Alan Slusarenko

Acquired resistance in Arabidopsis.

Acquired resistance is an important component of the complex disease resistance mechanism in plants, which can result from either pathogen infection or treatment with synthetic, resistance-inducing compounds. In this study, Arabidopsis, a tractable genetic system, is shown to develop resistance to a bacterial and a fungal pathogen following 2,6-dichloroisonicotinic acid (INA) treatment. Three proteins that accumulated to high levels in the apoplast in response to INA treatment were purified and characterized. Expression of the genes corresponding to these proteins was induced by INA, pathogen infection, and salicylic acid, the latter being a putative endogenous signal for acquired resistance. Arabidopsis should serve as a genetic model for studies of this type of immune response in plants.

Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica

Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)-[beta]-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific for the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.

Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava)

Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3′ and 5′ rapid amplification of cDNA ends. The sequence shows approximately 60–70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-β-d-thiogalactopyranoside and σ-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was≈2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.

Signalling molecules and the synthesis of alkaloids in Catharanthus roseus seedlings

The Madagascar periwinkle, Catharanthus roseus, produces numerous alkaloids, several of which have important pharmaceutical uses. Catharanthus seedlings rapidly accumulate the monomeric alkaloids, vindoline, catharanthine and tabersonine, during germination. Various plant signalling molecules were tested for their ability to enhance alkaloid synthesis in Catharanthus seedlings. The compounds tested included plant hormones, fatty acid-derived messengers and agents that can induce systemic-acquired resistance in plants. Of these compounds, only methyl jasmonate (MeJa) enhanced the synthesis of monomeric alkaloids. However, feeding of a MeJa biosynthetic precursor, or inhibition of the lipoxygenase pathway, had no effect on alkaloid production in the seedlings. We conclude that, although MeJa is able to enhance alkaloid synthesis when supplied exogenously, the lipoxygenase pathway probably does not play a role in the regulation of alkaloid synthesis during normal germination of Catharanthus. Furthermore, it was found that Catharanthus seedlings accumulate small quantities of the dimeric alkaloid, vinblastine, a valuable antitumour drug. Contrary to its effect on the accumulation of monomeric alkaloids, MeJa did not influence the accumulation of vinblastine.

Arabidopsis as a model host for studying plant-pathogen interactions

Because the molecular biology and genetics of Arabidopsis thaliana are so well defined, it is potentially a superb subject for research on plant-pathogen interactions. Viruses, bacteria and fungi that infect Arabidopsis and are representative pathogens of economically important plants have recently been described. The search now is for a pathogenic fungus with tractable genetics to combine with a direct analysis of plant resistance genes.

cDNA and derived amino acid sequence of a cytosolic Cu,Zn superoxide dismutase fromArabidopsis thaliana (L.) Heyhn

Copper,zinc superoxide dismutase (SOD, E.C. 1.15.1.1) has been found in all eukaryotic organisms so far examined. It greatly accelerates the dismutation of superoxide anions (2-) to molecular oxygen and hydrogen peroxide, and thus serves to protect cells from superoxide stress which can arise by a number of means, e.g. drought or herbicide treatment [7], pathogen attack [2, 6, 8] and ionizing radiation [9]. We are interested in the role SOD might play in the interaction between Arabidopsis thaliana and Peronospora parasitica, which causes downy mildew in crucifers [6] and to this end we have isolated cDNA and genomic clones from Arabidopsis libraries using SOD probes from tomato [10]. In this report we present statistical data for a cDNA encoding a putative cytosolic Cu,Zn SOD and compare its relatedness to other published SOD sequences. A 32p-labelled 450 bp fragment from the coding region of tomato cytoplasmic Cu,Zn SOD [10] was used to probe an A. thaliana ecotype Columbia 2gt l l library (Clontech) at tow stringency (2 x SSPE, 0.1~o SDS at 42 °C). Positive clones were subcloned into pBluescript (SK + ) and the longest inserts taken for further characterisation. One clone (pCSODRH) was sequenced by the dideoxy method using a Sequenase kit (United States Biochemical Corp.) with ssDNA rescued from the phagemid by helper phage infection. Both strands were completely sequenced using the KS and SK primers and synthetic oligonucleotides. The nucleotide and deduced amino acid sequence is shown in Fig. 1. The 774 nucleotide long cDNA (without the poly(A) tail) contains an open reading frame beginning with the first ATG initiation codon at position 113 and ending with a TAA stop codon at position 569. There is a putative poly(A) additional signal (AATAAA) from nucleotides 750755. The cDNA encodes a protein of 152 amino acids with a relative molecular weight (Mr) of 15 098 and a calculated pI of 5.09. The Cu,Zn SOD metal binding signatures [ 1 ] were found at amino acids 42 to 50 and 59 to 65 and are indicated in Fig. 1 by horizontal bars above the sequence. Sequence comparison of this protein with the entries of the Swiss-Prot database revealed that it is most similar to a cytosolic Cu,Zn SOD from Brassica oleracea [12]. The Arabidopsis sequence shows

Downy Mildew of Arabidopsis Thaliana Caused by Peronospora Parasitica: A Model System for the Investigation of the Molecular Biology of Host-Pathogen Interactions

The results of crosses between resistant and susceptible parents are reported and a strategy to clone and characterise genes associated with resistance in Arabidopsis is described. The use of mutants to elucidate the putative signal transduction pathway from perception of the pathogen to activation of defence genes is also discussed. Preliminary results of experiments to characterise Arabidopsis defence mechanisms are reported, for example the production of phytoalexins, hydrolytic and oxidative enzymes, and callose encasement of haustoria.

27 Microbial Pathogenesis of Arabidopsis

A PRIMER ON PLANT PATHOGENESIS In common with all other terrestrial angiosperms, Arabidopsis provides ecological niches for an array of microorganisms. They may inhabit the aerial parts of the plant or be confined to the roots; they may live within the plant or on its outer surfaces; and they may have detrimental, beneficial, or neutral effects. In this chapter we consider the relationships between Arabidopsis and its pathogens; that is, microorganisms causing overt symptoms of disease. Plant pathogens are in no sense a biologically homogeneous assemblage of organisms, and their diversity is well illustrated by the fungi and bacteria, listed in Tables 1 and 2, respectively, capable of parasitic growth on Arabidopsis. Despite this biological diversity, there is well-founded optimism that studies of Arabidopsis as a host to a variety of pathogens will facilitate a deeper understanding of common processes in microbial pathogenesis of plants. Modes of Parasitism Microbial plant parasites obtain their nutrients either “biotrophically” from living cells or “necrotrophically” from cells which they have killed (Lewis 1973). Examples of the former include fungi, such as powdery and downy mildews, and mollicutes, such as mycoplasma-like organisms which are obligately biotrophic and have not thus far been cultured on any synthetic medium. These obligate biotrophs invade and extensively colonize susceptible plants in such a way that host cells either remain alive or die only after the pathogen has grown on to exploit other living cells. Many of the most destructive plant pathogenic fungi (e.g., Botrytis spp. and Pythium spp.) and...