi and bacteria (reviewed in Tsugita, 1971; Joll~s and Joll~s, 1984). By definition, they are 1,4-fl-N-acetyl muramidases, cleaving the glycosidic bonds between the C-1 of Nacetylmuramic acid (MurNAc) and the C-4 of Nacetylglucosamine (GlcNAc) in peptidoglycan, the key material providing tensile strength in the cell wall of virtually all eubacteria. Various lysozymes display different enzymatic mechanisms and specificity requirements. Some lysozymes (g-type) act only hydrolytically, others as transglycosylases, and others (c-type) are capable of both hydrolysis and transglycosylation. This aspect has not been investigated with plant lysozymes. Phage lysozymes require a peptide-substituted substrate and cleave the glyco-

N.J. Panopoulos et a l . / Field Crops Research 45 (1996) 85-97

sidic linkages next to substituted MurNAc residues. A fungal lysozyme possesses a /3-1,4-N,6-O-diacetylmuramidase activity, in addition to the typical MurNAc activity, and the same appears to be true of a bacterial lysozyme. These enzymes are active against bacteria that are insensitive to other types of iysozymes. Many lysozymes are bifunctional enzymes, possessing in addition to N-acetyl muramidase activity endochitinase activity (random hydrolysis of 1,4-/3-N-GicNAc linkages in chitin, a structural polymer of the cell walls of many fungi and of the exoskeleton of insects). Usually, these bifunctional enzymes display stronger chitinase than lysozyme activity and only few enzymes with the opposite activity ratio are known. Plant lysozymes have chitinase as the dominant activity and cleave peptide-substituted peptidoglycan very poorly. These enzymes are strongly basic proteins located mainly in the vacuole (During, 1993, and references cited herein), except for tobacco, where an acidic protein with both lysozyme and chitinase activity is found in the intercellular spaces (Stintzi et al., 1993). Because of their location, the vacuolar lysozymes could not contribute in a defense reaction against bacterial infection. Attempts have been made to express foreign lysozyme genes in transgenic plants to increase their resistance against bacterial pathogens. The genes in question were derived from the hen egg-white lysozyme (HEWL, 'c' type lysozyme; Joll~s and Joll~s, 1984) and from the E. coli bacteriophage T4. Trudel et al. (1992) have used a HEWL cDNA clone bearing its own signal peptide to transform tobacco. The transformed plants produced mature active HEWL, but secreted the enzyme at low levels. In a later study, Trudel et al. (1993) created various HEWL constructs, some with alterations in the signal peptide and others with additions or deletions at the 3' end of the coding sequence, and compared their expression and the accumulation of active protein in tobacco and potato. The highest activity was observed in plants transformed by a HEWL gene with its native signal peptide and deleted of its noncoding 3'-end. Several fusions at the 3'-end of the gene that were tested did not allow recovery of active enzyme. HEWL was shown to be inhibitory in vitro to some bacterial and fungal pathogens including Clavibacter, Fusarium, Verticillium and Rhizoctonia species.

93

The published data do not indicate whether HEWLtransgenics exhibited resistance to bacteria. The bacteriophage T4 lysozyme was also expressed in transgenic tobacco. Hippe et al. (1989) fused the enzyme to a plant a-amylase signal peptide and demonstrated that it was secreted in the extracellular fluid of transgenic tobacco. During et al. (1993) recently introduced these chimeric lysozyme genes into transgenic potato plants. The authors reported effective resistance against infection by Erwinia carotovora under laboratory and greenhouse conditions, even though the levels of expression and secretion were low. 6.2. Cecropins and attachins Many insects respond to injection of live, nonpathogenic bacteria with the production of a potent spectrum of antibacterial peptides and proteins, which accumulate in large quantities in the insect hemolymph. Based on their molecular properties, they are classified into four general categories: lysozymes, cecropins, attachins, and diptericins. Another protein, melitin (the main component of the bee venom) also has antibacterial activity and a similar general design to cecropins but its two structural domains (see below) have reversed polarity relative to cecropins (Boman and Hultmark, 1987; Boman et al., 1991). As with the lysozymes, cecropins and attachins have recently been used in experiments involving transgenic plants that are resistant to bacterial pathogens (see below). Cecropins are a family of small, strongly basic, proteins with potent antibacterial activity, named from the insect Hyalophora cecropia, the giant silk moth, in which they were first described. Proteins with homology to cecropins that were isolated from other insects have often been given new names (e.g. bactericidin, lepidoptericin, sarcotoxin). Six different molecules, A - F , consisting of 35-37 amino acids have been isolated (Hultmark et al., 1980, 1982). Cecropins have some unusual structural features. For example, some of the molecular forms contain the modified amino acid hydroxylysine; others contain an additional C-terminal glycine residue. Together with the removal of the N-terminal signal peptide, at least some cecropins must be processed in two to three post-translational steps at both ends. The N-

94

N.J. Panopoulos et al. / Field Crops Research 45 (1996) 8 5 - 9 7

terminus is strongly basic, and can be folded into a nearly perfect amphipathic a-helix (cylindrical segment with charged groups on one longitudinal side and hydrophobic side chains on the opposite side). The C-terminus is hydrophobic, and is joined to the N-terminus by a hinge region containing glycine or proline or both (Steiner, 1982). This structure is probably responsible for the formation of ion channels in planar lipid membranes, as well as for cell membrane disruption and lethal activity (Christensen et al., 1988; Nordeen et al., 1992). In melitin, the C-terminus is basic and the N-terminus hydrophobic. Unlike melitin, cecropins seem to be specific for prokaryotic membranes, as they have no effect on mammalian cell lines or on yeast. Cecropin coding genes have been expressed in plants as a means for improving their resistance against phytopathogenic bacteria. Jaynes et al. (1989) synthesized and screened various analogs of cecropin B for differential activity against plants and bacterial pathogens. Cecropin SB37 is a construct designed to encode a protein similar to cecropin B with minor alterations (95% homology). The lethal concentration of this analog, measured in protoplasts of seven plant and in nine bacterial species that are pathogenic on these plants, was 4.5-41 /xM and 0.1-4.5 ktM, respectively (Nordeen et al., 1992), indicating that the introduction of the modified cecropin gene into the plants should not be phytocidal. In a later study, Jaynes et al. (1993) constructed a synthetic cecropin B analog, Shiva-1, which had 46% amino acid homology to the natural peptide and possessed more potent lytic activity in vitro against various plant pathogenic bacteria than SB37. The genes for both SB37 and Shiva-1 have been introduced in transgenic tobacco plants. The transformed plants producing the Shiva-1 analog had increased resistance against the pathogen Pseudomonas solanacearum compared to untransformed plants, as well as to those producing SB37. The resistance appeared as a delay of symptoms and reduction of plant mortality. Attachins are much larger than cecropins (180190 amino acids). Six different molecules ( A - F ) have been isolated from cecropia moth. Proteins A - D constitute a basic group, while E and F are acidic (Hultmark et al., 1983). All six proteins are believed to be derived from two genes, through different processing steps at the N- and C-terminus.

For example, from the two attachin cDNAs cloned from H. cecropia (Lee et al., 1983) the one corresponding to the acidic attachin E (184 amino acids) codes for a protein of 188 amino acids which include a C-terminal tetrapeptide (Ser-Lys-Tyr-Phe) that is missing in the mature protein. Unlike the cecropins, attachins have a narrow spectrum of action against bacteria, among them Escherichia coli, Acinetobacter calcoaceticus, and Pseudomonas maltophila (Hultmark et al., 1983). Their antibacterial action is mainly against growing Gram-negative bacteria and is not directly lytic. Rather, they disrupt the structure of the outer membrane by interfering with the synthesis of new outer membrane components (Engstrom et al., 1984). It is possible that attachins facilitate the action of the other two lytic peptide groups of the cecropia moth (lysozymes and cecropins). It is thought that effective insect defense derives from a triple action by different groups of proteins, which collectively can be effective against a range of infecting bacteria (Boman et al., 1991). One of these cDNAs coding for attachin E was coupled to plant promoters and used to transform apple (Norelli et al., 1993). Transformed plants of a susceptible apple rootstock N.26 possessed increased resistance to the fire blight pathogen Erwinia amylovora compared to the untransformed control, but were still more susceptible than the naturally resistant rootstock Liberty.

6.3. Thionins Thionins are a family of small (5-kD), cysteinerich proteins found in cereal endosperm and leaves (Garcia-Olmedo et al., 1987, 1989). Their expression in plants is subject to developmental control and is induced by both biotic and abiotic stresses (Bohlman et al., 1988; Fisher et al., 1989). Thionins are synthesized as precursors with a typical signal peptide and are toxic to plant pathogens in vitro (Fernandez de Caleya et al., 1972). Recently, Carmona et al. (1993) introduced thionin chimeric genes composed of the genomic a l-thionine gene from barley and a cDNA for the a-thionine from wheat in tobacco. Plants that expressed high levels of the barley a 1-thionine had enhanced resistance to two bacterial pathogens, Pseudomonas syringae pv. tabaci and Pseudomonas syringae, after artificial inoculation. The degree of

N.J. Panopoulos et al. / Field Crops Research 45 (1996) 85-97

resistance in individual transformants was proportional to thionine expression levels, determined by a thionine-specific antibody. Plants expressing the wheat a-thionine did not show resistance enhancement. This was attributed to the much lower levels of expression obtained rather than to any intrinsic differences between the two thionine proteins, which differ only at four positions and have the same MIC against the pathogen tested in vitro.

7. Perspective and s u m m a r y The engineering of resistance to bacterial diseases by molecular technologies potentially can tap many different sorts of genes, derived from the pathogens themselves, the plants they infect, and many other organisms. The examples discussed here already suggest a broad range of choices for potentially useful genes. The finding that bacterial a v r genes can function as avirulence determinants in heterologous pathogens indicates that corresponding resistance genes exist in nonhost plants. This will greatly facilitate the cloning of such genes from plants that are well characterized genetically and by modern genome mapping methods. A gene for resistance to a bacterial pathogen was recently cloned from tomato (Martin et al., 1993) and concerted efforts are underway to clone other similar genes from A r a b i d o p s i s and other plants by gene tagging, genome walking or other m a p - b a s e d strategies. Once cloned, these natural disease resistance genes can be introduced in other plants that lack genetic resistance to a given pathogen. Although most current approaches to obtain bacterial disease resistance in plants have yet to be evaluated under field conditions, the diversity of candidate genes provides a broad range o f engineering strategies and underlying resistance mechanisms. Combinatorial deployment of appropriate genes that provide mechanistically different forms o f resistance, could in theory insure both efficacy and durability. In some cases, transgenic plants displayed physiological, resistance, or other features, that could not have been predicted in advance. This gives basis for some concern for unanticipated properties that m a y affect performance and safety of transgenic plants in large-scale deployment. The impacts of transgenebased resistance on the ecology and evolution of the

95

pathogen, the host, and other organisms which interact with them or to which the transgene may potentially be transferred, even though many of these concerns also apply to classical breeding. There are several issues that are unique to transgenic resistance. For example, when expressed constitutively, resistance may affect nontarget organisms (e.g., symbiotic, commensal, herbivores) in different ways than conventional resistance. Furthermore, the transgenes may d i s r u p t / i n t e r r u p t the evolution of other natural resistance mechanisms in the plant. Due caution in deployment of transgenic plants on a large scale is essential for many reasons, and pertinent questions regarding biosafety will require case-by-case assessment and evaluation.

References Anzai, H., Yoneyama, K. and Yamaguchi, 1., 1989. Transgenic tobacco resistant to a bacterial disease by the detoxification of a pathogenic toxin. Mol. Gen. Genet., 219: 492-494. Barta, T.M., Kinscherf, T.G., Uchytil, T.F. and Willis, D.K., 1993. DNA sequence and transcriptional analysis of the tblA gene required for tabtoxin biosynthesis by Pseudomonas syringae. Appl. Environ. Microbiol., 59: 458-466. Bohlman, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V. and Apel, K., 1988. Leaf-specific thionins of barley - a novel class of cell wall proteins toxin to plant-pathogenic fungi and possibly involved in the defense mechanism of plants. EMBO J., 7: 1559-1565. Boman, H.G. and Huhmark, D., 1987. Cell-free immunity in insects. Annu. Rev. Microbiol., 41: 103-126. Boman, H.O., Faye, I., Gudmundson, G.H., Lee, J.-Y. and Lidholm, D.-A., 1991. Cell-free immunity in cocropia. Ear. J. Biochem., 201: 23-31. Boucher, C.A., Gough, C.L. and Arlat, M., 1992. Molecular genetics of pathogenicity determinants of Pseudomonas solanacearum with special emphasis on hrp genes. Annu. Rev. Phytopathol., 30: 443-461. Bowles, D.J., 1990. Defense-related proteins in higher plants. Annu. Rev. Biochem., 59: 873-907. Carlson, P.S., 1973. Methionine sulfoximine-resistant mutants of tobacco. Science, 180:1366-1368. Carmona, M.-J., Molina, A., Fernandez, J.A., Lopez-Fando, J.J. and Garcia-Olmedo, F., 1993. Expression of the a-thionine gene from barley in tobacco confers enhanced resistance to bacterial pathogens. Plant J., 3: 457-462. Christensen, B., Fink, J., Merrifield, R.B. and Mauzerall, D., 1988. Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc. Natl. Acad. Sci. USA, 85: 5072-5076. Daniels, M.J.. Dow, J.M. and Osburn, A.E., 1988. Molecular

96

N.J. Panopoulos et al. / Field Crops Research 45 (1996) 85-97

genetics of pathogencity in phytopathogenic bacteria. Annu. Rev. Phytopathol., 26: 285-312. De la Fuente-Martinez, Mosqueda-Cano, J.M., Alvarez-Moralles, A. and Herrera-Estrella, L., 1992. Expression of a bacterial phaseolotoxin-resistant omithyl transcarbamylase in transgenic tobacco confers resistance to Pseudomonas syringae pv. phaseolicola. Bio/Technology, 10: 905-909. De Witt, P.J.G.M., 1992. Molecular characterization of gene-forgene systems in plantfungus interactions and the applications of avirulence genes in control of plant pathogens. Annu. Rev. Phytopathol., 30: 391-418. Durbin, R.D. and Langston-Unkefer, P., 1988. The mechanisms of . self-protection against bacterial phytotoxins. Annu. Rev. Phytopathol., 26: 313-329. During, K., 1993. Can lysozymes mediate antibacterial resistance in plants? Plant Mol. Biol., 23: 209-214. During, K., Fladung, M. and Lorz, H., 1992. Antibacterial resistance of transgenic potato plants producing T4 lysozyme. 6th Int. Symp. Mol. Plant-Microbe Interactions, Seattle, Washington. Abstr. No. $72. During, K., Porsch, P., Fladung, M. and Lorz, H., 1993. Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. Plant J., 3: 587-598. Engstrom, P., Carlsson, A., Engstrom, A., Tao, Z.-J. and Bennich, H., 1984. The antibacterial effect of attachins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli. EMBO J., 3: 3347-3351. Fernandez de Caleya, R., Gonzalez, B., Garcia-Olmedo, F. and Carbonero, P., 1972. Susceptibility of phytopathogenic bacteria to wheat purothionins. Appl. Microbiol., 23: 998-1000. Fisher, R., Behnke, S. and Apel, K., 1989. The effect of chemical stress on the intercellular fluid of barley leaves. Planta, 178: 61-68. Flor, H.H., 1971. Current status of the gene-for-gene concept. Annu. Rev. Phytopathol., 9: 275-296. Garcia-Olmedo, F., Salcedo, G., Sanchez-Monge, R., Gomez, L., Royo, J. and Carbonero, P., 1987. Plant proteinaceous inhibitors of proteinases and a-amylases. Oxford Survey Plant Mol. Cell Biol., 4: 275-334. Garcia-Olmedo, F., Rodgrigues-Palenzuela, P., Hernandez-Lucas, C., Ponz, F., Marana, C., Carmona, M.J., Lopez-Fando, J., Fernandez, J.A. and Carbonero, P., 1989. The thionins: A protein family that includes purothionins, viscotoxins and crambins. Oxford Survey Plant Mol. Cell Biol., 6:31-60. Gross, D.C., 1991. Molecular and genetic analysis of toxin production by pathovars of Pseudomonas syringae. Annu. Rev. Phytopathol., 29: 247-278. Hatziloukas, E. and Panopoulos, N.J., 1992. Origin, structure and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression in transgenic tobacco. J. Bacteriol., 174: 5895-5909. Hippe, S., During, K. and Kreuzaler, F., 1989. In situ localization of a foreign protein in transgenic plants by immunoelectron microscopy following high pressure freezing. Freeze substitution and low temperature embedding. Eur. J. Cell Biol., 50: 230-234.

Hoffmann, J.A. and Hoffmann, D., 1990. The inducible antibacterial peptides of dipteran insects. Res. lmmunol., 141 : 910-918. Huang, Y., Keen, N.T., Whalen, M. and McBeath, J., 1993. Disease resistance in transgenic tobacco plants expressing bacterial avirulence gene. 6th Int. Congr. Plant Pathol., Montreal, Canada, Book of Abstracts, Poster 10.2.1 (276), p. 163. Hultmark, D., Steiner, H., Rasmuson, T. and Boman, H.G., 1980. Insect immunity: purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur. J. Biochem., 106: 7-16. Hultmark, D., Engstrom, A., Bennich, H., Kapur. R. and Boman, H.G., 1982. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components fron Cecropia pupae. Eur. J. Biochem., 127: 207-217. Hultmark, D., Engstrom, A., Andersson, K., Steiner, H., Bennich, H. and Boman, H.G., 1983. Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J., 2: 571-576. Jaynes, J.M., Julian, G.R., Jeffers, G.W., White, K.L. and Enringt, F.M., 1989. In vitro cytocidal effect of lytic peptides on several transformed mammalian cell lines. Pept. Res., 2: 157160. Jaynes, J.M., Nagpala, P., Destefano-Beltran, L., Huang, J.H., Kim, J.H., Denny, T. and Cetiner, S., 1993. Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Sci., 89: 43-53. Joll~s, P. and Joll~s, J., 1984. What's new in lysozyme research? Mol. Cell. Biochem., 63: 165-189. Klement, Z., 1988. Hypersensitivity. In: M.S. Mount and G.H. Lacy (Editors), Phytopathogenic Prokaryotes, Vol. 2. Academic Press, New York, pp. 149-177. Kotoujansky, A., 1987. Molecular genetics of pathogenicity in soft-rot Erwinias. Annu. Rev. Phytopathol., 25: 405-430. Lee, J.-Y., Edlund, T., Ny, T., Faye, 1. and Boman, H.G., 1983. Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia. EMBO J., 2: 577-581. Lehrer, R.J., Ganz, T. and Selsted, M.E., 1991. Defensins: endogenous antibiotic peptides of animal cells. Cell, 64: 229230. Leigh, J.A. and Coplin, D.L., 1992. Exopolysaccharides in plantbacterial interactions. Annu. Rev. Microbiol., 46: 307-346. Lindgren, P.B., Peet, R.C. and Panopoulos, N.J., 1986. Gene cluster of Pseudomonas syringae pv. phaseolicola controls pathogencity of bean and hypersensitivity on non-host plants. J. Bacteriol., 168: 512-522. Marek, E.T., and Dickson, R.C., 1987. Cloning and characterization of Saccharornyces cerevisiae genes that confer Lmethionine sulfoximine and tabtoxin resistance. J. Bacteriol., 169: 2440-1448. Martin, G.B., Brommonschenkel, S.H., Chunwongse, J., Frary, A., Ganal, M.W., Spivy, R., Wu, T., Earle, E.D. and Tanskley, S.D., 1993. Map-base cloning of a protein kinase gene conferring disease resistance in tomato. Science, 262: 1432-1436. Midland, S.L., Keen, N.T., Sims, J.J., Midland, M.M. Stayton, M.M., Burton, V., Smith, M.J., Mazzola, E.P., Graham, K.J.

N.J. Panopoulos et a l . / Field Crops Research 45 (1996) 85-97 and Clardy, J., 1993. The structures of syringolides 1 and 2, novel C-glycosidic elicitors from Pseudomonas syringae pv. tomato. J. Org. Chem., 58: 2940-2945. Mitchell, R.E., 1984. The relevance of non-host specific toxins in the expression of virulence by plant pathogens. Annu. Rev. Phytopathol,, 22: 83-245. Mitchell, R.E., 1991. Implications of toxins in the ecology and evolution of plant pathogenic microorganisms: Bacteria. Experientia, 47: 791-803. Mitchell, RE. and Bielski, R.L., 1977. Involvement of phaseolotoxin in halo blight of beans. Transport and conversion to functional toxin. Plant Physiol., 60: 723-724. Moore, R.E., Niemczura, W.P., Kwok, O.H.C. and Patil, S.S., 1984. lnhibitors of ornithine carbamoyltransferase from Pseudomonas syringae pv. phaseolicola. Revised structure of phaseolotoxin. Tetrahedron Lett., 25: 3931-3934. Mosqueda, G., Van den Broek, G., Saucedo, O., Bentley, A.M., Alvarez-Morales, A. and Herrera-Estrella, L., 1990. Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase. Mol. Gen. Genet., 222:461-466. Nordeen, R.O., Sinden, S.L., Jaynes, J.M. and Owens, L.D., 1992. Activity of cecropin SB37 against protoplasts from several plant species and their bacterial pathogens. Plant Sci., 82: 101-107. Norelli, J., Aldwinkle, H., Destefano-Beltran, L. and Jaynes, J.M., 1993. Increasing the fire blight resistance of apple by transformation with genes encoding lyric proteins. 6th Int. Congr. Plant Patbol., Montreal, Canada, Book of Abstracts, Poster 10.2.16 (288), p. 164. Patil, S.S., Kolatukudy, P. and Diamond, A.E., 1970. Inhibition of ornithine carbamyltransferase from bean plants by the toxin of Pseudomonas phaseolicola. Plant Physiol., 46: 752-753. Patil, S.S., Hayward, A.C. and Emmons, R., 1974. An ultraviolet induced nontoxigenic mutant of Pseudomonas phaseolicola of altered pathogencity. Phytopathology, 64: 590-595. Peet, R.C. and Panopoulos, N.J., 1987. Ornithine carbamoyl-transferase genes and phaseolotoxin immunity in Pseudomonas syringae pv. phaseolicola. EMBO J., 6: 3585-3591. Peet, R.C., Lindgren, P.B. and Panopoulos, N.J., 1986. Genetic analysis of phaseolotoxin production by Pseudomonas syringae pv. phaseolicola. J. Bacteriol., 166:1096-1105. Quigley, N.B., Lane, D. and Bergquist, P.L., 1985. Genes for phaseolotoxin synthesis are located on the chromosome of Pseudomonas syringae pv. phaseolicola. Curt. Microbiol., 12: 295-300. Sanford, J.C. and Johnston, S.A., 1985. The concept of parasitederived resistance deriving resitance genes from the parasite's own genome. J. Tbeor. Biol., 113: 395-405. Selsted, M.E., Brown, D.M., DeLange, R.G., Hatwig, S.S.L. and Lehrer, R.I., 1985. Primary structures of six antimicrobial peptides of rabbit peritoneal neutrophils. J. Biol. Chem., 260: 4579-4584. Shargool, P.D., Jain, J.C. and McKay, G., 1988. Oruithine biosynthesis, and arginine biosynthesis and degradation in plant cells. Phytochemistry, 27: 1571-1574. Smith, M.J., Mazzola, EP., Sims, J.J., Midland, S.L., Keen, N.T.,

97

Butron, V. and Stayton, M.M., 1993. The syringolides: bacterial C-glycosyl lipids that trigger plant disease resistance. Tetrahedron Lett., 34: 223-226. Steiner, H., 1982. Secondary structure of the cecropins: antibacterial peptides from the moth Hyalophora cecropia. FEBS Lett., 137: 283-287. Stintzi, A., Geoffroy, D., Bersuder, D., Fritig, B. and Legrand, M., 1993. cDNA cloning and expression studies of tobacco class ill chitinases-lysozymes. In: B. Fritig and M. Legrand (Editors), Developments in Plant Pathology, Vol. 2: Mechanisms of Plant Defense Responses. Proceedings of the 2nd EFPP Conference, Strasbourg, France. Kluwer Academic, Dordrecht, pp. 312-315. Surico, G. and lakobelis, N.S., 1992. Phytohormones and olive knot disease. In: D.P.S. Verma (Editor), Molecular Signals in Plant-Microbe Communication. CRC Press, Boca Raton, FL, pp. 209-227. Templeton, M.D., Mitchel, R.E., Sullivan, P.A. and Shepherd, M.G., 1985. The inactivation of ornithine transcarbamoylase by N'L(N'-sulphodiaminophosphinyl)-Lomithine. Biochem. J., 228: 347-352. Trudel, J., Potvin, C. and Asselin, A., 1992. Expression of active hen egg white lysozyme in transgenic tobacco. Plant Sci., 87: 55-67. Trudel, J., Potvin, C., Grenier, J. and Asselin, A., 1993. Expression of various hen egg white lysozyme constructs in transgenic tobacco and potato. 6th Int. Congr. Plant Pathol., Montreal, Canada, Poster 10.2.30. Tsugita, A., 1971. Phage lysozyme and other lytic enzymes. In: P.D. Boyer (Editor), The Enzymes, Vol. 5. Academic Press, New York, pp. 344-411. Turner, J.G. and Debbage, J.M., 1982. Tabtoxin-induced symptoms are associated with accumulation of ammonia formed during photorespiration. Physiol. Plant Pathol., 20: 223-233. Ucbytil, T.F. and Durbin, R.D., 1980. Hydrolysis of tabtoxin by plant and bacterial enzymes. Experientia, 47: 765-770. Vining, L.C., 1990. Functions of secondary metabolites. Annu. Rev. Microbiol., 44: 395-427. Vivian, A. and Mansfield, J., 1993. A proposal for a uniform genetic nomenclature for avirulence genes in phytopathogenic pseudomonads. Mol. Plant Microb. Interact., 6: 9-10. Willis, D.K., Barta, T.M. and Kinscherf, T.G., 1991a. Genetics of toxin production and resistance in phytopathogenic bacteria. Experientia, 47: 765-771. Willis, D.K., Rich, J.J. and Hraback, E.M., 1991b. hrp genes of phytopathogenic bacteria. Mol. Plant Microb. Interact., 4: 132-138. Yoder, O.C., 1980. Toxins in pathogenesis. Annu. Rev. Phytopathol., 18: 107-129. Zasloff, M., 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active froms, and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA, 84: 5449-5453. Zhang, Y., Rowley, K.B. and Patil, S.S., 1993. Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola. J. Bacteriol., 175: 6451-6458.

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.