John A. Lucas

Plant Proteins that Confer Resistance to Pests and Pathogens

Publisher Summary Throughout their lives, from dormant seed to maturity, plants are exposed to attack by a wide variety of potentially pathogenic microorganisms, predatory insects and other invertebrate pests. Plant resistance is often divided into constitutive defense, expressed as a normal feature of plant development, or inducible defense, switched on following contact with the invading organism. A fundamental distinction is often drawn between pre–existing or constitutive features of the plant, and inducible systems switched on following challenge by a pest or pathogen. Lectins bind to chitin and other glycoconjugates containing N–acetylglycosamine or N–acetylneuraminic acid, and also cause the agglutination of mammalian red blood cells. The most widely studied graminaceous lectin is wheat germ agglutinin, an M r 36000 protein consisting of two identical 171–residue chains. Thionins were first purified from wheat flour in the early 1940s and called purothionins. Lysozymes from egg white and bacteriophage are among the most widely studied proteins; they inhibit bacterial growth by hydrolysis of cell–wall peptidoglycans. In devising strategies for the manipulation of defense proteins in transgenic plants it is essential to consider the behavior of the target organism, as well as the construct to be used. The specificity of protective proteins for different groups of insects and other invertebrates provides an opportunity to develop resistances that are either highly specific or have wide spectra, based on individual proteins or combinations. A combination of protective proteins may also assist in preventing the development of resistance to individual components.

Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.

Somatic embryogenesis and plant regeneration in Floribunda rose (Rosa hybrida L.) cvs. Trumpeter and Glad Tidings

Abstract Somatic embryogenic callus was initiated from in vitro-derived petiole and root explants, but not leaves, of the Floribunda rose cultivars Trumpeter and Glad Tidings following repeated subculture of callus on Schenk and Hildebrandt (SH) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). The use of a high auxin pretreatment increased the frequency of somatic embryogenesis, whilst l -proline, as a media supplement, had a critical role in enhancing somatic embryogenesis in the early stages of culture but increased the frequency of abnormal embryo formation when used in the later stages of culture. Maturation of somatic embryos was achieved by transfer to SH medium with reduced concentrations of 2,4-D and incorporation of abscisic acid (ABA) and 6-benzylaminopurine (BAP). The use of SH medium containing BAP and indole-3-butyric acid (IBA) facilitated embryo germination which was also enhanced by cold treatment. Phenotypically normal plants were recovered from germinated somatic embryos and successfully transferred to the glasshouse for flowering.

Protection of Brassica seedlings against downy mildew and damping-off by seed treatment with CGA 245704, an activator of systemic acquired resistance

Control of seedling diseases is a major priority in many crop systems. Seed treatments that induce systemic resistance after seedling emergence may be an ideal way to provide protection against disease during the establishment of the crop. CGA 245704, a chemical activator of systemic acquired resistance, was tested as a seed treatment against two Brassica diseases with contrasting infection biologies, the airborne downy mildew pathogen, Peronospora parasitica, and the soilborne fungus, Rhizoctonia solani. Seeds of two Brassica spp. were either imbibed with various concentrations of the compound or imbibed and then dried. Both the imbibition treatment alone and the imbibition treatment followed by seed drying had a significant effect on the sporulation intensity of P. parasitica for all concentrations of the compound used, whereas the imbibition treatment provided some control of damping-off caused by R. solani, with the degree of control being highly dependent on the concentration applied to the seed. Seed treatment with the plant activator CGA 245704 might therefore simultaneously control several seedling diseases, thereby providing a novel option for management of these diseases.

Pathogenicity, host-specificity, and population biology of Tapesia spp, causal agents of eyespot disease of cereals.

Abstract Eyespot is an important stem-base disease of cereal crops in temperate regions. Cultural, genetic and molecular criteria have been used to separate the fungi responsible into two species, Tapesia yallundae (previously W-type) and T. acuformis (previously R-type). Discovery of the apothecial sexual stage (teleomorph) of Tapesia yallundae on straw stubble was a key factor in this change for a pathogen previously believed to be asexual (anamorph: Pseudocercosporella herpotrichoides ). Sexual reproduction is controlled by a two-allele heterothallic system in both species, although mating appears to be rare in T. acuformis . Infection of cereal hosts is achieved by formation of multicellular plaques, and the colonization process is described. The host range includes wild grass species as well as small-grain cereals, and new genetic sources of resistance to the disease have been identified in Triticum species and wild relatives. Recent advances in the molecular genetics of the pathogens will aid analysis of pathogenic variation in eyespot.

Plant immunisation: from myth to SAR†

The idea that plants might be able to develop a form of acquired immunity to infection following exposure to a pathogen has been current ever since discovery of the animal immune system in the later years of the nineteenth century. Early attempts to demonstrate a comparable system in plants focused on the detection of precipitating antibodies and hence were doomed to failure. Nevertheless, largely anecdotal evidence for plant immunisation continued to accumulate, culminating in the discovery of phytoalexins in the 1940s. Convincing evidence for systemic changes in plant resistance following an inducer inoculation was not available until 20 years later, when pioneering work on tobacco infected with blue mould (Peronospora tabacina) or tobacco mosaic virus (TMV) showed that tissues remote from the inoculation site were altered in disease reaction type. Increased resistance was expressed as a reduction in lesion numbers and size, and a reduced rate of pathogen reproduction. Systemic acquired resistance (SAR) has now been demonstrated in at least 20 plant species in at least six plant families, although detailed genetic or molecular analysis has mainly been confined to a few models, such as tobacco, cucumber and Arabidopsis. SAR is associated with the coordinate induction of genes encoding defence proteins which can be used as molecular markers of the response. The availability of Arabidopsis mutants altered in the induction and expression of SAR is now providing new insights into the signal transduction pathway(s) involved, and will enable comparison with the molecular mechanisms operating in other plant taxa. Important unresolved questions concern the nature of the translocated signal, the mechanism of defence priming, efficacy of the response against different pathogens, and practical exploitation of SAR in crop protection. The first generation of chemical plant defence activators is now commercially available and optimal use of these SAR inducers in integrated disease control requires further evaluation. The prospects for engineering transgenic crops altered in the regulation or expression of SAR is also a subject for further investigation.

Effects of plant defence activators on anthracnose disease of cashew

The plant defence activators acibenzolar-S-methyl (Benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester, ASM), 2,6-dichloro-isonicotinic acid (DCINA), salicylic acid (SA), and dibasic potassium phosphate (K2HPO4) were tested for their ability to protect cashew (Anacardium occidentale) seeds and leaves from anthracnose disease caused by Colletotrichum gloeosporioides. No inhibition of the early stages of pathogen development was caused by concentrations equal to or lower than 1.1mM a.i. ASM, 1.2mM a.i. DCINA, 5mM SA and 50mM K2HPO4. Maximum reduction of the disease in detached leaves, without phytotoxic effects, was obtained with 0.07mM a.i. ASM and DCINA, 5mM SA, and 50mM K2HPO4, with a time interval of at least 72h between application of the activator and inoculation with the pathogen. On attached leaves, foliar sprays were slightly more efficient than soil drench treatments, with 5mM SA being the most effective treatment, while 50mM SA as well as 0.3mM a.i. ASM and DCINA caused phytotoxic effects. In field-grown plants, protection was conferred by a soil drench of concentrations as low as 12.6μM a.i. ASM and DCINA and 2.6mM SA. These concentrations were not phytotoxic suggesting that plant defence activators have potential for control of anthracnose disease in the field.

Use of an isocitrate lyase promoter-GFP fusion to monitor carbon metabolism of the plant pathogen Tapesia yallundae during infection of wheat.

Abstract Green fluorescent protein (GFP) has been used as a vital marker in a variety of species. Here, we present the use of a GFP-promoter fusion to visualize carbon metabolism in a pathogenic fungus during growth on defined medium and during infection of plants. Isocitrate lyase (ICL), a key enzyme in carbon metabolism, is tightly regulated at the transcriptional level, with high levels of expression during 2-carbon growth and no expression during growth on glucose. A GFP-ICL promoter fusion was used to visualize carbon metabolism in the plant pathogenic fungus Tapesia yallundae during growth in vitro and in the host plant. The ICL promoter from Neurospora crassa retained its native induction and repression characteristics in T. yallundae. Loss of GFP fluorescence from hyphae after repression of the ICL promoter suggested a rapid turnover rate for GFP in T. yallundae. Regulation of this promoter was observed during infection, with expression occurring only on the plant surface, suggesting that 2-carbon metabolism occurs during this phase. These data suggest that GFP is a useful vital marker for the in planta imaging of fungal metabolism.

Genetic control of resistance to the sterol 14α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae.

ABSTRACT Sexual crosses were used to determine the genetic basis of resistance to the sterol 14 α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Three different crosses between sensitive parental strains (22-432 and 22-433 [the concentration required to inhibit growth by 50% {IG50} for each was ≤0.03 mg/liter]) and field isolates from France and New Zealand with differing levels of resistance (PR11 [IG50 = 0.5 mg/liter], PR1 [IG50 = 1.0 mg/liter], and 11-3-18 [IG50 = 2.4 mg/liter]) yielded progeny showing a bimodal distribution, with an even number of sensitive and resistant progeny. This indicated the segregation of a single major gene for resistance in each cross, which was confirmed by the use of backcrosses, crosses between F1 progeny, and control crosses between sensitive parents. However, there was also evidence of additional quantitative genetic components responsible for the increased IG50s of the more resistant isolates. A further cross was made between isolate PR11 and an F1 progeny arising from isolate 11-3-18, and this also yielded progeny which were entirely prochloraz resistant. This suggested that resistance genes were allelic in these two isolates, with resistance conferred by a gene at the same locus (or closely linked loci), despite the fact that the isolates (PR11 and 11-3-18) originated from different continents.

Cultivar resistance to anthracnose disease of cowpea (Vigna unguiculata (L.) Walp.) caused by Colletotrichum destructivum O'Gara

The infection process of Colletotrichum destructivum, a hemibiotrophic anthracnose fungus, was studied by light microscopy in two cowpea (Vigna unguiculata) cultivars which differ in disease reaction type. Large, multilobed, intracellular infection vesicles, followed by necrotrophic, radiating, secondary hyphae were produced in tissues of the susceptible cv. IT82E-60. In the resistant cv. TVxu20093236, both the production of appressoria and their melanisation were impaired, resulting in reduced penetration. Where penetration occurred, the initially-infected epidermal cells underwent a hypersensitive response, restricting the growth of multilobed vesicles and thereby blocking the destructive necrotrophic phase of disease development. The phytoalexins kievitone and phaseollidin accumulated earlier and more rapidly in stem tissues of the resistant cultivar, associated with the appearance of delimited, necrotic spots on inoculated surfaces. In contrast, delayed and slower accumulation of these compounds occurred in the compatible interaction, together with the development of typical spreading, water-soaked, anthracnose lesions.